C. Kalidas
M.V. Sangaranarayanan
Biophysical
Chemistry
Techniques and Applications
Biophysical Chemistry
C. Kalidas · M. V. Sangaranarayanan
Biophysical Chemistry
Techniques and Applications
C. Kalidas
Department of Chemistry
Indian Institute of Technology Madras
Chennai, India
M. V. Sangaranarayanan
Department of Chemistry
Indian Institute of Technology Madras
Chennai, India
ISBN 978-3-031-37681-8
ISBN 978-3-031-37682-5 (eBook)
https://doi.org/10.1007/978-3-031-37682-5
Jointly published with Ane Books Pvt. Ltd.
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ISBN of the Co-Publisher’s country edition: 978-93-90658-81-7
1st edition: © Authors 2022
© The Author(s) 2023
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Preface
The subject of Biophysical Chemistry is relatively new in the Indian context
although it is well established in the Western world. This perspective is
based on the experience of one of the authors viz. the institute where he
spent a year (1968–69) was “Max-Planck Institute für Physikalische
Chemie, Göttingen, Germany” but was later renamed as “Max-Planck Institute für Biophysikalische Chemie” by 1976–77 when he visited the same
institute again as a senior fellow of Alexander von Humboldt Foundation.
This reflects the importance of the subject which opened up a wide variety
of areas of research such as molecular biology, cellular biochemistry, physical biochemistry, neurobiology, and ultra fast dynamics to name a few.
Realizing the importance of this area, the authors embarked on the
preparation of this book.
The book “Biophysical Chemistry” provides a pedagogical outline of
various topics with a few selected applications. The essential objective in
this endeavour has been to include a large number of diverse topics customarily covered in any physical chemistry course, albeit with a biochemical flavour. In view of this, extensive courage of the entire amount of
biophysical chemistry is not pursued here. Nevertheless, it is hoped that
the present text will provide essential elements of biophysical chemistry
which will enable the reader to delve deeper into various advanced level
monographs.
There exist many classical textbooks in biophysical chemistry (for
example, Biophysical chemistry, D. Klostermeier and Markus G. Rudolph).
However, the present book is intended to cover most of the physical chemistry contents, essential for comprehending biophysical phenomena viz.
non-equilibrium thermodynamics (chapter 2), bioenergetics (chapter 11),
v
vi Biophysical Chemistry
chemical kinetics (chapters 18 to 21). It is quite difficult to decouple physical chemistry concepts from structural considerations of biological phenomena. In view of this, Part I, provides an elementary description of
the structure of the cell (cell and its biochemical setup (chapter 1)), carbohydrates (chapter 3), lipids (chapter 4), amino acids (chapter 5), peptides
(chapter 6), proteins (chapter 7), nucleosides and nucleotides (chapter 8),
enzymes (chapter 9), co-enzymes and vitamins (chapter 10). The emphasis
in these chapters consists in providing structural considerations pertaining
to diverse components of the cell.
In Part II, different bioanalytical techniques have been described with
illustrative applications viz. electrochemical methods (chapter 12), spectroscopic techniques (chapters 13, 16, 17, 22, 29 to 31), calorimetric methods
(chapters 23 and 24) analytical methods based on mobility (chapters 14, 15,
25 to 28). The recently discovered genome editing is briefly summarized
in chapter 32.
In each chapter, different types of questions have been provided. In
some cases, numerical questions with detailed solution have been provided. In the opinion of authors, this book will be of interest to students of
a variety of disciplines such as physical chemistry, biochemistry, medicine
and neurobiology, etc.
The authors and publishers have made sincere efforts to trace the copyright holders. If any copyright material has not been acknowledged, it may
please be brought to our notice so as to rectify this inadvertent omission in
future reprints. The authors request that such omissions be brought to their
attention so that due acknowledgements maybe made subsequently.
Thanks are due to D.V. Kirana, K.V. Akshaya, P.P. Archana,
Dr. Hema Chandra Kotamarthi and Prof. K.M. Muraleedharan of IIT
Madras, Dr. A. Muthukrishnan of IISER Thiruvananthapuram and Prof.
Nandita Madhavan of IIT Bombay for helpful suggestions. It is a pleasure to acknowledge Ane Books Pvt. Limited for their consistent support
during the entire preparation of the book.
The authors thank their families for the understanding, patience and
support given during this methodic, painstaking and formidable effort.
C. Kalidas
M.V. Sangaranarayanan
Table of Contents
List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
List of Symbols and Acronyms . . . . . . . . . . . . . . . . . . . . . . .
Part I
1
2
Different Types of Biomolecules
Cell and its Biochemical Setup
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2 Structure of an Animal Cell . . . . . . . . . . . . . . . . .
1.3 Composition of Human Cell . . . . . . . . . . . . . . . . .
1.4 Eukaryotic and Prokaryotic Cells . . . . . . . . . . . . . .
1.5 Membrane-Bound Organelles . . . . . . . . . . . . . . . .
1.6 Comparison Between Lysosome and Ribosome . . . . . .
1.7 Types of Cells in Human Body . . . . . . . . . . . . . . .
1.8 Peripheral Proteins . . . . . . . . . . . . . . . . . . . . . .
1.9 Transport Across Cell Membrane . . . . . . . . . . . . . .
1.10 Facilitated Diffusion . . . . . . . . . . . . . . . . . . . . .
1.11 Permeability of Molecules Across Phospholipid Bilayers
1.12 Thermodynamic basis of Transport . . . . . . . . . . . . .
1.13 Examples of Antiport and Symport System . . . . . . . .
1.14 Ca2+ ATPase . . . . . . . . . . . . . . . . . . . . . . . . . .
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xxix
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Thermodynamic Aspects of the Cell
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2 Enthalpy and Free Energy Changes in
Biochemical Processes . . . . . . . . . . . . . . . . . . . . .
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3
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5
8
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10
11
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25
27
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viii 2.3
2.4
2.5
2.6
2.7
2.8
2.9
2.10
2.11
2.12
2.13
2.14
2.15
2.16
2.17
2.18
2.19
2.20
2.21
2.22
2.23
3
Biophysical Chemistry
Effects of Electrochemical Changes . . . . . . . . . . . . . .
Distribution of Ions Across Membranes . . . . . . . . . . .
Distribution of Ions Near Charged Membranes
and Macromolecules . . . . . . . . . . . . . . . . . . . . . .
Osmotic Effects . . . . . . . . . . . . . . . . . . . . . . . . .
Role of Chemical Potential . . . . . . . . . . . . . . . . . . .
Effects of Different Molecular Environments and Phases
on Energy and Entropy . . . . . . . . . . . . . . . . . . . . .
Surface Free Energies and Surface Tension . . . . . . . . . .
Molecular Aggregation . . . . . . . . . . . . . . . . . . . . .
Non-equilibrium Thermodynamic Treatment of
Bacterial Cells . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacterial Cell as a Thermodynamic System . . . . . . . . .
Physical and Chemical Features of the Cell
and Environment . . . . . . . . . . . . . . . . . . . . . . . .
Concept of Steady State . . . . . . . . . . . . . . . . . . . .
Non-equilibrium Thermodynamics in Microbiology . . . .
Linear Laws . . . . . . . . . . . . . . . . . . . . . . . . . . .
Force and Flows in Living Systems . . . . . . . . . . . . . .
Chemical Potential and Mass Transfer: Activated Support
Applicability of Linear Laws . . . . . . . . . . . . . . . . . .
Non-linear Descriptions . . . . . . . . . . . . . . . . . . . .
Simple Cell Functions in Non-equilibrium
Thermodynamics . . . . . . . . . . . . . . . . . . . . . . . .
Batch Cultures . . . . . . . . . . . . . . . . . . . . . . . . . .
Thermodynamic Concepts . . . . . . . . . . . . . . . . . . .
Carbohydrates, their Reactions, Thermochemistry
and Energetics
3.1 Introduction . . . . . . . . . . . . . . . . . . . .
3.2 General Properties of Monosaccharides . . . .
3.3 Other Monosaccharides . . . . . . . . . . . . .
3.4 Glycosides . . . . . . . . . . . . . . . . . . . . .
3.5 Oligosaccharides . . . . . . . . . . . . . . . . .
3.6 Polysaccharides . . . . . . . . . . . . . . . . . .
3.7 Cell Walls of Bacteria . . . . . . . . . . . . . . .
3.8 Thermochemistry of Carbohydrates . . . . . .
3.9 Gibbs Free Energy Changes for Reactions . . .
3.10 Enolic Phosphate . . . . . . . . . . . . . . . . .
3.11 Guanidinium Phosphates . . . . . . . . . . . .
3.12 Carbohydrates and Microbial Fuel Cells . . . .
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Table of Contents 4
5
6
7
ix
Lipids
4.1 Introduction . . . . . . . . . . . . . . . . . . .
4.2 Properties of Lipids . . . . . . . . . . . . . . .
4.3 Bonding in Lipids . . . . . . . . . . . . . . . .
4.4 Classification . . . . . . . . . . . . . . . . . . .
4.5 Reactions . . . . . . . . . . . . . . . . . . . . .
4.6 Analysis of Lipids . . . . . . . . . . . . . . . .
4.7 Nomenclature of Phospholipids . . . . . . . .
4.8 Lipoproteins . . . . . . . . . . . . . . . . . . .
4.9 Role of Lipids in Cell Function . . . . . . . .
4.10 Distribution of Lipids . . . . . . . . . . . . . .
4.11 Physicochemical Data on Lipids . . . . . . . .
4.12 Lipid Bi-layers . . . . . . . . . . . . . . . . . .
4.13 Glycolipids on the Surface of all Membranes
4.14 Interfacial Studies of Lipid Bilayers . . . . . .
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87
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107
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Amino Acids
5.1 Introduction . . . . . . . . . . . . . . . . .
5.2 Classification of Amino Acids . . . . . . .
5.3 Chirality of Amino Acids . . . . . . . . . .
5.4 Acid-base Properties . . . . . . . . . . . .
5.5 Reactions of Amino Acids . . . . . . . . .
5.6 Biochemical Importance of Amino Acids
5.7 Electrochemical Studies of Amino Acids .
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115
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Peptides
6.1 Introduction . . . . . . . . . . . . . . . . . . . . .
6.2 Classes of Peptides . . . . . . . . . . . . . . . . .
6.3 Functions of Peptides . . . . . . . . . . . . . . . .
6.4 Acid-Base Properties of Peptides . . . . . . . . .
6.5 Peptides as Biosensors . . . . . . . . . . . . . . .
6.6 Current-Voltage Characteristics of Peptide Films
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137
Proteins
7.1 Introduction . . . . . . . . . . . . . .
7.2 Composition of Proteins . . . . . . .
7.3 Some Characteristics of Proteins . .
7.4 Classification of Proteins . . . . . . .
7.5 Nature of Bonds in Protein Structure
7.6 Structure of Proteins . . . . . . . . .
7.7 Role of Amino Acids in Proteins . .
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x
Biophysical Chemistry
7.8
7.9
7.10
7.11
7.12
7.13
7.14
7.15
7.16
7.17
7.18
8
9
Examples of Proteins . . . . . . . . . . . . .
Hemoglobin . . . . . . . . . . . . . . . . . .
Antibodies . . . . . . . . . . . . . . . . . . .
Hormones . . . . . . . . . . . . . . . . . . .
Denaturation of Proteins . . . . . . . . . . .
Helix-Coil Transitions in Proteins . . . . . .
Kinetics of Helix-Coil Transformation . . .
Membrane Proteins . . . . . . . . . . . . . .
Modelling of Tertiary Structure of Proteins
Levinthal Paradox . . . . . . . . . . . . . . .
Proteins in Nutrition . . . . . . . . . . . . .
Nucleosides and Nucleotides
8.1 Introduction . . . . . . . . . . . . . . . . .
8.2 Biological Function (DNA & RNA) . . . .
8.3 Examples of Nucleotides . . . . . . . . . .
8.4 Naming of Nucleosides and Nucleotides
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Enzymes
9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . .
9.2 Different Types of Enzymes . . . . . . . . . . . . . . . . .
9.3 Nature of Enzyme Action . . . . . . . . . . . . . . . . . .
9.4 Michaelis-Menten Mechanism . . . . . . . . . . . . . . . .
9.5 Effect of Temperature on Enzyme-catalysed Reactions . .
9.6 Specificity of an Enzyme . . . . . . . . . . . . . . . . . . .
9.7 Classification of Enzymes . . . . . . . . . . . . . . . . . .
9.8 Inhibitors of Enzymes . . . . . . . . . . . . . . . . . . . .
9.9 Reversible Inhibition . . . . . . . . . . . . . . . . . . . . .
9.10 Uncompetitive Inhibition . . . . . . . . . . . . . . . . . .
9.11 Allosteric Enzymes . . . . . . . . . . . . . . . . . . . . . .
9.12 Oligomeric Enzymes . . . . . . . . . . . . . . . . . . . . .
9.13 Isoenzymes . . . . . . . . . . . . . . . . . . . . . . . . . .
9.14 Bifunctional Oligomeric Enzymes . . . . . . . . . . . . . .
9.15 Multienzyme Complexes . . . . . . . . . . . . . . . . . . .
9.16 Modification of the Specificity of an Oligomeric Enzyme
9.17 Measurement of Enzymatic Activity of Lactose
Dehydrogenase (LDH) obtained from
Different Organisms . . . . . . . . . . . . . . . . . . . . .
9.18 Turn Over Rates (T.O.R) of Some Enzymes . . . . . . . .
9.19 Immobilisation of Enzymes . . . . . . . . . . . . . . . . .
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Table of Contents 10 Co-Enzymes and Vitamins
10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . .
10.2 Relation between Co-enzymes and Vitamins . . . .
10.3 Vitamins . . . . . . . . . . . . . . . . . . . . . . . . .
10.4 Biochemical Functioning of the B-Vitamin Group . .
10.5 Biochemical Function of Biotin . . . . . . . . . . . .
10.6 Adsorption of Riboflavin . . . . . . . . . . . . . . . .
10.7 Surface Tension Data of Complexes with Vitamin-A
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217
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11 Bioenergetics
11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . .
11.2 Metabolism . . . . . . . . . . . . . . . . . . . . . . . .
11.3 Coupled Reactions . . . . . . . . . . . . . . . . . . . .
11.4 ATP as an Energy Source . . . . . . . . . . . . . . . . .
11.5 High Phosphoryl Capacity of ATP . . . . . . . . . . .
11.6 Significance of Phosphoryl Transfer Potential (PTP) .
11.7 Intracellular Conditions Pertaining to ATP Hydrolysis
11.8 Methods by which ATP Transfers Energy . . . . . . .
11.9 Citric Acid Cycle . . . . . . . . . . . . . . . . . . . . .
11.10Reactions of the TCA Cycle . . . . . . . . . . . . . . .
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12 Biosensors
12.1 Introduction . . . . . . . . . . . . . . . . . .
12.2 Components of a Biosensor . . . . . . . . .
12.3 Different Types of Biosensors . . . . . . . .
12.4 Electrochemical Biosensors . . . . . . . . .
12.5 Enzymatic Sensing of Glucose . . . . . . . .
12.6 Estimation of Michaelis-Menten Constants
12.7 Non-enzymatic Sensing of Glucose . . . . .
12.8 Enzymatic Sensing of Urea . . . . . . . . . .
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271
Part II
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Biochemical Techniques
13 Surface Plasmon Resonance Spectroscopy
13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . .
13.2 Details of SPR Set Up . . . . . . . . . . . . . . . . . .
13.3 Surface Plasmon Resonance and Refractive Indices .
13.4 Kinetic Applications of SPR Spectroscopy . . . . . .
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xii Biophysical Chemistry
14 Affinity Chromatography
14.1 Introduction . . . . . . . . . . . . . . . . . . . . . .
14.2 Methodology . . . . . . . . . . . . . . . . . . . . .
14.3 Types of Affinity Chromatography (A.C.) . . . . .
14.4 Kinetic Applications of Affinity Chromatography
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287
287
288
294
297
15 Capillary Electrophoresis
15.1 Introduction . . . . . . . . . . . . . . .
15.2 Basic Instrumentation . . . . . . . . . .
15.3 Capillary Diameter and Joule Heating
15.4 Various Types of Electrophoresis . . .
15.5 Chiral Recognition . . . . . . . . . . .
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301
301
301
303
303
309
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313
313
314
314
315
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16 NMR Technique in the Elucidation of Biochemical Problems
16.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . .
16.2 Basics of NMR . . . . . . . . . . . . . . . . . . . . . . . . .
16.3 Applications of NMR . . . . . . . . . . . . . . . . . . . . .
16.4 Applications of NMR in Biomedical Research . . . . . . .
17 Applications of ESR
17.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .
17.2 Advances in Magic Angle Spinning (MAS) NMR . . . . . .
17.3 Protein Structure Determination . . . . . . . . . . . . . . .
17.4 Electron Nuclear Double Resonance Spectroscopy
(ENDOR), Electron Spin Echo Envelope Modulation
(ESEEM) and Hyperfine Sub-Level Correlation (HYScore)
Spectroscopic Techniques . . . . . . . . . . . . . . . . . . .
17.5 Structural and Dynamical Information of
Biological Systems . . . . . . . . . . . . . . . . . . . . . . .
17.6 Topology of Proteins . . . . . . . . . . . . . . . . . . . . . .
17.7 SDSLEPR Methods Under High Fields/
High Frequencies . . . . . . . . . . . . . . . . . . . . . . . .
17.8 Double Site-Directed Spin Labeling Methods . . . . . . . .
17.9 Other Important Biological Systems . . . . . . . . . . . . .
18 Flow Methods for the Kinetic Study of Fast
Biochemical Reactions
18.1 Introduction . . . . . . . . . . . . . . . .
18.2 Experimental Arrangement . . . . . . .
18.3 Reactions Studied Using this Technique
18.4 Applications to Unimolecular Reactions
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319
319
320
322
323
325
326
327
329
331
335
335
335
336
337
Table of Contents 18.5 Applications Involving Competitive Reactions . . . . . . .
18.6 Applications Involving Multi-step Reactions . . . . . . . .
19 Temperature Jump Relaxation Technique
19.1 Introduction . . . . . . . . . . . . . . . . . . .
19.2 Schematic Diagram of the Apparatus . . . . .
19.3 Follow Up of the Change in Concentration of
Reactants by Spectrophotometry . . . . . . .
19.4 Applications . . . . . . . . . . . . . . . . . . .
xiii
339
339
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345
345
346
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347
348
20 Flash Photolysis Technique
20.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .
20.2 Principle of the Method . . . . . . . . . . . . . . . . . . . .
20.3 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . .
351
351
351
352
21 Pressure Jump Relaxation Method
21.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .
21.2 Experimental Arrangement and Methodology . . . . . . .
21.3 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . .
355
355
356
357
22 Circular Dichroism as a Tool for the Analysis of
Biochemical Reactions
22.1 Introduction . . . . . . . . . . . . . . . . . . .
22.2 Principle . . . . . . . . . . . . . . . . . . . . .
22.3 Experimental Set Up . . . . . . . . . . . . . .
22.4 Methodology . . . . . . . . . . . . . . . . . .
22.5 Disadvantages as Limitations . . . . . . . . .
22.6 Applications . . . . . . . . . . . . . . . . . . .
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361
361
362
363
364
364
365
23 Applications of Isothermal Calorimetry
23.1 Introduction . . . . . . . . . . . . . .
23.2 Experimental Set Up . . . . . . . . .
23.3 Applications . . . . . . . . . . . . . .
23.4 Mutational Studies . . . . . . . . . .
23.5 Interaction of Transducer Fragments
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375
375
376
377
383
384
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387
387
387
389
390
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24 Principles of Differential Scanning Calorimetry
24.1 Introduction . . . . . . . . . . . . . . . . . . .
24.2 Experimental Set Up . . . . . . . . . . . . . .
24.3 Methodology . . . . . . . . . . . . . . . . . .
24.4 Illustrative Application of DSC . . . . . . . .
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xiv Biophysical Chemistry
25 Applications of Gel Filtration Technique in the
Separation of Biomolecules
25.1 Introduction . . . . . . . . . . . . . . . . . .
25.2 Methodology of Gel Filtration . . . . . . . .
25.3 Principle of Gel Filtration . . . . . . . . . .
25.4 Applications . . . . . . . . . . . . . . . . . .
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395
395
395
396
398
26 Gel Electrophoresis and its Applications to
Biochemical Analysis
26.1 Introduction . . . . . . . . . . . . . . . .
26.2 Nature of Gels Commonly Employed .
26.3 Experimental Arrangement . . . . . . .
26.4 Applications of Gel Electrophoresis . . .
26.5 Isoelectric Focusing (IEF) . . . . . . . . .
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401
401
402
403
404
407
27 Uses of Analytical Ultracentrifugation Methods
27.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . .
27.2 Details of the Apparatus . . . . . . . . . . . . . . . . .
27.3 Detection Method and Data Collection . . . . . . . . .
27.4 Rayleigh Interference Optics . . . . . . . . . . . . . . .
27.5 Applications . . . . . . . . . . . . . . . . . . . . . . . .
27.6 Determination of Sedimentation Coefficient . . . . . .
27.7 Effect of Association on Sedimentation Coefficient . .
27.8 Active Enzyme Sedimentation . . . . . . . . . . . . . .
27.9 Estimation of Diffusion Coefficients . . . . . . . . . .
27.10Estimation of Molar Mass . . . . . . . . . . . . . . . .
27.11Thermodynamic Parameters of Association Reactions
27.12Sedimentation in Biological Environments . . . . . . .
27.13Density Gradient Sedimentation Equilibrium . . . . .
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409
409
409
410
411
411
413
414
415
415
416
417
418
418
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421
421
421
423
426
426
426
427
28 Ion Exchange Chromatography
28.1 Introduction . . . . . . . . . . . . . . . .
28.2 Mechanism of Ion Exchange . . . . . . .
28.3 Applications . . . . . . . . . . . . . . . .
28.4 Purification of Adenovirus . . . . . . . .
28.5 Separation of Membrane Phospholipids
28.6 Separation of Soyabean Proteins . . . .
28.7 Choice of Column Media . . . . . . . . .
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Table of Contents 29 Surface Enhanced Raman Scattering
29.1 Introduction . . . . . . . . . . . .
29.2 Principle of SERS . . . . . . . . .
29.3 Experimental Aspects . . . . . . .
29.4 Applications of SERS . . . . . . .
xv
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429
429
429
431
433
30 Mass Spectrometry and its Applications in the
Analysis of Biomolecules
30.1 Introduction . . . . . . . . . . . . . . . . . .
30.2 Principle of the Technique . . . . . . . . . .
30.3 Basic Components of a Mass Spectrometer
30.4 Ionisation Methods in Mass Spectrometry .
30.5 Applications . . . . . . . . . . . . . . . . . .
30.6 Analysis of Glycoproteins . . . . . . . . . .
30.7 ESI of Equine Apomyoglobin . . . . . . . .
30.8 Analysis of Phosphoproteins . . . . . . . .
30.9 Protein Ladder Sequencing . . . . . . . . .
30.10Specific Examples of Biomolecules . . . . .
30.11Peptides, Proteins and Polynucleotides . .
30.12Polysaccharides . . . . . . . . . . . . . . . .
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441
441
441
442
442
443
444
444
445
445
446
447
448
31 X-Ray Studies in the Elucidation of Structure of Biomolecules
31.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .
31.2 Bragg’s Law . . . . . . . . . . . . . . . . . . . . . . . . . . .
31.3 Structural Determination of Proteins . . . . . . . . . . . . .
31.4 X-ray Structures of Haemoglobin and Myoglobin . . . . . .
31.5 Photosynthetic Reaction Centers . . . . . . . . . . . . . . .
31.6 Ribosomal Subunit . . . . . . . . . . . . . . . . . . . . . . .
451
451
452
456
456
457
457
32 CRISPR-CAS-9, A Method for Genome Editing
32.1 Introduction to CRISPR-CAS-9 . . . . . . . .
32.2 Genesis of the Discovery . . . . . . . . . . . .
32.3 Details on Enzyme “CAS-9” . . . . . . . . . .
32.4 CAS-9 Mechanism . . . . . . . . . . . . . . .
32.5 Target DNA Binding and Cleavage by CAS-9
459
459
460
460
461
464
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Appendix A: Donnan Membrane Potential
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465
xvi Biophysical Chemistry
Appendix B: Nernst Planck Equation
B.1 Nernst–Planck Equation from Onsager’s Linear
Flux-Force Relation . . . . . . . . . . . . . . . . . . . . . . .
469
Appendix C: Goldman-Hodgkin-Katz Voltage Equation
473
Appendix D: Salient Aspects of COVID-19
D.1 Introduction . . . . . . . . . . . . . . . . . .
D.2 Composition of the COVID-19 Virus . . . .
D.3 Symptoms and Other Effects of COVID-19
D.4 Remedial Measures . . . . . . . . . . . . . .
Notes and Bibliography
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470
475
475
475
476
476
479
List of Figures
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
1.10
1.11
1.12
1.13
1.14
1.15
1.16
1.17
1.18
1.19
2.1
Components of animal cell. . . . . . . . . . . . . . .
Parts of the human cell. . . . . . . . . . . . . . . . . .
Two types of cells: (a) Eukaryotic cell and
(b) Prokaryotic cell. . . . . . . . . . . . . . . . . . . .
Components of the cell. . . . . . . . . . . . . . . . . .
Lysosome. . . . . . . . . . . . . . . . . . . . . . . . .
Structure of phospholipid. . . . . . . . . . . . . . . .
Structure of a phospholipid bilayer. . . . . . . . . . .
Representation of a cell membrane. . . . . . . . . . .
Simple diffusion across the cell. . . . . . . . . . . . .
Facilitated diffusion. . . . . . . . . . . . . . . . . . .
Na+ /K+ pump. . . . . . . . . . . . . . . . . . . . . .
Three forms of endocytosis. . . . . . . . . . . . . . .
Exocytosis is endocytosis in reverse. . . . . . . . . .
Pancreatic acinar cells. . . . . . . . . . . . . . . . . .
Relative permeability of a phospholipid bilayer. . .
Mediated transport: (a) Passive transport and
(b) Active transport. . . . . . . . . . . . . . . . . . . .
Secondary active transport. . . . . . . . . . . . . . .
Scheme for transport of Na+ and K+ by Na+ /K+
ATPase. . . . . . . . . . . . . . . . . . . . . . . . . . .
Scheme for the active transport of Ca2+ by the Ca2+
ATPase. . . . . . . . . . . . . . . . . . . . . . . . . . .
Boltzmann distribution of permeating cations
and anions. . . . . . . . . . . . . . . . . . . . . . . . .
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11
12
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16
18
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25
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27
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35
xvii
xviii 2.2
2.3
2.4
2.5
4.1
4.2
4.3
4.4
5.1
5.2
6.1
7.1
7.2
7.3
7.4
7.5
7.6
7.7
8.1
8.2
8.3
8.4
8.5
8.6
8.7
9.1
9.2
9.3
9.4
Biophysical Chemistry
Distribution of ions on negatively charged surfaces. . .
Permeability of cations and anions. . . . . . . . . . . . .
The surface free energy of water. . . . . . . . . . . . . .
(a) The equilibria among a lipid monomer, (b) a vesicle
containing N molecules and (c) a microscopic
lipid bilayer. . . . . . . . . . . . . . . . . . . . . . . . . .
Schematic representation of hydrophobic and
polar groups in soaps. . . . . . . . . . . . . . . . . . . .
Schematic depiction of lipid bilayers. . . . . . . . . . . .
Energetically favourable structures of bilayers. . . . . .
Structure of cholesterol in free state and fluid region. . .
Fischer projection and ball-stick model. . . . . . . . . .
Potentiometric titration of aqueous alanine solution. . .
Structure of tetrapeptide containing Val-gly-Serine-Ala
with L-Valine being at left end and L-Alanine at the
right end. . . . . . . . . . . . . . . . . . . . . . . . . . . .
Structure of α-helix. . . . . . . . . . . . . . . . . . . . . .
Quaternary structure of a complex globular protein
(dimer). . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Structure of hemoglobin. . . . . . . . . . . . . . . . . . .
Schematic depiction of helix-coil transitions in proteins.
Shapes of proteins under different coordinates. . . . . .
An energy diagram of a two-state folding event. . . . .
(i) Native and (ii) a compact structure of amino acid
chain lengths 16. . . . . . . . . . . . . . . . . . . . . . . .
Structure of nucleoside, nucleotide, nucleoside di and
tri phosphates. . . . . . . . . . . . . . . . . . . . . . . . .
Structures of purines. . . . . . . . . . . . . . . . . . . . .
Structures of pyrimidines. . . . . . . . . . . . . . . . . .
The structural elements of the nucelosides and the
phosphate bearing nucleotides. . . . . . . . . . . . . . .
Scheme depicting electrochemical reduction of adenine.
Electro-oxidation of guanine. . . . . . . . . . . . . . . .
Helical structure of DNA. . . . . . . . . . . . . . . . . .
Schematic depiction of enzyme - substrate complex. . .
Energy diagram with and without the
presence of enzymes. . . . . . . . . . . . . . . . . . . . .
Dependence of the reaction rate on concentrations of
(a) enzyme and (b) substrate. . . . . . . . . . . . . . . .
Lineweaver-Burke plot. . . . . . . . . . . . . . . . . . . .
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36
37
40
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106
106
106
115
118
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149
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155
156
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161
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182
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185
186
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xix
List of Figures
9.5
Variation of the rate of enzyme-catalysed
reaction with pH. . . . . . . . . . . . . . . . . . . . . . . . .
9.6
Positioning of a substrate to an active site. . . . . . . . . . .
9.7
Graph showing the variation of reaction rate with
substrate concentration. . . . . . . . . . . . . . . . . . . . .
9.8
Graph showing the variation of reaction rate:
(i) without inhibitor; (ii) competitive and
(iii) non-competitive inhibitor. . . . . . . . . . . . . . . . . .
9.9
Variation of the reciprocal velocity with reciprocal
substrate concentration. . . . . . . . . . . . . . . . . . . . .
9.10 Dependence of the maximum velocity on the substrate
concentration. . . . . . . . . . . . . . . . . . . . . . . . . . .
10.1 Conversion of an apoenzyme to a holoenzyme. . . . . . . .
10.2 Conversion of an active enzyme with substrate to yield
an enzyme with products. . . . . . . . . . . . . . . . . . . .
12.1 Sketch of a typical biosensor. . . . . . . . . . . . . . . . . . .
12.2 Applications of biosensors. . . . . . . . . . . . . . . . . . . .
12.3 Schematic diagram of thermometric biosensor. . . . . . . .
12.4 Sketch of an optical biosensor. . . . . . . . . . . . . . . . . .
12.5 Schematic variation of amperometric current with time. . .
13.1 Air-Solution interface. . . . . . . . . . . . . . . . . . . . . .
13.2 Reflection of wave for gold film in air. . . . . . . . . . . . .
13.3 Variation of SPR angle with refractive index. . . . . . . . .
13.4 Binding of rabbit lgG to protein A and anti-rabbit
to lgG to FAB . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.5 Illumination-induced activity of G-protein and its
desorption form the membrane. . . . . . . . . . . . . . . . .
14.1 Separation procedure in affinity chromatography. . . . . .
14.2 Variation of absorbance with time. . . . . . . . . . . . . . .
14.3 Structure of agarose. . . . . . . . . . . . . . . . . . . . . . .
14.4 Structure of silica. . . . . . . . . . . . . . . . . . . . . . . . .
14.5 Dependence of eluted amount with elution volume. . . . .
14.6 Different types of immobilization methods. . . . . . . . . .
14.7 Immobilization by N-hydroxy succinimide method. . . . .
14.8 Immobilization using carbonyl diimidazole method. . . . .
14.9 Target protein identification using affinity
chromatography. . . . . . . . . . . . . . . . . . . . . . . . .
14.10 Application of high performance affinity chromatography
in estimating rate constants. . . . . . . . . . . . . . . . . . .
.
.
190
192
.
195
.
196
.
196
.
.
197
217
.
.
.
.
.
.
.
.
.
218
263
264
265
266
269
280
282
282
.
282
.
.
.
.
.
.
.
.
.
283
288
289
290
290
292
292
293
294
.
297
.
297
xx
Biophysical Chemistry
14.11 Binding of D-tryptophan with a HPAC column containing
immobilized HSA. . . . . . . . . . . . . . . . . . . . . . . . .
15.1 Diagram of a capillary electrophoresis unit. . . . . . . . . . .
15.2 Effect of pH on electro osmotic flow. . . . . . . . . . . . . . .
15.3 Separation of components using electrophoretic. . . . . . . .
15.4 Migration of charges using isoelectric focusing method. . . .
15.5 Separation of proteins using isoelectric focusing method. . .
15.6 Size separation using capillary gel electrophoresis. . . . . . .
15.7 Physical and chemical gels. . . . . . . . . . . . . . . . . . . .
15.8 Capillary gel electrophoresis of thymidine
synthetic polymer. . . . . . . . . . . . . . . . . . . . . . . . . .
15.9 Isotachophoresis of a mixture of anions. . . . . . . . . . . . .
15.10 Separation of chiral amino acids using MECC. . . . . . . . .
16.1 Basic features of NMR. . . . . . . . . . . . . . . . . . . . . . .
16.2 Protein sequence formed by connecting peptide bonds. . . .
17.1 Energy-changes in electron spins in the absence and
presence of a magnetic field. . . . . . . . . . . . . . . . . . . .
17.2 Magnetic Resonance Imaging principle of body
and tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
17.3 Protein structure determination using solid state NMR. . . .
17.4 Block diagram of a EPR spectrometer. . . . . . . . . . . . . .
17.5 Structure of MTSL and the resulting side chain produced
by reaction with cysteine residue of the protein. . . . . . . . .
17.6 Molecular structure of vimentin. . . . . . . . . . . . . . . . .
17.7 Three pulse ESEEM spectra with T = 200 ns of the I + 2
and I + 3 labelled Leu for AchR M-282 helical peptide in lipid
bilayer and Ubiquitin β-sheet peptide in solution. . . . . . .
17.8 Identification of transmembrane region of α-helical nature
of C99 amyloid precursor protein in proteoliposomes. . . . .
17.9 EPR spectra and corresponding simulations at three fields
(or frequencies) of the radical intermediate in the
Phycocyanobilin—Ferredoxin oxidoreductase system. . . . .
18.1 A simple experimental set up for flow methods. . . . . . . .
18.2 Technique of stopped flow method. . . . . . . . . . . . . . . .
18.3 Rapid interaction between apotransferrin and Zn2+ . . . . . .
18.4 (a) Variation of intensity of fluorescence with time in the
kinetic study of high mobility protein with cisplatin modified
DNA and (b) Variation of k obs with concentration of
HMGI domain. . . . . . . . . . . . . . . . . . . . . . . . . . .
298
302
303
304
305
305
306
306
307
308
310
314
315
320
322
323
323
325
326
328
329
331
335
336
338
338
List of Figures
19.1
19.2
20.1
21.1
22.1
22.2
22.3
22.4
22.5
23.1
23.2
23.3
24.1
24.2
24.3
25.1
25.2
25.3
25.4
26.1
26.2
26.3
27.1
27.2
27.3
27.4
xxi
Schematic diagram of a temperature jump apparatus
developed by Eigen and his group. . . . . . . . . . . . . . . .
Variation of concentration of species with time following
a temperature jump. . . . . . . . . . . . . . . . . . . . . . . .
A flash photolytic unit. . . . . . . . . . . . . . . . . . . . . . .
Experimental arrangement of a pressure jump unit. . . . . .
Different types of CD spectra. . . . . . . . . . . . . . . . . . .
CD spectra of flavoproteins in the near UV
and visible region. . . . . . . . . . . . . . . . . . . . . . . . . .
CD spectra of isocitrate lyase from E.coli. . . . . . . . . . . . .
CD spectra of the same species in the near UV region. . . . .
CD spectra of α-lactalbumin (a) and (b) represent far UV
and near UV spectra at pH = 7.0. . . . . . . . . . . . . . . . .
Experimental set up of an isothermal heat
flow calorimeter. . . . . . . . . . . . . . . . . . . . . . . . . . .
Binding curve obtained from unprocessed data. . . . . . . .
Thermodynamic profiles of HIV proteanase inhibitors. . . .
Block diagram of a heat flux differential
scanning calorimeter. . . . . . . . . . . . . . . . . . . . . . . .
Single heat flux source in DSC. . . . . . . . . . . . . . . . . .
Curve trace obtained with the solution of Arg96 → His mutant
of the lysozymes of T4 phage. . . . . . . . . . . . . . . . . . .
Schematic picture of (A) a bead with enlargement,
(B) sample molecules diffusing into bead and
(C) separation process. . . . . . . . . . . . . . . . . . . . . . .
Schematic diagram of elution. . . . . . . . . . . . . . . . . . .
Typical elution pattern of dialysis (on Sephadex G25). . . . .
Separation of RNAase from protease in pancreatic extract
using Sephadex G-75 column. . . . . . . . . . . . . . . . . . .
Experimental set-up of gel electrophoresis. . . . . . . . . . .
Agarose gel electrophoresis pattern for cleavage of
supercoiled PUC DNA. . . . . . . . . . . . . . . . . . . . . . .
Scheme depicting the separation of molecules
by charge differences. . . . . . . . . . . . . . . . . . . . . . . .
Schematic depiction of ultracentrifuge pattern. . . . . . . . .
Movement of the boundary in a sedimentation
experiment. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Estimation of the sedimentation coefficient. . . . . . . . . . .
Concentration dependence of weight average “S” for
DIP-α chymotrypsin. . . . . . . . . . . . . . . . . . . . . . . .
346
347
352
356
362
366
366
367
369
376
378
381
388
388
390
396
397
398
398
403
405
407
410
412
414
415
xxii 27.5
28.1
28.2
28.3
28.4
28.5
29.1
29.2
29.3
29.4
29.5
29.6
29.7
29.8
30.1
30.2
30.3
30.4
30.5
30.6
30.7
30.8
31.1
31.2
31.3
31.4
Biophysical Chemistry
Dependence of the apparent molar mass of DNA
on concentration. . . . . . . . . . . . . . . . . . . . . . . . . .
Separation based on the binding of analytes to positively
or negatively charged groups on the stationary phase. . . . .
Separation mechanism in ion exchange. . . . . . . . . . . . .
Schematic depiction of charged variants of monoclonal
antibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Two examples illustrating the use of BioMAB column for
identification of c-terminal truncation on heavy chains. . . .
High resolution of a mixture of proteins with a wide range
of isoelectric points. . . . . . . . . . . . . . . . . . . . . . . . .
Electromagnetic and chemical enhancement mechanism. . .
SERS enhancement and enhancement mechanism. . . . . . .
Metals exhibiting SERS and their wave length ranges. . . . .
Schematic depiction of SERS for spherical aggregates. . . . .
Field of a point dipole. . . . . . . . . . . . . . . . . . . . . . .
SERS of dopamine in silver colloidal solution. . . . . . . . . .
(A) Scheme of an immuno assay system using two different
SERS Labels. (B) SERS signatures of three types of reporter
labeled immuno gold colloid (a) MBA/goat anti-rat IgG,
(b) NT/goat antirat IgG, (c) TP/goat anti rabbit IgG and
(d) goat anti rat IgG. . . . . . . . . . . . . . . . . . . . . . . .
Cleavage of single nucleotides and attachment
to colloidal silver or gold clusters. . . . . . . . . . . . . . . . .
Components of a mass spectrometer. . . . . . . . . . . . . . .
ESI of equine apomyoglobin. . . . . . . . . . . . . . . . . . .
Distribution of charged states characteristic of native
and denatured proteins. . . . . . . . . . . . . . . . . . . . . .
Denaturation of myoglobin in an acidic environment. . . . .
ESI of cytochrome-C and glucagon. . . . . . . . . . . . . . . .
Fragmentation pattern of protonated peptide
in FAB/MS. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mass spectrometry of nucleotide structures. . . . . . . . . . .
Pattern of mass spectrum in polysaccharides. . . . . . . . . .
Schematic diagram of an X-ray diffractometer. . . . . . . . .
Reflection of X-rays in ( hkl ) planes of a crystal. . . . . . . . .
Diffraction angle of X-rays. . . . . . . . . . . . . . . . . . . . .
(a) X-ray camera and (b) X-ray film showing
diffraction lines. . . . . . . . . . . . . . . . . . . . . . . . . . .
417
422
423
424
425
425
430
430
431
432
433
434
435
437
442
444
445
446
447
447
448
449
452
452
453
453
List of Figures
31.5
31.6
32.1
32.2
32.3
32.4
32.5
A.1
D.1
Lines corresponding to planes of different
cubic structures. . . . . . . . . . . . . . . . . . . . . . . . .
X-ray structure of Myoglobin obtained
at high resolution. . . . . . . . . . . . . . . . . . . . . . . .
Parts of a bacterial immune system: Genomic DNA,
CAS-9, target sequence and guide RNA. . . . . . . . . . .
The six domains of CAS-9. . . . . . . . . . . . . . . . . . .
Single strand of RNA forming a T-shaped molecule. . . .
CAS-9 complex (inactive) and target complimentary
region of guide RNA. . . . . . . . . . . . . . . . . . . . . .
CAS-9/guide RNA and target DNA leading to
CAS-9/guide RNA complex bound to target DNA. . . . .
Schematic depiction of the Donnan membrane potential.
Schematic depiction of Corona virus. . . . . . . . . . . . .
xxiii
. .
455
. .
457
. .
. .
. .
459
461
462
. .
462
. .
. .
. .
463
465
475
List of Tables
3.1
3.2
3.3
3.4
4.1
5.1
5.2
6.1
6.2
6.3
6.4
6.5
7.1
7.2
7.3
7.4
7.5
7.6
Enthalpy of formation (ΔH ◦f ) and ΔHc◦ at 298K and S◦ . . . .
Gibbs energy of formation (ΔG ◦f ) of
carbohydrates. . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thermodynamic data for anomeric conversions at 298 K. . .
Redox reactions and their E◦ and ΔG values. . . . . . . . . .
Examples of a few fatty acids. . . . . . . . . . . . . . . . . . .
Classification of amino acids based on polarity. . . . . . . . .
Isoelectric point of a few amino acids. . . . . . . . . . . . . .
Residues, their sources and amino acid sequences. . . . . . .
Critical Aggregation Concentration (CAC) of a few peptides
and their diffusion coefficients. . . . . . . . . . . . . . . . . .
Variation of the MB peak current with time. . . . . . . . . . .
Rate constant data for the effective cleavage rate of MB in the
peptides TA-1 and MA-1. . . . . . . . . . . . . . . . . . . . . .
Current potential response of peptides from cyclic
voltammetry at a scan rate of 500 mv sec−1 at 25◦ C. . . . . .
Classification of proteins based on structure. . . . . . . . . .
Equilibrium constants, Kh−c for helix-coil transformation. . .
Electrophoretic mobility data of a few typical proteins. . . .
Kinetic parameters for unfolding of S6 in SDS. . . . . . . . .
Thermodynamic data of polypeptides in proteins relating to
helix-coil transformation in aqueous solutions. . . . . . . . .
Equilibrium constants (Khel −nonhel ) for some natural
amino acids in nine globular proteins. . . . . . . . . . . . . .
75
75
75
80
90
116
119
132
134
138
138
138
144
153
155
157
158
159
xxv
xxvi 7.7
8.1
8.2
8.3
8.4
8.5
8.6
8.7
8.8
9.1
9.2
9.3
9.4
9.5
9.6
9.7
9.8
9.9
9.10
10.1
10.2
10.3
10.4
10.5
10.6
10.7
Biophysical Chemistry
A( p, q) values which arise from the counting of
‘hydrophobic-polar’ contacts for a square lattice of
16 sites, assuming periodic boundary conditions. . . . . . . .
Comparison of nucleosides and nucleotides. . . . . . . . . .
Typical polarographic data for the reduction of adenine,
cytosine and guanine. . . . . . . . . . . . . . . . . . . . . . . .
Functional importance of a few nucleotides. . . . . . . . . . .
Sites of protonation and pKa ’s of four nucleobases and the
ribose-phosphate backbone. . . . . . . . . . . . . . . . . . . .
pKa data and ΔG ◦ values of nucleotides. . . . . . . . . . . . .
Conductivity data of nucleotides and nucleotides
at 400 K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thermodynamic parameters for DNA duplex formation in
4 MNaCl and 4 M choline dihydrogen phosphate
(Choline dhP). . . . . . . . . . . . . . . . . . . . . . . . . . . .
Stability constant data of the complexes formed between the
nucleotides and polyamines in aqueous solution at 25◦ C. . .
Kinetic data on some reactions catalysed by enzymes
(or inhibited in presence of inhibitors). . . . . . . . . . . . . .
Kinetic expressions for different inhibition types. . . . . . . .
Conversion of zymogens to active enzymes. . . . . . . . . . .
Data on glycolic enzymes. . . . . . . . . . . . . . . . . . . . .
Activation parameters for the reactions of some enzymes. . .
Enzymatic activity of lactose dehydrogenase. . . . . . . . . .
Turn over numbers for selected enzymes. . . . . . . . . . . .
Some industrial processes using enzymes. . . . . . . . . . . .
Illustrative examples of industrial catalysts. . . . . . . . . . .
Michaelis-Menten constants for some enzyme
substrate reactions. . . . . . . . . . . . . . . . . . . . . . . . .
Typical combinations of enzyme-co-enzyme systems
in presence of vitamins for their effective functioning. . . . .
Water soluble vitamins along with their co-enzymes
and their physiological functions. . . . . . . . . . . . . . . . .
Fat soluble vitamins and their physiological function. . . . .
Typical examples of fat-soluble vitamins and their source. . .
Water soluble Vitamins: Vitamins B-1 to B-7, B-9, B-12,
Vitamin C and their source. . . . . . . . . . . . . . . . . . . .
List of reactions catalysed by nicotinamide nucleotides. . . .
Redox potential data on water soluble B-Vitamins. . . . . . .
160
165
168
170
173
175
175
177
178
187
195
199
201
204
207
208
209
210
212
219
221
222
223
223
225
227
List of Tables
10.8
10.9
10.10
10.11
10.12
10.13
10.14
10.15
10.16
10.17
10.18
10.19
10.20
10.21
11.1
11.2
11.3
11.4
11.5
11.6
12.1
12.2
xxvii
Typical examples of enzymes (belonging to group of
flavoproteins) and reactions catalysed by them. . . . . . . . .
Half wave potential data of a few vitamins and
related compounds. . . . . . . . . . . . . . . . . . . . . . . . .
Average dissociation constant of thiamine at
different temperatures. . . . . . . . . . . . . . . . . . . . . . .
Kinetic data on the reaction of hydrated e−
with cobalamine. . . . . . . . . . . . . . . . . . . . . . . . . .
Redox potential of cobalamines. . . . . . . . . . . . . . . . . .
Redox potential data against S.H.E. at pH = 7.0. . . . . . . . .
Kinetic data on the oxidation of pyridoxine. . . . . . . . . . .
Protonation constants of folic acid and stability constants
of metal complexes. . . . . . . . . . . . . . . . . . . . . . . . .
Conductances of the metal complexes formed at two points
of addition in the titration of 25 ml of metal ions
(1 × 10−3 M) with folic acid (0.01 M). . . . . . . . . . . . . . .
Values of surface tension and conductivity at the inflection
point pertaining to the formation of 1: 1 complex. . . . . . .
Association constant (Ka ) and thermodynamic parameters of
different Vit-β-cyclodextrin inclusion complexes. . . . . . . .
Distribution constants (K) and Gibbs free energy changes (ΔG ◦ )
of Vitamin E partitioned between dry reversed micelles of
surfactants and non-polar organic solvents. . . . . . . . . . .
Optical data of Vit-K2 and Vit D3 in ethanol. . . . . . . . . . .
Zeta potential data of loaded liposomes. . . . . . . . . . . . .
Standard Gibbs energies of hydrolysis (ΔG ◦ ) of some
phosphorylated compounds. . . . . . . . . . . . . . . . . . .
Standard Gibbs free energies of phosphoesters. . . . . . . . .
Gibbs free energy changes for ATP hydrolysis in various
organisms under different physiological conditions. . . . . .
Enthalpy changes of reactions involving ATP and
related compounds. . . . . . . . . . . . . . . . . . . . . . . . .
Dissociation constants and enthalpy changes for reactions
involving ATP. . . . . . . . . . . . . . . . . . . . . . . . . . . .
Gibbs free energy changes of reference compounds. . . . . .
Typical range of detection accomplished for analytes
using chemically modified electrodes. . . . . . . . . . . . . .
Illustrative examples of analytes and corresponding
enzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
229
234
235
236
236
237
238
238
239
239
239
239
241
241
249
250
251
256
257
257
268
270
xxviii 14.1
14.2
14.3
16.1
23.1
23.2
23.3
23.4
23.5
23.6
26.1
30.1
Biophysical Chemistry
Examples of pre-activated products for immobilizing
ligands. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Examples of commonly used lectins for isolation of
carbohydrates and polysaccharides. . . . . . . . . . . . . .
List of chelating agents and corresponding metal ions. . . .
Mass numbers, atomic numbers and spin
quantum numbers. . . . . . . . . . . . . . . . . . . . . . . .
Enthalpy–entropy compensation in the binding of F4E
to 7 methyl-GpppG. . . . . . . . . . . . . . . . . . . . . . . .
Binding constants and thermodynamic data of tyrosyl
phosphopeptides with different SH2 domains at 298 K. . .
Thermodynamic data for binding in ATP in presence
of various mutants. . . . . . . . . . . . . . . . . . . . . . . .
Thermodynamic parameters of citrate binding to CitAPhis
at 298 K in 50 mM phosphate buffer at different pH’s
as determined by ITC. . . . . . . . . . . . . . . . . . . . . .
Thermodynamic parameters of citrate binding at different
concentrations of MgCl2 . . . . . . . . . . . . . . . . . . . . .
Thermodynamic parameters pertaining to
binding affinities. . . . . . . . . . . . . . . . . . . . . . . . .
Illustrative examples of various gels. . . . . . . . . . . . . .
General range of applications of different MS methods. . .
.
291
.
.
295
296
.
314
.
380
.
382
.
383
.
384
.
384
.
.
.
385
402
446
List of Symbols and
Acronyms
ΔH ◦
γ
Ψ
ε
A
aX
dζ
dq
Ji
(4 Fe-4S)+
1,2, diacyl DPPC
1,3-BPG
A,B,C,D NP’s
A.C
ADP3−
Acetyl CoA
ACTH
ADP
Ala
AMP
AN
Arg
AoT
Enthalpy change between the products and reactants
surface tension
Electrical potential
dielectric constant
Affinity
Activity of X
Degree of advancement of a reaction
Heat exchanged by a system
heat flux of species Ji
iron-sulfur cofactors
1,2 diacylphosphatidylcholine
1,3-bisphosphoglycerate
A, B, C, D natriuretic peptides
Affinity chromatogrphy
Adenosine diphosphate anion
Acetyl coenzyme A
Adrenocorticotropic hormone
Adenosine diphosphate
Alanine
Adenosine monophosphate
Acetonitrile
Arginine
Sodium bis (2-ethylhexyl) sulfosuccinate
xxix
xxx
Biophysical Chemistry
Asn
ATP
B0
BCCP
bipy
BLES
C.M.R.F
CAC
CDTA
DEER
DHAP
DHF
DNA
DPPC
DSPE
e−
hyd
E.coli
EDTA
EAK-16-II
ECF
E1/2
ENDOR
ENO
EOF
EPC
EPR
ER
FAD+
FADH
FBA
FMN
FUM
GLn
GLP-1
Glu
Gly
GMP
GSH
GSSG
Asparagine
Adenosine triphosphate
Strength of magnetic field
Biotin carboxyl carrier protein
Bipyridyl
Bovine lipid extract sulfactant
Carbomethoxy riboflavin
Critical aggregation concentration
Trans,1,2-cyclohexyl ethylene dinitrilo tetraacetic acid,
C14 H22 N2 O8
Double electron-electron resonance
Dihydroxyacetone phosphate
Dihydrofolic acid
Deoxyribonucleic acid
Dipalmitoyl phosphatidylcholine
Distearoyl (Phosphatidylethanolamine)
hydrated electron
Escherichia coli
Ethylenediaminetetraacetic acid
(Ala-Gln-Ala-Gln-Ala-Lys-Ala-Lys)2
Extracellular fluid
Half wave potential
Electron nuclear double resonance spectroscopy
Enolase
Electroosmotic flow
Egg phosphatidylcholine
electron paramagnetic resonance
Endoplasmic reticulum
Flavin adenine dinucleotide
Reduced form of Flavin adenine dinucleotide
Fructose 1,6-bisphosphate aldolase
Flavin mononucleotide
Fumarate hydratase
Glutamine
Glucose like peptide 1
Glutaric acid
Glycine
Guanosine-5’-monophosphate
Reduced form of glutathione
Oxidised form of glutathione
List of Symbols and Acronyms
HA
IEF
IF
Ilc
IMP
In
IPP
ITP
Km
L.A
LDH
Lecithin
Leu
LipS2
LTAB
LTAC
malonyl coA
MAS
Mb
mC
MDH
MECC
MRI
NAD+
NADH
NADP+
NAG
NC
NHS
Nm−1
NoE
NRPS
ODN
OEC
PTP
PAGE
Pc
PE
PEG
PELDOR
Dehydroascorbic acid
Isoelectric focusing
Interstitial fluid
Isoleucine
Inosine-5’-monophosphate
Inhibitor (also Imb)
Isoleucine-Proline-Proline
Isotachophoresis
Michaelis-Menten constant
Lipoic acid
Lactic dehydrogenase
L-α-phosphatidylcholine
Leucine
LipoylS2
Lauryl trimethyl ammonium bromide
Lauryl trimethyl ammonium chloride
malonyl coenzyme A
Magic angle spinning
Myoglobin
Millicoulomb
malate dehydrogenase
Micellar electrokinetic capillary chromatography
Magnetic resonance imaging
Nicotinamide adenine dinucleotide
Reduced form of NAD+
Nicotinamide adenine dinucleotide phosphate
N-acetyl glucosamine
Noncompetitive inhibition
N-hydrosuccinimide
Newton per meter
Nuclear Overhauser effect
Nonribosomal peptide synthetase
Oligodeoxyncleotide
Oxygen evolving complex
Phosphoryl transfer potential
Polyacrylamide gel electrophoresis
Phosphatidylcholine
Phosphotidylethanolamine
polyethylene glycol
Pulse electron double resonance spectroscopy
xxxi
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Biophysical Chemistry
PEP
PET
PFK
PGD
PGI
PGM
Phe
PHI
PI
Pi
PPY
R5PI
RBP
RDC
RNR
rRNA
SA
SAM
SDS
SDSL
Ser
SM
Succinyl CoA
5THF
Thr
TOR
TPI
TPP
Tyr
UC
Vitamin A
Vitamin B12
Vitamin B1
Vitamin B2
Vitamin B5
Vitamin B6
Vitamin B7
Vitamin B9
Vitamin C
Vitamin D3
Vitamin E
Vitamin K
Phosphoenolpyruvic acid
3’-ethyl phosphate
Phosphofructokinease
Phosphogluconate dehydrogenase
Phosphoglycerate isomerase
Phosphoglycerate mutase
Phenylalamine
peptide histidine isoleucine
pH at isoelectric point
Phosphate group
Pancreatic polypeptide
Ribose-5-phosphate isomerase
Ribflavin binding protein
Residual dipolar coupling
Ribonucleotide reductase
ribosomal Ribonucleic acid
Stearic acid
Self assembled monolayer
Sodium dodecyl sulfate
Site Directed Spin Label
Serine
Sphingomyelin
Succinyl coenzyme A
Tetrahydrofolic acid (or Tetrahydrofuran)
Threonine
Turn over rate
Triosephosphate isomerase
Thiamine pyrophosphate
Tyrosine
Uncompetitive inhibition
Retinol
Cyanocobalamine
Thiamine
Riboflavin
Pantothenic acid
Pyridoxin
Biotin
Folic acid
Ascorbic acid
cholecalciferol
Tocoferol
Phylloquinone
Part I
Different Types of
Biomolecules
1
Cell and its Biochemical
Setup
1.1
Introduction
All living things are composed of cells, which are bound by a membrane
and which contain the fundamental molecules of life. A single cell is a
complete organism in itself examples of which being yeast or a bacterium.
When cells mature they acquire special functions. When these cells cooperate with other special cells, they become building blocks of large multi cellular organisms such as humans and other animals. Cells are much larger
than atoms and some single celled organisms are spheres with a diameter
of 0.2 μm. Human cells have a mass 4 × 105 larger than the mass of a single
micoplasma bacterium, have a diameter of about 20 μm and have a mass
of one nanogram.
1.2
Structure of an Animal Cell
The main components of an animal cell are shown in the following diagram (refer Fig. 1.1).
1.2.1
Principal Structures of an Animal Cell
Cytoplasm surrounds the cells specialized structures or organelles. Ribosomes, the site of protein synthesis are found free in the cytoplasm or
attached to the endoplastic reticulum through which materials are
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_1
3
4
Biophysical Chemistry
Figure 1.1 Components of animal cell.
transported throughout the cell. Energy needed by the cell is released
by the mitochondria. The Golgi complex, stacks of flattened sacs, processes and packages materials to be released from the cell in secretory vesicles. Digestive enzymes are contained in lysosomes. Peroxisomes contain
enzymes that detoxify dangerous substances. The centrosome contains the
centriols which play a role in cell division. The microvilli are finger-like
extensions found on certain cells. Cilia, hair-like structures that extend
from the surface of many cells, can create the movement of surrounding
fluid. The nuclear envelope, a double membrane surrounding the nucleus,
contains pores that control the movement of substances into and out of
nucleoplasm. Chromatin, a combination of DNA and proteins that coil
into chromosomes make up much of the nucleoplasm. The dense nucleolus is the site of ribosome production.
1.2.2
Structure of a Human Cell and its Functions
A cell is a fully operational living entity. Human beings are composed of
multicellular organisms with different types of cells operating together to
sustain life. The human body contains other noncellular components such
Cell and its Biochemical Setup
5
as water, macronutrients like carbohydrates, lipids, proteins and micronutrients like minerals, vitamins and electrolytes. Tissue is composed of a
collection of cells and they perform many activities apart from other specific functions of the body.
1.3
Composition of Human Cell
The cell contains a variety of structural components, it known as organelles
required to maintain life. The organelles are suspended in a gelatinous
matrix known as cytoplasm, which is contained within the cell membrane.
The main organelles of the cell may be listed as
(i) cell membrane,
(ii) nucleus,
(iii) mitochondria,
(iv) endoplastic reticulum,
(v) golgi apparatus,
(vi) lysosomes,
(vii) peroxisomes,
(viii) microfilaments, and
(ix) microtubules.
The red blood cells of the human body lack organelles. A sketch of the
human cell is given in the Figure 1.2. The functions of the various components are briefly described below cell membrane. It is an outer coating of
the cell and contains cytoplasm and its contents and organelle. It is a double layered membrane containing proteins and lipids. The lipid molecules
on the outer and inner part of the lipid bilayer allow it to transport substances in and out of the cell.
1.3.1
Human Cell
The functions of the human cell vary depending on the type of cell and
its location in the body. Organelles are the most basic functional units and
they cannot exist or operate without a cell. All organelles work together
to keep the cell alive and help it to carry out its specific function. They
are highly specialized and have various sizes, shapes. The functions of
organelles include intake of nutrients and other substances, their processing, production of new substances, cell replication and energy generation.
6
Biophysical Chemistry
Figure 1.2 Parts of the human cell.
In motile cells like sperm cells, tail like projections allow for cellular locomotion.
1.3.2
Nucleus
The nucleus controls the cell. It contains genes, bunches of DNA which
determine all aspects of human anatomy and physiology. The DNA which
is organized into chromosomes also contains the blue print specific for each
type of cell. It also allows proliferation of the cell.
There is an area known as nucleolus in the nucleus, which is an accumulation of RNA and protein. It is also a site where the ribosomal RNA is
transcribed from DNA and assembled.
1.3.3
Mitochondria
They are power houses of the cell and break down nutrients to yield energy.
They also produce the high energy compound ATP. They are composed of
two membranous layers: (i) an outer membrane that surrounds the structure and (ii) an inner membrane that provides sites for energy production. The inner membrane has many folds forming shelves where enzymes
attach and oxidize nutrients. The mitochondria also contain DNA which
replicates when necessary.
Cell and its Biochemical Setup
1.3.4
7
Endoplastic Reticulum (ER)
It is a membranous structure that contains a net work of tubules and vesicles. Its structure permits substances to move through it and keep isolated from the rest of the cell until all the manufacturing processes within
it are completed. Two types of ER are present: (i) rough (granular) and (ii)
smooth (agranular).
The smooth ER does not have any attached ribosomes. Its function is to
synthesize different types of lipids and it also plays a role in carbohydrate
and drug metabolism.
1.3.5
Golgi Apparatus
It is a stacked collection of flat vesicles. Substances produced in ER are
transported as vesicles in Golgi apparatus. Products from ER are stored
in it and converted into different substances necessary for cell’s various
functions.
1.3.6
Lysosomes
They are vesicles that break off from Golgi apparatus. They vary in size
and function depending on the type of cell. Lysosomes contain enzymes
which help in the digestion of nutrients in the cell. They also break down
any cellular debris or invading microorganisms like bacteria. Secretory
vesicle is a structure similar to lysosome and it contains enzymes which are
used outside the cell. For example, secretory vesicles of pancreatic acinar
cell release digestive enzymes which help in the digestion of nutrients in
the gut.
1.3.7
Peroxisomes
These organelles are similar to lysosomes and contain enzymes. The
enzymes act in the form of H2 O2 to neutralize substances toxic to the cell.
Perioxisomes are formed from endoplastic reticulum.
1.3.8
Microfilaments and Microtubules
These are rigid proteinous substances that form the internal skeleton of the
cell known as cytoskeleton. Some microtubules also make up centrioles
and mitotic spindles within the cell. They are responsible for the division
of the cytoplasm when the cell divides. The microtubules are the central
component “Cilia” which are small hair like projections that protrude from
the surface of certain cells. It is also the central component of specialized
8
Biophysical Chemistry
celia like the tail of sperm cells which acts in a manner as to allow the cell
to move in a fluid.
1.4
Eukaryotic and Prokaryotic Cells
Eukaryotes are organisms whose cells possess a nucleus enclosed within a
cell membrane. They include multicellular organisms such as plants, animals and fungi. Prokaryotic cells do not possess membrane bound cellular
components such as nuclei. They include bacteria and Archaea. The structures of eukaryotic and prokaryotic cells are given in the Figure 1.3.
1.4.1
Similarities and Differences Between the
Two Types of Cells
Similarities
(i) Cell membrane: Both types of cells have a lipid bilayer which is an
arrangement of phospholipids and proteins that acts as a selective
barrier between the internal and external environment of the cells.
(ii) Genetic material: Eukaryotic and prokaryotic cells use DNA as the
basis for their genetic information. The genetic material is needed to
regulate the cell function through creation of RNA by transcription
followed by the generation of proteins through translation.
Figure 1.3 Two types of cells: (a) Eukaryotic cell and (b) Prokaryotic cell.
Cell and its Biochemical Setup
9
(iii) Ribosomes: They facilitate RNA translation and creation of protein
which is essential to both types of cells.
(iv) Cytoplasm: The cytoplasm is the medium in which the biochemical
reactions of the cell take place, of which cytosol is the primary component. In eukaryotic cells, the cytoplasm covers all material between
plasma membrane and the nuclear envelope including organelles.
The material within the nucleus is known as nucleoplasm. In prokaryotes, the cytoplasm encompasses everything within the plasma membrane including the cytoskeleton and genetic material.
Differences
(i) Cell size: Eucaryotic cells have a size of 10–100μm while prokaryotic
cells have size in the range 1–10μm. (μm = 1 micrometer = 10−6 m).
(ii) Cell arrangement: Eucaryotes are multicellular whereas prokaryotes
are unicellular. However, some unicellular eukaryotes exist such as
amoebas, yeast and paramecium.
(iii) Membrane bound nucleus: Eucaryotic cells have a true nucleus bound
by a double membrane. It contains DNA related functions of the large
cell in a small enclosure. This ensures close proximity of materials
and increases efficiency for cellular communication and functions. In
contrast, the smaller prokaryotic cells have no nucleus. The materials
are close to each other and there is a “nucloid” which is the central
open region of the cell where the DNA is located.
(iv) DNA structure: Eukaryotic DNA is linear and complexed with proteins called “histones” which organize into a number of chromosomes
Prokaryotic DNA is circular and is not associated with histones or
chromosomes. This cell is simpler and requires far fewer genes to
function than an eukaryotic cell. Therefore, it contains only one circular DNA molecules and various smaller DNA circlets (Plasmids).
1.5
Membrane-Bound Organelles
Eukaryotic cells contain many membrane enclosed organelles, which are
large and complex and in the cytoplasm. Prokaryotic cells do not contain membrane bound organelles. This is an important difference because
it allows to a large level of intracellular division and contributes to the
highly complex character of eukaryotic cells. The larger size of eukaryotic cells and the confinement of certain cellular processes to a smaller area
increases the efficiency of communication and movement within the cell.
10
Biophysical Chemistry
Eukaryotes only possess a membrane bound nucleus and organelles such
as mitochondria, lysosomes, golgi apparatus, peroxisomes and ER.
(i) Size of Ribosomes: Both types of cells contain many ribosomes but
the ribosomes of eukaryotic cells are larger than prokaryotic cells.
Eukaryotic ribosomes also show more complexity than prokaryotic
cells. They are formed from five kinds of ribosomal RNA and about
eighty kinds of proteins. Prokaryotic ribosomes are composed of
three kinds of RNA and about fifty kinds of proteins.
(ii) Cytoskeleton: This is a multi-component system in eukaryotes composed of microtubules, active filaments and intermediate filaments.
It is required to maintain cell shape, provide internal organization
and mechanical support. It is also important for cell division and
movement.
(iii) Sexual reproduction: Most eukaryotes undergo sexual reproduction
whilst prokaryotes reproduce asexually. Prokaryotes reproduce
clones of themselves via binary fission and relies more on horizontal genetic transfer for variation.
(iv) Cell division: This occurs by mitosis for eukaryotic cells and binary
fission for prokaryotic cells. Eukaryotic cells undergo mitosis and
then cytokinesis but this involves several stages. The nuclear membrane disintegrates and then the chromosomes are sorted and separated to ensure that each daughter cell receives two sets of chromosomes. Following this, the cytoplasm divides to form two genetically
identical daughter cells a process known as Cytokinesis. On the other
hand, prokaryotes undergo a simple process of binary fission. This is
faster than mitosis and involves DNA replication, chromosomal segregation and ultimately cell separation into two daughter cells genetically identical to the parent cell. Unlike mitosis, this process does not
involve the nuclear envelope and centromere and spindle formation.
1.6
Comparison Between Lysosome and Ribosome
To understand the above, we briefly consider the cell and its components.
Let us now consider the differences between lysosome and ribosome. A
cell contains different types of organelles which perform different roles in
the cell and they help in the survival of living organisms. As cell components, lysosomes and ribosomes perform different functions in the cell.
Lysosomes are found only in eukaryotic cells but ribosomes are found
in both eukaryotic as well as prokaryotic cells.
Cell and its Biochemical Setup 11
Figure 1.4 Components of the cell.
Lysosomes are membrane bound cell organelles and contain different
types of digestive enzymes. Lysosome is also known as garbage disposal
system of cells which destroy unwanted materials from the cell. Lysosomes
are capable of degrading all types of polymer of the cell because they have
a variety of digestive enzymes.
1.7
Types of Cells in Human Body
The diverse types of cells in the body may be listed as: (i) stem cells, (ii)
bone cells, (iii) blood cells, (iv) muscle cells, (v) fat cells, (vi) skin cells, (vii)
nerve cells, (viii) endothelial cells, (ix) sex cells, (x) pancreatic cells, and
(xi) cancer cells. The cells in human body are innumerable and come in
all shapes and sizes. These tiny structures are the basic unit of all living
organisms comprise of tissues, tissues make up organs and organs form
12
Biophysical Chemistry
Figure 1.5 Lysosome.
organ systems. The organ systems work together to create an organism
and keeps it alive.
Each type of cell in the body is specially equipped for its role. For
example, the cells of digestive system are very different in structure and
function from cells of the skeletal system. Cells of the body depend on
each other to keep the body functioning as one unit. A brief account of
types of cells is given below:
(i) Stem cells: Stem cells originate as unspecialized cells and have the
ability to develop into specialized cells which can build specific
organs or tissues. They can divide and replicate many times in order
to replenish and repair the tissue.
(ii) Bone cells: Bones are a type of mineralized connective tissue which is
a major component of skeletal system. Bones are made up of a matrix
of collagen and calcium phosphate minerals. There are three types
of bone cells in the body i.e., osteoclasts, osteoblasts and osteocytes.
Osteoclasts are large cells that decompose bone for resorption and
assimilation while they heal. Osteoblasts regulate bone mineralization and produce osteoid, an organic substance of the bone matrix,
which mineralizes to form bone. Osteoblasts mature to form osteocytes which aid the formation of bone. Osteocytes help maintain calcium balance.
(iii) Blood cells: They transport oxygen throughout the body to fight infection. They are produced by bone marrow. There are three major
types of cells in blood: (a) red blood cells, (b) white blood cells and
Cell and its Biochemical Setup Figure 1.6 Structure of phospholipid.
Figure 1.7 Structure of a phospholipid bilayer.
13
14
Biophysical Chemistry
(c) platelets. Red blood cells determine blood type and are responsible for transporting oxygen. White blood cells are immune system
cells that destroy pathogens and provide immunity. Platelets help
in clotting of blood and prevent blood loss due to damaged blood
vessels.
(iv) Muscle cells: They form muscle tissue which enables the body movement. There are three types of muscle cells—(i) Skeletal, (ii) Cardiac
and (iii) Smooth.
• Skeletal muscle tissue attaches to bones to facilitate voluntary
movement. The muscle cells are covered by connective tissue
which protects and supports muscle fibres.
• Cardiac muscle cells form involuntary muscle and are found in
the heart. These cells aid in heart contraction and are joined to
one another by intercalated discs that allow for heart beat synchronization.
• Smooth muscle is an involuntary muscle that lines body cavities
and forms walls of many organs like kidneys, intestines, blood
vessels and lung airways.
(v) Fat cells: These cells are a major cell component of adipose tissue and
they are also called “adipocytes”. Adipocytes contain drops of stored
fat (triglycerides) that can be used for energy. Adipose cells have
also a critical endocrine function. They produce hormones, influence
sex hormone metabolism, blood pressure regulation, insulin activity,
blood clotting and cell signaling.
(vi) Skin cells: The skin is composed of a layer of epithelial tissue (epidermis) that is supported by a layer of connective tissue (dermis) and
an underlying subcutaneous layer. The outer most layer of the skin
is composed of flat, squamous epithelial cells that are closely packed
together. The skin covers a wide range of roles, such as protecting
internal structures of the body from damage, prevents dehydration,
acts as a barrier against germs, stores fat, and produces vitamins and
hormones.
(vii) Nerve cells: Nerve cells or neurons are most basic for the nervous system. Nerves send signals between the brain, spinal cord and other
body organs via nerve impulses. Structurally, a neuron consists of
cell body and nerve processes. The central cell body contains neuron’s nucleus, associated cytoplasm and organelles. Nerve processes
are finger like projections that extend from the cell body and transmit
signals.
Cell and its Biochemical Setup 15
(viii) Endothelial cells: These form the inner lining of the cardiovascular system and lymphatic structures. They make up the inner layer of blood
vessels, lymphatic vessels and organs like the brain, lungs, skin and
heart. Endothelial cells are responsible for the creation of new blood
vessels. They also regulate the movement of macromolecules, gases
and fluid between the blood and surrounding tissues as well as manage the blood pressure.
(ix) Sex cells: Sex cells are reproductive cells created in male and female
gonads that bring new life into existence. Male sex cells (or sperm)
are motile and have long, tail like projections called flagella. Female
sex cells or ova are non-motile and relatively large in comparison to
male gametes. In sexual reproduction, sex cells unite during fertilization to form a new species, while other body cells replicate by mitosis,
gametes reproduce by miosis.
(x) Pancreatic cells: Pancreas functions both as exocrine and endocrine
organ indicating that it discharges hormones both through ducts and
directly into other organs. Pancreatic cells regulate blood glucose levels and also are important for digestion of proteins, fats and carbohydrates. Exocrine acinar cells, produced by pancreas, secrete digestive enzymes that are carried by ducts into small intestine. A small
percentage of pancreatic cells have an endocrine function or secrete
hormones into cells and tissues. Pancreatic endocrine cells are found
in small clusters. Hormones produced by these cells include insulin,
glucagon and gastrin.
(xi) Cancer cells: Cancer cells work to destroy the body. Cancer results
from the development of abnormal properties that cause cells to
divide uncontrollably and spread to other locations. Cancer cell development originates from mutations arising from exposure to chemicals, radiation, UV light. Cancer can also have genetic origins such
as chromosome replication errors and cancer causing viruses of the
DNA. Cancer cells spread rapidly because they develop decreased
sensitivity to anti-growth signals and proliferate quickly. They lose
the ability to undergo programmed cell death making them even
more formidable.
The cell membrane is a very adjustable structure composed primarily of
phospholipids (lipid bilayers). Cholesterol I also present in the membrane
and it contributes to the fluidity of the membrane. There are also other
proteins embedded in the membrane which have different functions.
16
Biophysical Chemistry
A simple phospholipid molecule has a ‘phosphate’ head group and
two side-by-side chains of fatty acids that make up the lipid tails as shown
in Figure 1.7. The phosphate group is negatively charged and is thus polar,
hydrophilic. The lipid tails are uncharged, nonpolar and hydrophobic.
They consist of saturated and unsaturated acids, and contribute to the fluidity of the tails which are in motion. Phospholipids are amphipathic (containing both hydrophilic and hydrophobic groups). The cell membrane
consists of two adjacent layers of phospholipids. The lipid tails of one layer
face the lipid tails of the other layer, joining at the interface. The phospholipid heads face out and one layer exposed to the interior of the cell, with
the other layer being exposed to the exterior.
The lipid bilayer forms the basis of the cell membrane and also contains various proteins. The cell membrane is associated with two different
types of proteins, namely integral and peripheral proteins. An integral
protein is one that is embedded in the membrane. A channel protein selectively allows certain species such as ions to pass in or out of the cell.
Another important group of the integral proteins is known as ‘recognition proteins’, which serve to mark a cell’s identity. A receptor is a cell
recognition protein. Recognition proteins can selectively bind a specific
molecule outside the cell and this binding induces a chemical reaction
within the cell. A ligand is a specific molecule that binds to and activates a
receptor. Some integral proteins serve as both a receptor and an ion channel. An example of a receptor-ligand interaction is the receptors on nerve
cells that bind neurotransmitters such as dopamine. When a dopamine
Figure 1.8 Representation of a cell membrane.
Cell and its Biochemical Setup 17
molecule binds to a dopamine receptor protein, a channel within the transmembrane protein opens to allow certain ions to flow into the cell.
Some integral membrane proteins are glycoproteins which have carbohydrate molecules attached that extend into extracellular matrix. The
attached carbohydrates aid in cell recognition. The carbohydrates that
extend from membrane proteins and from membrane lipids form what is
known as “Glycocalyx”. The “glycocalyx” is a coating around the cell from
glycoproteins and other carbohydrates attached to the cell. It has several
roles (i) it allows the cell to bind to another cell, and (ii) it may contain
receptors for hormones or it may have enzymes to break down nutrients.
1.8
Peripheral Proteins
Peripheral proteins are generally found on the inner or outer surface of the
lipid bilayer but may also be attached to the internal or external surface of
an integral protein. These proteins perform a specific function for the cell.
For example, some peripheral proteins on the surface of intestinal cells act
as digestive enzymes to break down nutrients to sizes such that they can
pass through the cells into blood stream.
1.9
Transport Across Cell Membrane
An important aspect of cell membrane is its ability to regulate the concentrations of substances inside the cell, such as ions like Na+ , K+ , Ca2+ , Cl− ;
nutrients like sugars fatty acids, amino acids and waste products like CO2
(which must leave the cell.)
The membrane’s lipid bilayer structure provides the 1st layer of control. The phospholipids are tightly packed together, and the membrane has
a hydrophobic interior. This structure causes the membrane to be selectively permeable. Because of this, only certain substances meeting specific
criteria can pass through it unaided. Thus only relatively small non-polar
materials like the gases O2 , CO2 and also some lipids can move through the
lipid bilayer. Water soluble substances like glucose, electrolytes and amino
acids require assistance to cross through the membrane because they are
repelled by the hydrophobic tails of the lipid bilayer. The passage of all
substances across the membrane occurs by two (general) methods depending upon whether energy is required or not for such a process; (i) Passive
transport, and (ii) Active transport. For passive transport, no cellular energy
need be expended for the movement of substances across the membrane.
18
Biophysical Chemistry
Figure 1.9 Simple diffusion across the cell.
But for active transport i.e., the movement of substances across the membrane, energy from ATP is used. Some details for passive or active transport of substances across the membrane are given below.
1.9.1
Passive Transport
This transport occurs via diffusion across a semipermeable cell membrane
whenever there is a concentration gradient across the membrane. Substances like O2 , CO2 , which can easily diffuse across the bilayer, do so
with the O2 diffusing into the cell (because it is more concentrated outside the cell) and CO2 diffuses out of the cell (because it is more concentrated inside the cell). It may be mentioned that O2 is rapidly used up
during metabolism and hence there is a lower concentration of the same
within the cell while CO2 is a product of metabolism and its concentration
is higher with in the cell.
Large polar or ionic species (which are hydrophilic) cannot easily cross
the phospholipid bilayer. Charged species of any size cannot cross the cell
membrane by diffusion because they are repelled by the hydrophobic tails
in the interior of the lipid bilayer.
1.10
Facilitated Diffusion
Facilitated diffusion is the diffusion process used for such substances that
cannot cross the lipid bilayer due to their size, charge or polarity. This diffusion of substances crossing cell membrane takes place with the help of
proteins such as channel proteins and carrier proteins. Channel proteins
Cell and its Biochemical Setup 19
Figure 1.10 Facilitated diffusion.
are less selective than carrier proteins and discriminate mildly between
their cargo based on size and charge carrier proteins are more selective
often allowing only one particular type of molecule to cross. A good example of facilitated diffusion is the movement of glucose into the cell where
it is used to make ATP. Its simple diffusion through the cell is not possible because of its size and polarity but is made possible via a specialized
carrier protein called glucose transporter.
As an example, even though Na+ are highly concentrated outside the
cells, they cannot pass through the non-polar lipid layer due to their charge.
Their diffusion is facilitated by membrane proteins that form sodium channels (or pores) so that these ions can move down their concentration gradient from outside to inside the cells. Facilitated diffusion is a passive
process and does not require any energy expenditure from the cell.
Water can also move freely across the cell membranes of all cells either
through protein channels or by slipping between the lipid tails of the membrane. The movement of water molecules is not regulated by cells, hence,
it is important that cells are exposed to an environment in which the concentration of solutes outside the cells (i.e., in the extracellular fluid) is equal
to the concentration of the solutes inside the cells (in the cytoplasm).
Another mechanism (besides diffusion) to passively transport materials between compartments is filtration. Filtration uses a hydrostatic pressure gradient that pushes the solution from a higher pressure area to a
lower pressure area and is an important process in the body. For example, the circulatory system uses filtration to move plasma and other substances across the endothelial lining of capillaries and into surrounding tissues supplying cells with nutrients. Filtration pressure in kidney provides
the mechanism to remove wastes from blood stream.
20
Biophysical Chemistry
Figure 1.11 Na+ /K+ pump. It is found in many cell membranes. It is
powered by ATP, moves Na+ and K+ ions in opposite directions, each
against its concentration gradient. In a single cycle of the pump, 3Na+
ions are extruded and 2K+ are imported into the cell.
1.10.1
Active Transport
During active transport, ATP is required to move a substance across a
membrane with the help of protein carriers against its concentration gradient. One of the common types of active transport involves proteins
that serve as “pumps”. Energy from ATP is required for these membrane
proteins to transport substances (molecules or ions) across a membrane
against their concentration gradient.
The Na+ /K+ pump, also called Na+ /K+ ATPase transports Na+ out
of a cell while moving potassium ions into the cell. The Na+ /K+ pump
is found in the membranes of many cells. These pumps are abundant in
nerve cells which constantly pump out sodium ions and pull in K+ ions to
maintain an electrical gradient across the cell membranes. In nerve cells,
the electrical gradient exists between the inside and outside of the cell with
the inside being −vely charged (at about −70 mV) relative to the outside.
The −ve electrical gradient is maintained because each Na+ /K+ pump
moves three Na+ ions out of the cell and pushes in two K+ ions into the
cell for each ATP molecule that is used. This process is important for nerve
cells and it accounts for the majority of their usage.
Active transport pumps can work together with other active or passive
transport systems to move substances across the membrane. For example,
the Na+ /K+ pump maintains a high concentration of Na+ ions outside the
Cell and its Biochemical Setup 21
cell. If the cell needs the ions, it has to open a passive sodium channel and
drive Na+ ions into the cell which is facilitated by the concentration gradient. When active transport powers the transport of another substance, it is
called secondary active transport.
Symporters are secondary active transport systems that move two substances in the same direction. For example, the sodium-glucose symporter
uses Na+ ions to pull glucose molecules into the cell. Because cells store
glucose for energy, it is at a higher concentration inside the cell than outside. But, due to the action of Na+ /K+ pump, Na+ ions diffuse easily into
the cell when the symporter is opened. The flood of Na+ ions through the
symporter provides the energy that allows glucose to move into the cell
against its concentration gradient.
Antiporters are secondary active transport systems that transport substances in opposite directions. For example, the Na− hydrogen ion
antiporter uses the energy from the inward flow of ions to move H+ ions
out of the cell. This antiporter is used to maintain pH of the cell interior.
Other forms of active support do not involve membrane carriers.
Endocytosis is the process of a cell ingesting material by enveloping
it in a portion of its cell membrane and pinching off that portion of the
membrane. Once pinched off, the portion of the membrane and its contents become an independent, intracellular vesicle. (A vesicle is a membranous sac-a spherical and hollow organelle bounded by a lipid bilayer
membrane) Endocytosis brings materials into the cell that must be broken
down or digested.
Phagocytosis (cell eating) is the endocytosis of large particles. Many
immune cells engage in phagocytosis of invading pathogens. In contrast to
phagocytosis, pinocytosis, (cell drinking) brings fluid containing dissolved
substances into a cell through membrane vesicles.
Phagocytosis and pinocytosis take in large portions of extracellular material and they are not highly selective in the substances they bring in. Cells
regulate the endocytosis of specific substances via receptor mediated endocytosis (receptor mediated endocytosis is endocytosis by a portion of cell
membrane that contains many receptors that are specific for a given substance). Once the surface receptors have bound sufficient amounts of the
specific substance, the cell will endocytose the part of the cell membrane
containing receptor-ligand complexes. Example: Iron is bound to a protein
called transferrin in the blood. Specific transferrin receptors on red blood
cell surfaces bind the ion-transferrin molecules and the cell endocytoses
the receptor-ligand complexes.
22
Biophysical Chemistry
Figure 1.12 Three forms of endocytosis. Endocytosis is a form of active
transport in which a cell envelops extra cellular materials using its cell
membrane. (a) In phagocytosis, which is relatively non-selective, the cell
takes in a large particle, (b) In pinocytosis, the cell takes in small particles
in fluid, (c) Receptor mediated endocytosis is quite selective. When external receptors bind a specific ligand, the cell responds by endocyting the
ligand.
In contrast with endocytosis, exocytosis (taking out of the cell) is the
process of a cell exporting material using vesicular transport. Many cells
manufacture substances that must be secreted. These substances are packaged into membrane bound vesicles with in the cell. When the vesicle
membrane fuses with the cell membrane, the vesicle release its contents
into the interstitial fluid. The vesicle membrane then becomes part of
the cell membrane. Cells of the stomach and the pancreas produce and
secrete digestive enzymes through exocytosis. Endocrine cells produce
and secrete hormones that are sent throughout the body and certain
immune cells produce and secrete large amounts of histamine, a chemical important for immune responses. Pancreatic cells enzyme secretion
products. They produce and vesicles secrete many enzymes.
1.11
Permeability of Molecules Across
Phospholipid Bilayers
Most molecules diffuse across a protein free lipid bilayer down its concentration gradient over a period of time. The diffusion rate depends on the
size of the molecule and its relative solubility in oil. Generally, a smaller
Cell and its Biochemical Setup 23
Figure 1.13 Exocytosis is endocytosis in reverse. Material to be exported
is packaged into a vesicle inside the cell. The membrane of the vesicle fuses
with the cell membrane and the contents are released into the extracellular
space.
Figure 1.14 Pancreatic acinar cells.
molecule which is more soluble in oil (i.e., more hydrophobic and nonpolar) diffuses rapidly across a cell membrane. Small, non-polar molecules
such as O2 , CO2 dissolve in cell membrane and thus diffuse rapidly across
where as small, uncharged polar molecules like water or urea also diffuse
but at a slower rate (ethanol diffuses readily). It may however be said that
24
Biophysical Chemistry
lipid bilayers are highly impermeable to ions and this is due to their charge,
size and hydration. Because of these factors, such molecules are prevented
from entering the lipid bilayer. These bilayers are 109 times more permeable to water than even to Na+ or K+ ions.
1.12
Thermodynamic basis of Transport
The diffusion of a substance X, across a membrane represented by x (outside membrane) → x (inside membrane) is associated with the Gibbs free
energy change in the standard state
[μ̄ x − μ̄0 ( x )] = RT ln a x
( a x = activity of x )
(1.1)
where μ0 ( x ) is the standard chemical potential
μ x (in) − μ x (out) = ΔGx = RT ln
a x (in)
a x (out)
(1.2)
If μ x (out) > μ x (in) , Δ̄Gx is negative, the spontaneous flow of x will be from
outside to inside.
If μ x (in) > μ x (out) , Δ̄Gx is +ve, and an inward only if an exergonic
process, such as ATP hydrolysis is coupled to it make the overall free
energy change negative.
The transmembrane movement of ions also depends on charge differences across the membrane, there by generating an electrical potential
difference,
Δψ = ψ(in) − ψ(out)
(1.3)
where Δψ is termed as the membrane potential. Therefore, if x is ionic,
the equation for ΔG( x) must be corrected to include the electrical work
required to transfer a mol of x from outside to inside as
Δ Ḡx = RT ln
[ a x ](in)
+ z x FΔψ
[ a x ](out)
(1.4)
where a’s are the activities and Zx is the charge on x and F is the Faraday
constant. ΔGx is now referred as the electrochemical potential of X.
1.12.1
Secondary Active Transport
This type of transport (also called co-transport uses energy to transport
molecules across a membrane; however, there is no direct coupling to ATP
(unlike in primary active support), instead the electrochemical potential
difference created by pumping out of true cell is instrumental.
Cell and its Biochemical Setup 25
Figure 1.15 Relative permeability of a phospholipid bilayer.
Figure 1.16 Mediated transport: (a) Passive transport and (b) Active
transport.
1.13
Examples of Antiport and Symport System
An example of an antiport system is the Sodium–Calcium exchanger which
allows three Na+ ions into the cell to transport one Ca2+ ion out.
An example of symport system is glucose symporter SGLTI which cotransports one glucose (or galactose) molecule into the cell for every two
Na+ ions it imports into the cell.
26
Biophysical Chemistry
Figure 1.17 Secondary active transport.
1.13.1
(Na+ /K+ ) ATPase
This active transport system is found in the plasma membranes of higher
eukaryotes. This transmembrane protein consists of two types of subunits: a 110 kD non glycosylated sub unit that contains the enzyme’s catalytic activity and ion binding sites and a 55 kD glycoprotein β-sub unit
of unknown function. The α-sub unit has eight transmembrane α-helical
segments and two large cytoplasmic domains. The β-sub unit has a single
transmembrane helix and a large extracellular domain. The protein many
function as an (αβ) tetramer in vivo.
The Na+ /K+ ATPase is also called as the Na+ /K+ pump because it
pumps 3Na+ out of and 2K+ in both directions across the membrane in
presence of hydrolyzing ATP. The overall reaction is
3Na+ (in) + 2K+ (out) + ATP + H2 O → 3Na+ (out) + 2K+ (in) + ADP + Pi
(1.5)
+
+
The important feature of Na /K ATPase is the phosphorylation of
a specific Aspartate residue of the transport protein which phosphorylates
only in the presence of Na+ whereas the aspartyl phosphate residue is subject to hydrolysis only in the presence of K+ . Hence it has two conformations named E1 and E2 .
They operate in the following way:
(1) The protein in the E1 state has three high affinity Na+ binding sites
and two low affinity K+ binding sites accessible to the cytosolic surface of the protein. Hence E1 binds 3Na+ ions inside the cell and then
binds ATP to yield an E1 . ATP.3Na+ complex.
Cell and its Biochemical Setup 27
Figure 1.18 Scheme for transport of Na+ and K+ by Na+ /K+ ATPase. Ion
pumping by Na+ /K+ ATPase involves phosphorylation, dephosphorylation and conformational change. In this case, hydrolysis of E2 -intermediate
powers E2 → E1 conformational change and simultaneous transport of
two K+ ions inward.
(2) ATP hydrolysis produces ADP and a ‘high energy’ aspartyl phosphate intermediate E1 -P.3Na+ .
(3) This ‘high energy’ intermediate relaxes to its ‘low energy’ conformation E1 ∼ P.3Na+ and relaxes its bound Na+ outside the cell.
(4) E2 -P binds two K+ ions from outside the cell to form E2 -P.2K+ complex.
(5) The phosphate group hydrolyses to yield E2 .2K+ (P = Phosphate
group)
(6) E2 .2K+ changes conformation, releases its 2K+ ions inside the cell
and replaces them with three Na+ ions thereby completing the transport cycle.
1.14
Ca2+ ATPase
Eucaryotic cells maintains a low conc of free Ca2+ in the cytosol (10−7 M)
where as extra cellular concentration is very high on the opposite face
28
Biophysical Chemistry
Figure 1.19 Scheme for the active transport of Ca2+ by the Ca2+ ATPase.
Here, (in) refers to the cytosol and (out) refers to the outside of the cell for
plasma membrane Ca2+ ATPase on the lumen of the endoplastic reticulum
(or sarcoplastic reticulum) for the Ca2+ ATPase of the membrane.
(10−3 M). Thus a small influx of Ca2+ significantly increases the conc of free
Ca2+ is the cytosol and the flow of Ca2+ ions down its steep concentration
gradient in response to extra cellular signals is one of the means of transmitting these signals across the plasma membrane. The Ca2+ ATPases are
commonly found in muscle cells and neurons.
Ca2+ transporters are the common examples of P-type transport of
ATPase. It is also known as Ca2+ pump or Ca2 ATPase. These transporters actively pump Ca2+ out of the cell and help in maintaining the
gradient. The structure of Ca2+ pump has an asymmetrical arrangement
of transmembrane and cytosolic domains that undergo movements during Ca2+ transport. It contains ten transmembrane α-helices and two cytoplasmic loops between transmembrane α-helices. The transmembrane αhelices form Ca2+ binding site which binds two Ca2+ ions from cytosol.
The two cytoplasmic loops form three separate domains nucleotide binding domains that binds ATP, actuator domain that contains catalytic phosphorylation site and P-domains which is important for transmission of
conformational changes between cytosolic and transmembrane domains.
In unphosphorylated state, the two helices are disturbed and form a cavity for binding of two Ca2+ ions from the cytosolic side of the membrane.
ATP also binds to a binding site on the same side of the membrane and
Cell and its Biochemical Setup 29
the subsequent transfer of the terminal phosphate group of ATP to an
Aspartic acid of an adjacent domain lead to a drastic rearrangement of the
transmembrane helices. This rearrangement disturbs the Ca2+ ion binding
site and releases Ca2+ ions on the other side of the membrane i.e., into the
lumen of SR (SR = Sarcoplastic Reticulum). The mechanism of Ca2+ ATPase
in the SR membrane may be understood through the following steps:
(1) The protein is E1 conformation has two high affinity binding sites for
Ca2+ ions accessible from the cytosolic side and ATP binds to a side on
cytosolic surface.
(2) In the presence of Mg2T , the bound form of ATP is hydrolysed to ADP
and phosphate. Subsequently, the liberated phosphate is transferred to
a specific aspartate residual in the protein forming the high energy acyl
phosphate bond given by El ∼ P.
(3) The protein then undergoes a conformational change and forms E2 ,
which has two low affinity Ca2+ binding sites accessible to the SR
human.
(4) The free energy of E1 ∼ P is greater than E2 − P and this leads to
E1 → E2 conformational change. Simultaneously, the Ca2+ ions also
dissociate from the low affinity sites to enter the SR humen following
which the aspartyl phosphate bond is hydrolysed.
(5) De-phosphorylation then leads to E2 → E1 conformational change and
E1 is ready to transport two more Ca2+ ions.
Questions
(1) The mass of a E.coli organism me.c yeast my and mh.c mass cell are in
the order
(a) me.c > my > mh.c
(c) mh.c > my > me.c
(b) my > me.c > mh.c
(d) mh.c > me.c > my
(2) (a) Eukaryotes are cells enclosed in a cell membrane
(b) Prokaryotes are cells enclosed in a cell membrane
(c) Eukaryotes are not multicellular organisms
(d) Prokaryotes are larger in size than eukaryotes
30
(3) (a)
(b)
(c)
(d)
Biophysical Chemistry
Mitochondria do not produce ATP
There is only a single membrane layer in mitochondria
Mitochondria do not contain DNA
DNA is present in mitochondria and replicates as needed.
(4) (a) The rough endoplastic reticulum contains only carbohydrates.
(b) The rough endoplastic reticulum synthesizes difference types of
lipids.
(c) The rough endoplastic reticulum does not synthesize proteins.
(d) The smooth endoplastic reticulum does not have ribosome with
in it.
(5) (a)
(b)
(c)
(d)
Ribosome is a membraneous of organelle
Lysosome is a membraneous organelle
Lysosomes are smaller in size than ribosomes
Lysosomes are present in the cytoplasm of eukaryotic and prokaryotic cells
(6) (a) Ribosome contains an enzyme
(b) Protein synthesis does not occur in ribosomes
(c) Lysosomes always remain separate from each other while ribosomes are grouped together
(d) None of the above statements are true.
(7) (a) Cholesterol is not part of lipid bilayer
(b) In a lipid bilayer, the phosphate group is negatively charged and
hydrophilic
(c) Lipid molecules in bilayer are amphiphilic
(d) Phospholipids contain only hydrophilic groups
(8) (a) Glycoproteins are integral proteins
(b) Peripheral proteins do not act as digestive enzymes
(c) Dopamine attached to a receptor is an example of a peripheral
protein
(d) Cell recognition proteins are peripheral proteins
(9) (a) For permeability across a membrane through passive transport,
cellular energy is spent.
(b) For active transport across a membrane energy from ATP is
required.
(c) Na+ /K+ pump is an example of passive transport.
(d) Filtration is not a method adopted by a cell for passive transport.
Cell and its Biochemical Setup 31
(10) (a) Sodium glucose symporter uses Na+ ions to move substances like
sugar and KCl from inside to outside of cell.
(b) Na+ -K+ pump is a hindrance for transport of Na+ into the cell.
(c) Na-H+ antiporter maintains pH of the interior of a cell.
(d) Antiporters are primary active systems for moving sugar.
(11) (a) Endocytosis requires membrane carriers for trimming part of
sugar of glucose outside cell.
(b) Phagocytosis is not synonymous with the endocytosis of large
particles.
(c) Immune cells do not use endocytosis to identify pathogens.
(d) Endocytosis of large molecules is referred as phagocytosis.
(12) (a) Iron bound to the protein transferrin uses endocytosis for transfer
of the complex into or out of cell.
(b) Exocytosis refers to the process of a cell bringing material into it.
(c) Pancreatic and stomach cells do not create digestive enzymes
through exocytosis.
(d) Immune cells secret histone through the process of endocytosis.
(13) The Gibbs free energy ΔGx of a substance from outside to the inside
of a cell given by
a x (in)
Δ Ḡx = RT ln
a x (out)
(a’s are the “activities” of the cell inside (a x (in)) and outside (a x (out))
indicates the spontaneous of the process in the cell when
(a)
(b)
(c)
(d)
μ x (inside) = μ x (outside)
μ x (inside) = μ x (outside)
μ x (outside) > μ x (inside)
none of the above
(14) (a) Sodium-Calcium exchanger is an example of antiport system.
(b) Glucose symporter is not an example of port system.
(c) Na+ -K+ ATPase is not present in plasma membrane of higher
eukaryotes.
(d) Corticosteroids do not bind to the surface of (Na+ -K+ ) ATPase
(15) The study of ion transport across membranes is a frontier area of
research in biochemistry. In a lipid bilayer membrane, the magnitude
of the concentration gradient is 50 mol m−4 . Estimate the ionic flux.
The diffusion coefficient of K+ ions is 8.7 × 10−5 cm2 s−1 .
32
Biophysical Chemistry
Solution: According to Fick’s first law of diffusion, the flux ( J ) can be
written as
J = − D (∂C/∂x ) x=0
= −8.7 × 10−5 cm2 s−1 (−50) mol m−4
= 4.35 × 10−3 mol m−2 s−1
(16) Draw a sketch of a human cell and indicate the parts. Explain their
function.
(17) What are eukaryotic and prokaryotic cells? Describe the similarities
and differences between the two types of cells.
(18) Explain what are Liposomes and Ribosomes? Bring out a comparative
account of these organelles.
(19) List out the types of bone cells in human body and give a brief description of their role in the body.
(20) Show the structures of phospholipid and lipid bilayers. Name the
proteins in the bilayer and their function.
(21) What are the general modes of transport of non-polar and polar
molecules across lipid bilayer and explain their mode of operation.
(22) Briefly describe the following: (i) Na+ /K+ pump (ii) Endothelial cells
(iii) Phagocytosis and (iv) Exocytosis.
(23) Give an example each of symport and antiport systems and explain
their function.
(24) Explain the transport of Ca2+ ions across the lipid bilayer with Ca2+
ATPase by means of a diagram and give details of the transport
mechanism.
2
Thermodynamic Aspects
of the Cell
2.1
Introduction
The variables such as temperature, concentration (more correctly, activity), nature of ions in electrolyte solutions dictate diverse phenomena viz.
osmosis across semipermeable membranes, diffusion of reactants, phase
changes, molecular aggregations etc. These processes are essential for comprehending the structure and function of human or animal cells.
Both thermodynamic and non-equilibrium thermodynamic (or thermodynamics of steady state) studies are useful to an understanding of the
cell functions as well as its interaction with the surroundings. The following examples in plants and animals may be considered:
(2.1)
(2.2)
A consideration of the entropy changes in the biosphere reveals that the
earth absorbs the solar energy at a rate of 5 × 1016 W (J/sec) so that the rate
of entropy input is 5 × 1016 W/6000 K ≈ 8 × 1012 WK−1 (assuming that
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_2
33
34
Biophysical Chemistry
the sun’s core temperature is 6000 K). If the earth radiates heat at the same
rate, the rate of output of entropy is 5 × 1016 /293 ≈ 2 × 1014 (assuming
an average temperature of earth as 20◦ C). Thus, the rate of entropy production in the biosphere is about 2 × 1014 WK−1 . Living cells of all types
contribute only a small part of this entropy.
2.2
Enthalpy and Free Energy Changes in
Biochemical Processes
In biochemical processes at 1 atm pressure without any gas phase, the P −
V work is negligible. In a biochemical context, a molecular volume change
of one (nanometer)3 [i.e., (10−9 m3 ) = 10−27 m3 ] is quite large but the
P − V work is only 10−23 J molecule−1 .
Biochemical reactions often take place at constant temperature T. From
the relation
G = H − TS = U + PV − TS
any change in the state of the system, can be expressed as
ΔG = ΔH − TΔS
(at constant T)
The term TΔS refers to the heat gained at a given temperature and in
a biochemical process it is the entropic contribution to the Gibbs energy
change ΔG.
2.3
Effects of Electrochemical Changes
In an electrolyte solution, positive ions tend to accumulate in the regions
of low electrical potential Φ where their electrical potential energy is low.
For an ion of energy q the electrical potential energy is q × Φ. Under equilibrium conditions, +ve ions accumulate preferentially in regions of low
electrical potential, whereas negative ions (anions) have higher concentrations at high values of Φ. The equilibrium distribution of ions is given by
Boltzmann distribution law, given by
C = C0 ε−zFΦ/RT
(2.3)
where z represents the ionic charge, F indicates the Faraday constant and
C0 denotes the concentration of the ions at Φ = 0.
Thermodynamic Aspects of the Cell 2.4
35
Distribution of Ions Across Membranes
The cytoplasm of a cell has a lower electrical potential than the external
solution. Considering the Cl− ions in the cytoplasm, their concentration
is lower in the cell than outside at equilibrium. In the case of cations permeating into the cytoplasm of a cell, their concentration is higher in the
cytoplasm than outside. Large molecules such as glucose and proteins do
not permeate while others cross the membrane under certain conditions.
The negative potential in the cytoplasm is maintained in part by pumps
i.e., mechanisms that by expending energy produce a flux of specific ions
in a particular direction. This distribution (or mechanism) is illustrated in
the following diagram.
Figure 2.1 Boltzmann distribution of permeating cations and anions.
Boltzmann distribution explains the equilibrium distribution of permeating cations and anions across a membrane. For a permeating cation,
the left side has lower electric potential (low Φ) while for permeating
anions it is on the right. The highest entropy state would require equal
concentrations every where. Many of the intracellular anions required to
36
Biophysical Chemistry
approach electro-neutrality are found on non-permeating species including macromolecules while the extra cellular solution will also contain many
cations that are expelled from the cell by active energy using processes.
2.5
Distribution of Ions Near Charged Membranes
and Macromolecules
In the vicinity of a negatively charged surface of a membrane or a macromolecule, the concentration of cations in the solution is higher than in
the bulk of the solution. Since the surfaces of many membranes are −ve
charged, there is usually an excess of cations and a depletion of anions near
the membrane. Many macromolecules also have a small excess of negative
charge and are therefore surrounded by excess cations as shown in the Figure 2.2.
Figure 2.2 Distribution of ions on negatively charged surfaces.
In figure 2.2(a) the layer of excess +ve charges (i.e., electrical double
layer) near the interface counteracts the above at sufficiently large distance.
(b) and (c) show how the electrical potential Φ and the ionic concentrations
C+ and C− vary near the surface but approach their bulk concentration at
large distance X from the charged surface. The electric charge of the ions
has another effect that contributes to their distribution i.e., the Born energy.
The Born energy of an ion is the energy associated with the field of the ion
and it depends on the nature of the medium, being lower in polar media
(like water) than in non-polar media (such as the hydrophobic regions of
lipids or other macromolecules). Thus ions are found in negligible concentrations in the hydrocarbon interior of membranes where Born energy is
high. This energy also affects the distribution inside the pores in aqueous
medium. If the ion is sufficiently close (in nm range) to a non-polar region
of the membrane it has still a high Born energy because of the penetration
Thermodynamic Aspects of the Cell 37
of the field into non-polar regions. The Born energy and Boltzmann distribution act in such a way that the concentration of either cations or anions
is lower both in non-polar regions and also in polar aqueous solution near
such regions. Thus the equilibrium distribution has a small excess of negative charge and is therefore surrounded by excess cations as shown in the
Figure 2.2.
Thus, the equilibrium distribution of the ions is a compromise between
minimizing the Born energy (i.e., ions keeping away from non-polar
regions) and maximizing entropy (uniform distribution of ions).
Electrical interactions are weaker in solution than in pure water
because ions of opposite sign tend to accumulate near charges and screen
their effects. Further electrical effects in pure water are much weaker than
in vacuum because of the dipolar nature of water.
2.6
Osmotic Effects
Osmosis refers to the movement of a solvent through a semipermeable
membrane from a region of low solute concentration to a region of high
solute concentration. Note that a semipermeable membrane is one in which
Figure 2.3 Permeability of cations and anions.
38
Biophysical Chemistry
the pores are so small that water molecules can pass through them but not
solute particles (because of their much larger size). Many membranes are
permeable to water but impermeable to macromolecules, sugars and some
ions as well.
In figure 2.3(a) the membrane is permeable to water but not to solutes.
Water flows from the solution of low concentrations into that of higher concentrations. This flow stops when the osmotic pressure in the concentrated
solution equals to that on the other side. (b) The entropy of the system will
be maximized if the membrane bursts and the solutions mix freely. For
the osmotic equilibrium, the value of the hydrostatic pressure difference
required to stop from their flow into the solution side:
The equilibrium pressure difference = P = Osmotic pressure = RTCs
where Cs denotes the concentration of the solute in mol liter−1 (or kmol
m−3 ). If the solute concentration = 1 mol liter−1 of non-ionic solute, I I ≈
P = 0.082 × 300 × 1 = 24.63 atm = 24.63 × 1011.325 = 24.9kPa = 2.5 MPa
at 300 K.
The semi permeability of membranes is not the only factor that prevents solutes and water mixing completely. Solutes of large size cannot
enter the narrow spaces between closely packed macromolecules or membranes. When cells are dehydrated (by extracellular freezing or air-drying)
the highly concentrated solutions produce very large osmotic effects.
2.7
Role of Chemical Potential
The Boltzmann equation is given by
C = C0 e−ΔE/RT
(2.4)
or
ln C = ln C0 − ΔE/RT
RT ln C0 = RT ln C + ΔE
(2.5)
(2.6)
where ΔE is the energy difference between the given state and the reference
states. ΔE may also include P − V work or electrical energy etc. Considering the P − V work as the only energy term, the chemical potential μ may
be expressed as
μ = μ◦ + RT ln C + PV
= μ0 + RT ln C0
(2.7)
(2.8)
Thermodynamic Aspects of the Cell 39
where μ◦ = chemical potential in the standard state. It is the value of μ in
some reference state with a concentration C0 .
2.8
Effects of Different Molecular Environments
and Phases on Energy and Entropy
For a solid, the average intermolecular energy of attraction is kT; in a gas, it
is kT; in a liquid, it is comparable to kT. The entropy of the three phases of
a substance Ssolid < Sliq < Sgas . In the case of energy also, it increases from
solid to gas due to supply of the latent heats of fusion and evaporation.
Thus, the coexistence of two phases in equilibrium such as ice aqueous
solution or solution vapour are examples of entropy-energy compromise. The energy of a solute molecule is different in different solvents. If
the molecule is non-polar, its energy is different when it is in aqueous solution than when it is partitioned in the interior of a membrane. These differences may be considered in terms of the chemical potential. If molecules
of a substance are in equilibrium at a temperature T and pressure P.
According to Gibbs phase rule,
μ (phase 1) = μ (phase 2)
μ1◦
+ RT ln C1 + PV1 =
μ2◦
+ RT ln C2 + PV2
(2.9)
(2.10)
When the energy in terms of electrostatic potential energy is also included,
the equation for chemical potential may be written as
μ = μ◦ + RT ln C1 + PV + ZeΦ
(2.11)
Molecules diffuse from a region of high chemical potential to regions of
low chemical potential at a rate that increases with the gradient in Φ.
2.9
Surface Free Energies and Surface Tension
An important free energy difference in cellular biology is that between
water molecules at the surface and the water molecules in the interior of
the solution. At surfaces between two pure fluids having an interfacial tension, γ, the extra work done in introducing a molecule of area “a” to the
interface is “γa” i.e., the surface free energy per unit area.
In the bulk solution, water molecules are H-bonded to their neighbours. A water molecule at an air-water interface has fewer neighbours
with which to form H-bonds. So, its energy is higher. Because the molecules
40
Biophysical Chemistry
Figure 2.4 The surface free energy of water.
will tend to rotate to form bonds with their neighbours, the surface is more
ordered and thus have a lower entropy per molecule. These effects give
the water surface a relatively large free energy per unit area which gives
rise to hydrophobic effect. The work done per unit area of a new surface is
numerically equal to γ.
2.10
Molecular Aggregation
A single molecule has an energy of translation which may be related to
the entropy associated with its freedom to diffuse through at the available volume. When molecules aggregate to form polymers, the entropy of
translation is shared among N participating molecules. Hence entropy per
molecule is obtained by dividing the number by N. Consider a simple lipid
molecule in water with its standard chemical potential μ◦ , in equilibrium
with a micelle or vesicle containing N molecules. The standard chemical
potential μ◦N in the aggregate is much lower because the aggregate has a
smaller hydrocarbon-water interface per molecule. The membrane may
be subject to a tensile force per unit length, Y, and this may do work by
changing the area.
Figure 2.5 (a) The equilibria among a lipid monomer, (b) a vesicle containing N molecules and (c) a microscopic lipid bilayer.
Thermodynamic Aspects of the Cell 41
The equilibrium in terms of change in chemical potential may be given
as
μ1◦ + kT ln X1 = μ◦N +
kT
ln
N
XN
N
= μ◦m − γa /2
(2.12)
where μ◦m is the standard chemical potential of lipids in the membrane,
X1 , X N are the number fraction of lipids in the monomer and vesicle state
respectively. The factor 12 in eqn. (2.8) arises because each lipid molecule
has an area “a” in only one face of the membrane. If the lipid has more
or longer hydrocarbon tails, then the extra energy difference due to the
hydrophobic effect will increase (μ1◦ − μ◦N ) resulting in a lower monomer
concentration. Increasing the tensile stress in the membrane (larger Y)
makes the last term (equation 2.12) more −ve, so monomers and vesicle
will move into the membrane increasing the area and relaxing the stress.
Alternatively changing the area of a membrane causes a transient mechanical change in Y and results in the transfer of membrane contents between
the membrane and a reservoir of material. It is possible that such processes
occur in some cell membranes where regulation or homeostasis of membrane tension and cell area. Homeostasis is the state of steady internal,
physical and chemical conditions maintained by living systems. It is a condition of optical functioning for the organism and includes many variables
such as body temperature and fluid balance being kept within certain preset limits. Humans rely on homeostasis to keep this temperature around
98.4◦ F.
2.11
Non-equilibrium Thermodynamic Treatment
of Bacterial Cells
Bacteria in their natural habitat have an environment whose physical and
chemical nature change little with time. Living higher animals in whose
organs or tissues various bacterial species are found, have been endowed
with a homeostatic capability. A bacterium is an open system from a thermodynamic standpoint. The theory of non-equilibrium thermodynamics
(which applies to open systems) is also applicable to bacteria.
2.12
Bacterial Cell as a Thermodynamic System
An individual bacterial cell bounded by the outer cell wall is the thermodynamic system under consideration. A living bacterium grows and at
some point, during growth it divides into two bacteria whose size varies
42
Biophysical Chemistry
during growth. The relative size of the two daughter cells varies but there
are upper and lower limits to cell volume. Bacterial life is thus a cyclic process beginning with the newly separated daughter cell and starting again
when the next generation grows as a free species. A single cell or the sum
of all cells in a culture is an open system in a thermodynamic sense. Matter,
energy and entropy flow into the system followed by physical and chemical changes within the system which are accompanied by net production
of entropy. Conversely matter, energy and entropy flow from the system
to the environments. The environment includes aqueous solutions of cell
nutrient, any substances discarded by the cells and any gases above the
solution.
2.13
Physical and Chemical Features of the
Cell and Environment
2.13.1
Physical Features
The system is composed of three major physical components: (i) the outer
most part of the cell wall which limits the volume of the cell; (ii) a very
thin membrane lying beneath the cell wall, its major function being to control the flow of molecules into and out of the cytoplasm; (iii) the cytoplasm
which is an aqueous solution containing molecules such as glucose, ions
like Na+ , K+ and Cl− in the interior of the cell. Other organelles of different kinds are also present in the cell.
2.13.2
Chemical Features
The bacterial cell wall, cytoplasmic membrane, the cytoplasm contain
many substances in the environment of growing cells. The concentrations of some molecules (e.g., glucose), electrolytes (e.g., NaCl, KCl) in the
cell’s environment decrease steadily with time. It is therefore necessary
that nutrient molecules enter the bacterial cell so that chemical reactions
occur to produce additional bacterial material in the cell. Within the cell
system, nutrients undergo reactions that produce macromolecular components resulting in an increase of bacterial cell mass and volume. The
energy required for the syntheses is obtained from the reactions of nutrient molecules in which some energy is lost as heat. Such chemical reactions that produce complex molecules (from relatively simple ones occur
in a coordinated sequence of reactions), each producing one molecule more
complex than the one utilized. The entropy produced in the cells flow into
the environment.
Thermodynamic Aspects of the Cell 43
As the thermodynamic system (i.e., the bacterial cell) functions, it is
observed that some low molecular weight substances also appear and their
concentration increases with time. This is due to cell metabolism. For
example, CO2 produced by metabolism of carbohydrates does not accumulate in the system but is released to the environment. A bacterial mass
(composed of many individual cells) must contain many cells in every
physiological state between two cell divisions. Due to this, the available
data are averages over cell growth and division cycle. If one has cultures in
which growth of cells is perfectly synchronized with respect to the physiological state of individual cells, they could be chemically analyzed (at any
chosen physiological state). For a mass of synchronously growing cells,
it would in effect be amplifying the response of a single cell by a certain
factor i.e., the number of cells per unit volume.
Even though one works with average values of the desired physical
and chemical properties of a system, some thermodynamic properties of
the system can be employed to study the system. Hinshelwood et al., have
considered the chemical species in the bacteria, the reactions producing
them and the kinetics of the reactions. These studies led to the proposals of reaction sequences starting with nutrient molecules and ending with
the formation of bacterial substances and waste products. Many molecular entities appear as members of more than one sequence thus providing
a way for the coupling of sequences. The sequence of physical and chemical processes comprising bacterial growth (derived from kinetic studies) is
associated with a decrease of Gibbs free energy.
When a system of bacteria is studied under growth as a batch culture,
it has the disadvantage of living under continually changing environment.
The supply and environmental concentration of nutrient molecules (except
O2 ) diminish rapidly as the system enlarges cell division while concentration of waste molecules rises rapidly. Thus, the system is (thermodynamically) an open system and the effect of these rapid, drastic environmental changes requires constant changes in the system to maintain life. Ultimately cell replication ceases and the disintegration of the system begins.
2.13.3
Continuous Culture
Continuous culture of bacteria under time-invariance provides chemostatic
conditions. These conditions provide only the desired approximate environmental conditions. It is preferable to use chemically defined media to
obtain accurate and continuous control of nutrient chemical species in the
environment so that the identification and quantification of molecules discarded by the system is easy.
44
Biophysical Chemistry
As bacteria multiply in a continuous culture apparatus, the population
density of the cells become established at a fixed level in a short time. New
cells replace those that leave with the effluent because the thermodynamic
system (i.e., the bacterial cell) has attained a steady state of growth and
replication.
2.14
Concept of Steady State
In steady state, there are no changes of intensive variables of the system
with time. Thermodynamic potential differences between the system and
environment are maintained at a positive value because of constraints
applied through the system. Some examples of the constraints are chemical potential difference of solutes, pressure and temperature gradients.
As stated above, time-invariance of the constraints is necessary if the system is to achieve a steady state. Altering the magnitude of an external
constraint after the system reaches steady state results in changes of the
thermodynamic parameters of the system until the system reaches a new
steady state. The distinction between steady state condition and equilibrium may be resolved by considering an isolated system, where it can neither exchange matter or energy with its environment. A system in a steady
state would undergo changes for a while after it was isolated but for a system at equilibrium there would be no change at all.
2.15
Non-equilibrium Thermodynamics
in Microbiology
The condition for the spontaneity of a reaction ΔGT,P < 0 is not applicable to bacteria because reactions associated with bacterial cells such as
cell growth require variation of pressure or temperature as external constraints. The only criterion of spontaneity in such cases is the entropy
change accompanying such processes. It is known that this quantity always
increases in a spontaneous (or irreversible) processes. The entropy change
can occur in two ways: (i) entropy flow between the system and the
environment, and (ii) production of entropy within the system. Nonequilibrium thermodynamics concerns with the quantitative evaluation of
entropy production within the system. Entropy per unit time permit volume is a sum of terms, each of which is defined as the product of a flow and
the conjugate force that drives the flow. For example, entropy production
accompanying the flow of heat has a temperature gradient as the conjugate force. Similarly, affinity is the driving force for a chemical reaction.
Thermodynamic Aspects of the Cell 45
Simple free-living microorganisms have numerous metabolic pathways to
follow. The utilization of these pathways implies flows of matter and heat
into and out of the system that is also associated with entropy production. The variety of chemical reactions occurring within the organism is
a accompanied by further production of entropy. To apply the principles
of non-equilibrium thermodynamics to the study of these organisms, it is
necessary to assume simple possible sequence of metabolic reactions of all
those that occur in cell species.
2.16
Linear Laws
It is possible to apply linear laws relating flows and forces whose product is
the entropy of the total process of bacterial life. The set of phenomenological equations that describe the bacterial growth must include: (i) equations
for chemical reactions within the cell, (ii) equations describing the entry of
nutrient materials and exit of waste materials, and (iii) equations relating
energy exchange with the environment. Consider the functioning of some
cells maintained in a time invariant nutritional environment as described
by the following set of linear laws.
J1 = L11 X1 + L12 X2 + L13 X3 + L14 X4
(2.13)
J2 = L21 X1 + L22 X2 + L23 X3 + L24 X4
(2.14)
J3 = L31 X1 + L32 X2 + L33 X3 + L34 X4
(2.15)
J4 = L41 X1 + L42 X2 + L43 X3 + L44 X4
(2.16)
where Ji = flow of heat, chemical substance (diffusion) or rate of a chemical reaction (i = 1, z, . . . , n). X j = force conjugate to flow Ji (temperature
gradient), difference of chemical potential [or affinity ( J = 1, z, . . . , n)]. Lij
= Onsager’s (straight) coefficients if i = j; Onsager’s cross coefficients if
i = j, Lij = L ji .
Equations (2.13 to 2.16) indicate that one must determine experimentally (and simultaneously) each of the flow’s Ji occupying when each force
X j has a known value that one may calculate the values of the constants Lij .
Onsager’s reciprocal relations are useful in this context. Experimentally,
one requires the use of a continuous flow culture apparatus to fulfil the
conditions of time invariant change of nutritional environment. When one
46
Biophysical Chemistry
deals with bacteria, it is difficult to determine directly the chemical potential of any substance inside a bacterial cell. Such difficulties can be circumvented by employing equations reciprocal to equations (2.13 to 2.16), i.e.,
X1 = R11 J1 + R12 J2 + R13 J3 + R14 J4
(2.17)
X2 = R21 J1 + R22 J2 + R23 J3 + R24 J4
(2.18)
X3 = R31 J1 + R32 J2 + R33 J3 + R34 J4
(2.19)
X4 = R41 J1 + R42 J2 + R43 J3 + R44 J4
(2.20)
where
Rij =
[ Lij ]
, [ Lij ]
[ L]
being the minor determinant corresponding to the coefficient Lij and [ L]
the determinant of the matrix of all the coefficients Lij . The Onsager reciprocal relation apply to Rij (i = j). Equations (2.17) to (2.20) give the forces
as functions of the flows. The fluxes of particular chemical species into or
out of the cell can be determined with suitable chemical techniques. The
importance of equations (2.13 to 2.16) and (2.17 to 2.20) arises from the fact
that by proper manipulation of the experimental of condition one or more
of the flows or forces can be held to zero value. This leads to simpler relationships among constants and experimental quantities and it is easier to
evaluate specific constants. If, for example, the temperature of the environment (and hence of the cell) be set at set new value in a chosen sequence, it
may be determined that one or more specific numerical constants, Lij , take
on new values for each temperature. With these changes, the same forces
and flows may serve to describe the functioning of the bacteria. If one
uses the steady states sustained by bacterial growth in a continuous culture (as affected by the concentration of the nutrient in the environment)
to determine the phenomenological coefficients for a single organism, one
is able to compare the efficiency of utilization of those carbon sources by
the organism under the specified conditions. Changes in numerical values of phenomenological coefficients would signal alterations of relative
importance of metabolic reactions.
2.17
Force and Flows in Living Systems
2.17.1
Temperature and Heat Flow
There is a temperature difference between the interior of a living bacterial
cell and the medium that surrounds the cell. Heat moves from a point
Thermodynamic Aspects of the Cell 47
at higher temperature to a point of lower temperature, nutrient medium
surrounding growing bacteria becomes heated. It is the driving force of
a small amount of heat flow and the product of the force and heat flow
contribute to entropy production.
The rate of heat flux flow with in the cell will be affected by the temperature drop across cell boundaries and the heat conductivity of the cell and
its boundary layers. Consequently a steady state of internal temperature
will be attained after a sufficient length of time. The internal temperature
at the steady state characteristic of a bacterial species will govern the rates
of reactions with in the cell and the metabolic diffusion rates (with in the
cell) and across the boundary.
The temperature difference between the environment and the cell interior can be considered in terms of non-equilibrium thermodynamics and it
can also explain the observations that bacteria are able to live at temperatures where most enzymes are inactive.
By the temperature drop across cell boundaries and the heat conductivity of the cell and its boundary layers. Consequently, a steady state of
internal temperature will be attained after a sufficient length of time. The
internal temperature at the steady state characteristic of a bacterial species
will govern the rates of reactions within the cell and the metabolic diffusion rates (within the cell) and across the boundary.
The temperature difference between the environment and the cell interior can be considered in terms of non-equilibrium thermodynamics and it
can also explain the observations that bacteria are able to live at temperatures where most enzymes are inactive.
Thermophilic organisms (i.e., heat living organisms which can thrive
above 55◦ C) are stable at their observed temperature because at that temperature the thermodynamic force (related to temperature difference
between the environment and the cell interior) is compatible with other
thermodynamic forces, e.g., affinity of the system. The contribution of temperature gradient to the flow of substances through the cell membranes of
bacteria and its influence on metabolic processes differs widely for different membranes.
2.18
Chemical Potential and Mass Transfer:
Activated Support
Another combination of flows and forces contributing to entropy productions by microorganisms is mass flow and difference in chemical potential. Nutrients and metabolic products pass through the exterior layers of
48
Biophysical Chemistry
the cell at a rate that depend, on the nature of the cell wall, the specific
substances and their chemical potential differences across the exterior layers. The amount of flow of any one substance caused by non-conjugate
forces may be small or large and the flow of the substance driven by nonconjugate forces may be in the same or opposite direction as that of the
force.
2.19
Applicability of Linear Laws
Biological membranes have been examined using the theory of nonequilibrium thermodynamics. This formalism has been employed to
describe the function of ascites tumor cell membranes and also to transport through toad skin. Synthetic membranes are capable of demonstrating some of the properties of living membranes. Linear equations with
constant phenomenological coefficient have been used to describe their
behavior. These equations help in describing the coupling of an enzymatic
reaction to transmembrane flow of electric current in a synthetic active
transport system involving enzymes.
2.20
Non-linear Descriptions
The linear kinetic equations yield results in agreement with experiments
in cases near equilibrium (near equilibrium assumption). In such cases,
affinities of the involved chemical reactions are small.
2.20.1
Systems Involving Large Affinities
Prigogine et al., have shown that in some systems, there are steady states
both near equilibrium and far from equilibrium. In these cases, the same
chemical reactions account for events under both circumstances.
In one model proposed, two substances A and B are initially transformed into two final products D and E, through two intermediates X
and Y by the action of the catalysts C, W, V and V following the general
scheme:
(2.21)
(2.22)
Thermodynamic Aspects of the Cell 49
The following reactions are assumed to take place
(2.23)
(2.24)
(2.25)
The above model is satisfactory in a steady state near equilibrium. The
requirements of linear irreversible thermodynamics are met with in this
case.
Consider equations (2.17) and (2.18). The carbon source (analogous to
B) serves as a precursor to bacterial mass “D” and for discarded molecules
(E) from which energy is extracted for combining (A) and (B) and for all
other reactions requiring energy. The reactant A represents the nitrogen
source and other substances required for cell function. In order that the
system may function, certain catalysts in enzymes (C, W, V, V ) must be
present as one produces new bacterial mass only from preexisting living
bacterial mass.
Prigogine et al., have shown that oscillations around an unstable steady
state is a typical phenomenon of systems far away from equilibrium. It is
believed that bacterial cell growth and division is a single process which
can be interpreted as a process of oscillation about an unstable steady state.
Cyclic processes in open systems with constant external constraints can be
understood in terms of the theory of non-equilibrium thermodynamics.
2.21
Simple Cell Functions in Non-equilibrium
Thermodynamics
Non-equilibrium thermodynamics provides a reasonable basis for understanding the operation of a living cell. The chemical reactions that occur in
cell growth are not at equilibrium. The appearance of non-homogeneities
i.e., the production of cell membranes, cell walls as a result of these processes indicates that these phenomena involve non-equilibrium stationary
states far from equilibrium. In different growth media, chemical and physical properties of bacteria within a strain (of one species) show marked
differences.
50
Biophysical Chemistry
Growth temperature is a significant variable in continuous culture of
a yeast and bacteria. The E.Coli B observations on S. cerevisiae LBGH 1022
and on a bacterium Aerobactor cloacae demonstrate that for a given set of
time-independent external constraints, there results (in continuous cultures) a single characteristic cell population density.
2.22
Batch Cultures
The theory of non-equilibrium thermodynamics has been developed under
the postulate of time independent boundary conditions and its practical
application to bacteria cell growth, divisions and function is only possible
when such conditions are present. The “lag phase” in batch cultures is that
period during which the condition of time in variant external constraints is
nearly met. The “lag” ends with the first division of the cells of the inoculum. Prior to division, the cells of the inoculum have attained an unstable
stationary state of growth; departure from the unstable state is initiated by
some perturbation such as an enzyme function. The “exponential phase”,
the stationary phase and the phase of decline of batch cultures are artifacts
of the batch culture. The exponential phase is a steady state of growth. The
fundamental condition of time invariant external constraints is not met in
batch cultures in all the phases.
In batch cultures, the reduction in nutrient concentration in extracellular environment (as a result of cellular growth) will cause a reduction
in affinities of some substances which causes cessation of growth. In some
cases, end products of metabolism appear outside the cell as –COOH
anions. The apparent toxicity of metabolic end products increases with
decreasing pH in batch cultures. The uncharged carboxylic acid molecules
pass more easily through the cell’s negatively charged exterior layers than
does the anions. The cell’s internal concentration of the acid or anion will
rise as –COO and reenters leading to lowered affinity of the metabolic reaction producing it.
2.23
Thermodynamic Concepts
(1) Entropy change: The change in entropy of a closed system is greater
than zero for all irreversible processes and may be expressed as
di S ≥ 0
(2.26)
with equality sign being valid for a reversible process The entropy
changes of an open system (Example: a single bacterial cell) may be
Thermodynamic Aspects of the Cell 51
given as
dS = de S + di S
(2.27)
where dS = total entropy change of the system; de S = the entropy
change resulting from exchange of energy and matter between the system and environment, di S = total entropy change due to physical or
chemical changes within the defined system, it may be zero or positive. The entire time span of cell life is a sum of incremental time periods during which the incremental changes occur. If a bacterial cell is
divided into two subsystems such as cell cytoplasm (I) and cell covering (II) the entropy production of the entire system is
di S = di ( S I + S I I ) ≥ 0
= di S + di S
I
II
(2.28)
(2.29)
The entropy production by every macroscopic region of the system
must separately be greater than zero i.e.
di S I ≥ 0,
di S I I ≥ 0
(2.30)
Entropy production must occur in a single volume element. It is manifest in metabolizing systems through the coupling of reactionsproduction by one reaction in a sequence of a molecule required in
the next reaction of the sequence. Coenzymes are thus thermodynamic
necessities; because they exist it is possible to carry out chemical reactions in controlled steps, the sites of the steps being well separated as in
all living system. Non-equilibrium thermodynamics is concerned with
the quantitative evaluation of d1S for spontaneous processes. Changes
of this nature can be exemplified by conformational changes in proteins. Such reactions are involved in the growth and function of
bacteria.
(2) The Gibbs equation: It is given by
dS =
1
P
∑ μr
du + dv −
dnr
T
T
r T
(2.31)
where the terms have their usual significance. The Gibbs equation is
developed for systems at equilibrium. In non-equilibrium thermodynamics (systems not in equilibrium), it is postulated that there exists at
every point a state of local equilibrium for which the local entropy is
given by the Gibbs equation.
52
Biophysical Chemistry
(3) Entropy production: The total entropy change of a system dS varies
with time as dS/dt. But
dS
de S di S
=
+
dt
dt
dt
(2.32)
where ddte S = entropy exchanged with environment (in unit time).
Entropy produced within the system per unit time. If the whole volume of a system is considered as the sum of many volume elements,
di S may be considered as the sum of internal entropy production in
their volume elements. In each volume element the entropy produced
is called the “local entropy production” symbolized by σ. It can be
divided into three parts: (i) Production of pure entropy = heat flow ×
gradient of temperature function, (ii) production of entropy by diffusion of one or more substances present, and (iii) production of entropy
due to chemical reactions occurring with in the system. Each of these
three parts (components) is a product of two terms, one a flow and the
other a force. (i) production of pure entropy = heat flux × gradient of
temperature function, (ii) production of entropy by diffusion process =
flow of matter by diffusions × gradient of chemical potential, and (iii)
production of chemical reaction entropy = rate of a chemical reaction ×
a function of the affinity of the chemical reaction. We may summarize
the above by writing
σ=
∑ Ji Xi
i = 1, 2, 3, . . . , n
(2.33)
Once a flow is described, the primary force associated with it is fixed by
certain thermodynamic requirements. Ji is said to be the flow conjugate
to the force Xi . The primary idea of the local entropy production is
to establish the flows and forces involved in the irreversible processes
occurring in the system under consideration.
(4) Linear Law: They may be illustrated by an example of “thermo couple”. It measures a potential difference to measure the temperature difference between two junctions. This might seem strange, for a potential difference between two points by Ohm’s law, is associated with
the flow of electricity between two points and it does not say anything
about temperature differences. Further, Fourier’s law relates temperature difference to heat flow and does not consider electrical potential
differences. The conclusion is that when there is a difference there is
a heat flow and when there is a potential difference, there is a current flow. It is observed that if one forces an electric current (DC)
through a thermocouple whose junctions are at the same temperature,
Thermodynamic Aspects of the Cell 53
the junctions do not remain at the same temperature, one becomes
warmer and the other becomes cooler. The interrelationship of these
four quantities–heat flow, electric current flow, temperature gradient
and potential difference may be expressed by the following equations:
( T2 − T1 )
(φ − φ1 )
+ L12 2
T
T2
( T2 − T1 )
(φ2 − φ1 )
Electric current = L12 ×
+ L12
T
T2
Heat flow = L11 ×
(2.34)
(2.35)
These equations indicate the following: (1) Each flow in the system is
a function of both forces operating in the system. Both forces involve
the average temperature: one is ΔT/T2 and the other is Δφ/T. These
forces meet the requirement of being those conjugates to the flow to
which they are primarily related. (2) The dependence of the flow on
each force is linear (i.e., it depends on the first power of the source).
(3) There is a proportionality constant connecting each flow with the
corresponding driving force. These equations can be summarized in
the following mathematical form:
Ji =
n
∑ Lik Xk ,
k =1
i = 1, 2, 3, . . . , n
(2.36)
Since these equations are based on experimental observations an
describe the chosen phenomena, these are often designated as phenomenological relations.
(5) Phenomenological coefficients and Onsager reciprocal relations:
From the postulates of non-equilibrium thermodynamics, the phenomenological coefficients are constant over the range where the linear
laws hold good. In equations (2.34 and 2.35), the coefficient L11 is identified as the thermal conductivity of the metal junction and L22 is the
electrical conductivity. The other two coefficients L12 and L21 are called
“cross coefficients”. They are independent of both L11 and L22 but are
not independent of each other. Infact L12 = L21 (as proved by Onsager)
and are called Onsager reciprocal relations.
(6) Degree of advancement of a reaction: This concept applies to both
physical and chemical processes. It is a number between zero and one
that expresses how far the process has gone from inception to completion. It is given the symbol ξ. Consider the reaction
CO2 + H2 O FGGGB
GGG H2 CO3
(2.37)
54
Biophysical Chemistry
The degree of advancement of the reaction at the time of instant exposure of CO2 to water is zero. As CO2 dissolves in water, the degree of
advancement reaches unity as a steady state is established under the
given experimental conditions. The change in degree of advancement
unit time is the reaction velocity
dζ
= Velocity
dt
(2.38)
(7) Affinity: For a spontaneous or irreversible reaction occurring at constant T and P, it is possible that a certain amount of heat is released to
the surroundings which is proportional to ξ. For this, one may write
the equation
dQ = Adξ ≥ 0
(2.39)
The proportionality constant A is called affinity. The heat produced by
the irreversible reaction is a direct measure of the entropy produced.
The affinity A may be defined as
A=−
∑
vγ μγ
v
(2.40)
in which vγ is the stoichiometric coefficient (negative for reactants and
positive for products) and μγ is the chemical potential of the species
participating in the given reaction.
As an example, consider the reaction
N2 (g) + 3H2 (g) FGGGB
GGG 2NH3 (g)
(2.41)
The stoichiometric coefficient for N2 = −1, H2 = −3 and NH3 = +2A
is given by
A = − ∑(−μN2 − 3μH2 + 2μNH3 )
(2.42)
The properties of A are: (i) it is a function of state, (ii) always has the
same algebraic sign of the rate of reaction, (iii) if A = 0, the system is
in equilibrium, and (iv) it is possible for A to be very high but the rate
is very close to zero. This is a case of false equilibrium.
(8) Coupling of reactions: If there are several reactions going on at the
same time in a system, the total entropy produced per unit time per
Thermodynamic Aspects of the Cell 55
unit volume is the sum of the entropies produced of all individual reactions i.e.,
P=
di S
1
=
dt
T
∑ Ae Ve
e = 1, 2, 3, . . .
(2.43)
e
where e refers to each reaction involved. In a system where two reactions occur in a local region, it is possible to have
A1 V1 < 0, A2 V2 > 0 provided that A1 V1 + A2 V2 ≥ 0
(2.44)
The first reaction is called the coupled reaction and the second reaction
is the coupling reaction and because of thermodynamic coupling, the
coupled reaction may proceed in a direction, opposite to that dictated
by its affinity. An example is the synthesis of carbohydrate coupled
to the combustion of elemental hydrogen in Bacillus pycnoticus. The
coupled reactions involved are:
Coupled reaction
CO2 + 0.931H2 O →
1
C6 H12 × 0.93106 + 1.931O2
6
(2.45)
Coupling reaction
2H2 + O2 → H2 O
(2.46)
The coupled reaction indicated by subscript 1 in equation (2.44) is so
designated because a carbohydrate similar in formula weight to glucose was observed to accumulate with time in the bacterium when it
grew in a solution of H2 , O2 and CO2 .
Hydrogen plays the role of a metabolite, for the mass present decreases
as a result of combustion with oxygen. The hydrogen combustion is
the coupling reaction [indicated by subscripts in equation (2.44)]. The
affinities of the reaction and their velocities were found to be
A1 = −105.1 kcal, V1 = 0.543 × 10−3
A2 = +108.5 kcal, V2 = 2.377 × 10−3
(2.47)
If inequality of equation (2.44) holds, there is an upper limit on A1
V1 ≤
| A2 |V2
| A1 |
(2.48)
56
Biophysical Chemistry
substituting the experimental value given in equation (2.47) the equation (2.48) is verified accordingly.
0.543 × 10−3 <
108.5
× 2.377 × 10−3
|105.1|
(2.49)
Questions
(1) Which of the following is true regarding the Onsager coefficients for
a symmetric matrix when thermal and electrical effects are coupled?
The matrix is
(a) negative definite
(c) positive semi definite
(b) negative semi definite
(d) sign indefinite
(2) In an aqueous solution containing glucose, fructose and sucrose, the
number of independent Onsager coefficients is
(a) 16
(c) 6
(b) 9
(d) 3
(3) Calculate the osmotic pressure of a solution containing 3.42 grams of
sucrose (C12 H22 O11 ) in one liter of water. Express the result in (i) atm
(ii) in kPa (The temperature is 27◦ C).
Solution: π = CRT, concentration of sucrose = 3.42/342 = 0.01,
therefore
π = 0.01 × 0.0821 × 300
= 0.2463 atm
here 1 atm = 101.325 kPa, therefore
π = 0.2463 × 101.325
= 24.96 kPa
(4) Calculate the osmotic pressure of 0.1 M solution of zinc sulphate a 2:2
electrolyte at 30◦ C if the salt forms ion pairs to the extent of 80%.
Solution:
ZnSo4 Zn2+ + So24− (ion pair)
degree of ion-paring = 80/100 = 0.8. Therefore concentration of
Zn2+ is equal to concentration of So24− in ion-pair 0.1 × 0.8 = 0.08.
Thermodynamic Aspects of the Cell 57
Concentration of unpaired salt = (1 − α) a = 0.2 × 0.1 = 0.02. Therefore total number of particles (ions+free salt) = 0.08 + 0.08 + 0.02 =
0.18.
π = n × c × RT = 0.18 × 0.1 × 0.0821 × 303 = 4.48 atm
(5) Explain briefly the difference in terms of free energy and entropy of
water molecules in the bulk and the surface.
(6) What is homeostasis and how is it important for humans?
(7) Explain the concept of steady state in non-equilibrium thermodynamics. How is it different from the equilibrium state.
(8) Why is the concept of Gibbs free energy ΔGT,P ≤ 0 not applicable in
the case of bacterial cells. What other criterion is applicable in such a
case and why?
(9) What are electrokinetic phenomena and describe the experimental
approaches to study them.
(10) Write down the phenomenological relations governing the heat flow
and current flow using the thermocouple an example.
(11) For the reaction
H2 O(l) + CO2 (aq) H2 CO3 (aq)
the equilibrium constant, K is 3.1 × 10−5 at 298 K. Calculate the uncompensated heat and the entropy produced in the system.
(12) A membrane permeable for anions separates two solutions of NaCl,
with differing concentrations. The concentrations are 1 mM and
50 mM in the two phases (I and II). The membrane potential is −125
mV. Calculate the activity of Cl− in the phase II at 310 K.
3
Carbohydrates, their
Reactions, Thermochemistry
and Energetics
3.1
Introduction
Many carbohydrates have the general formula Cn (H2 O)n when n ≥ 3.
They may be classified as monosaccharides, oligosaccharides (which may
be considered to be polymeric forms of monosaccharides that can be
hydrolyzed to simple sugars containing two to six molecules) and polysaccharides. Oligosaccharides consist of 2 to 10 monosaccharide units joined
together. They are disaccharides. Polysaccharides are very large and consist of 100’s or 1000’s of monosaccharide units joined together. They act as
fuel and their main biological function is to supply energy. As examples
of monosaccharides, one may cite glucose (C6 H12 O6 ), Ribose (C5 H10 O5 ),
galactose and fructose (C6 H12 O6 ) whose structures are given below:
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_3
59
60
Biophysical Chemistry
Among the disaccharides, the following compounds may be mentioned
viz. Sucrose (C12 H22 O11 ), Maltose (C12 H22 O11 ) and Lactose (C12 H22 O11 ),
whose structures are:
H
HO
CH2OH
O
H
OH
H
H
OH
CH2OH O
H
O
H
OH
HO
OH
CH2OH
H
Sucrose
3.2
General Properties of Monosaccharides
They are water soluble, white and are sweet to taste. They are classified as
aldopentoses and ketohexoses.
Glyceraldehyde is the simplest among aldoses. It exists in D- and Lforms having the structures shown below:
CHO
H
C
OH
CH2OH
D-glyceraldehyde
CHO
HO
C
H
CH2OH
L-glyceraldehyde
They have asymmetric carbon atoms. They are mirror images of each
other but can not be superimposed. Considering the 4-carbon aldoses, we
Carbohydrates, their Reactions, Thermochemistry and Energetics
61
may mention D- and L-erythroses, D- and L-threoses. They may be represented as:
CHO
CHO
CHO
H
C
OH
HO
C
H
HO
C
H
H
C
OH
HO
C
H
H
C
OH
CH2OH
D-erythrose
CH2OH
CHO
H
C
OH
HO
C
H
CH2OH
L-erythrose
CH2OH
D-threose
L-threose
Among the aldopentoses, the important ones are D-ribose, D-arabinose,
D-xylose and D-lyxose whose structures are:
Among aldopentoses, D-ribose is biologically important while
D-glucose and D-galactose are important among aldohexoses. They differ
on their configuration on C-4 and such compounds are known as epimers.
3.2.1
Ketoses
Dihydroxy acetone is the simplest 3-carbon ketone and has the structure
given below. It has no stereospecific carbon (chiral).
CH2OH
C
O
CH2OH
when a 4th carbon is added, a stereospecific center is formed. Their D- and
L-structures may be represented as
CH2OH
CH2OH
C
O
C
OH
∗
C H
∗
H C
CH2OH
D-erythrulose
HO
O
CH2OH
L-erythrulose
62
Biophysical Chemistry
In the case of ketopentoses, there are four stereoisomers because there
are two chiral carbons. They are:
3.2.2
CH2OH
CH2OH
CH2OH
CH2OH
C
O
C
O
C
O
C
O
H
C
OH
HO
C
H
H
C
OH
HO
C
H
H
C
OH
H
C
OH
HO
C
H
HO
C
H
CH2OH
CH2OH
CH2OH
CH2OH
D-ribulose
D-xylulose
L-xylulose
L-ribulose
Oxidation
Oxidising agents like Fehling’s solution (or Benedict’s solution) bromine
water, nitric acid, periodic acid may be employed for this purpose.
Fehling’s solution (Cupric ions complexed with tartrate ion in alkaline
medium) oxidizes the sugar according
Thus aldoses are oxidized directly to the acid (as shown above). In the
case of ketoses, the carbon chain breaks and the acid is formed α-hydroxy
ketones give positive test with Fehling’s solution (but not all ketones). This
is due to tautomerisms. Let us consider the α-hydroxy ketone CH3 COCH2 OH. Due to tautomerism,
An aldehyde group is formed and reaction with Fehling’s solution
takes place. The reduction of Cu2+ to Cu+ occurs with the precipitation
of Cu2 O.
Carbohydrates, their Reactions, Thermochemistry and Energetics
63
Bromine water, being a weaker oxidizing agent, oxidizes aldoses but not
ketoses. The reaction in the general case of a ketose is
CHO
(CHOH)n
CH2OH
COOH
Br2 water
(CHOH)n
CH2OH
Using this reaction, aldoses can be distinguished from ketoses.
Nitric acid oxidizes both aldoses and ketoses and the reactions may be
generalised as
For example, fructose is oxidized by concentration HNO3 to give a
mixture of glycolic acid and tartaric acid together with some trihydroxy
glutaric acid, which is formed by oxidative decarboxylation.
The oxidation of aldoses and hexoses by periodic acid occurs in a specific way that it may be used to identify them as well as determine their
structure. A compound with a −C−C− bond attached to oxidizable groups
such as −C=O or −C−OH, on reaction with IO4− , results in the cleavage
of the −C−C− bond.
64
Biophysical Chemistry
Since a monosaccharide has a hydroxyl group on every carbon, all
−C−C− bonds are ruptured and a single one carbon compound is formed.
It is possible to generalize the oxidation product by HIO4 in the following
way.
In the case of oxidation of glucose by HIO4 , breakage of 4–OH groups
results in the formation of 4 formic acid molecules and oxidation of −CHO
group yields another molecule of formic acid. The oxidation of −CH2 OH
group gives HCHO.
3.2.3
Reduction
Using sodium borohydride, aldoses are reduced to alcohols according to
CHO
(CHOH)n
CH2OH
3.2.4
CH2OH
NaBH4
(CHOH)n
CH2OH
Reaction with Phenylhydrazine
D-glucose (or D-mannose) on reaction with phenylhydrazine gives the
osazone according to
Carbohydrates, their Reactions, Thermochemistry and Energetics
3.2.5
65
Reaction with Acids and Bases
While a dilute acid does not affect a monosaccharide, strong acids act as
dehydrating agents or break the chain. Bases break or fragment the chain.
An aldohexose can be converted to an aldopentose by Ruff degradation.
3.2.6
Unusual Reactions of Monosaccharides
Some properties of monosaccharides are not consistent with the properties of a carbonyl group. For example, many aldoses do not give a positive Schiff test. Further addition of HCN (Kiliani-Fischer synthesis) occurs
slowly with monosaccharides but rapidly with normal aldehydes.
In the case of D-glucose, it can exist in two forms with different specific
rotations. The α-form of D-glucose (crystallized from water) has a specific
rotation of +112◦ while the β-form (D-glucose crystallized from pyridine)
has a specific rotation of +19◦ . When either of the forms is dissolved in
water and allowed to stand for some time, the specific rotation changes to
+52.7◦ . This mutual conversion is referred as mutarotation of glucose.
A new asymmetric center is formed (C-1) with −OH pointing down
or upwards as shown. α- and β-forms are interconvertible since the hemiacetal form is unstable.
α-D-glucose
β-D-glucose
→
63%
37%
The two forms of glucose are called anomers and C-1 is the anomeric
carbon.
The above observed properties can be explained as arising from the
formation of a hemiacetal. For example, in the case of glucose the hemiacetal formation can be shown as:
66
3.2.7
Biophysical Chemistry
Conversion to Haworth Structures
A D-sugar can be converted to a Haworth structure starting from the
straight chain form. For example,
CHO
H
C
OH
HO
C
H
H
H
C
OH
C
HOH2C
C
H
OH
OH
d-glucose
CH2OH
CH2OH
C
O
H
OH
H
C
C
H
OH
C
C
H
HO
H
OH
β -d-glucose
C
O
H
OH
H
C
C
H
C
OH
H
OH
α -d-glucose
Other aldohexoses will react similar to glucose.
3.3
Other Monosaccharides
3.3.1
Aldopentoses
They undergo a similar reactions (to glucose) to form a five membered ring
called furanose based on the compound furan
Carbohydrates, their Reactions, Thermochemistry and Energetics
H
C
OH
H
C
OH
H
C
OH
HOH2C
C
H
67
O
1
CHO
2
H
C
H
C
3
4
H
C
OH
(a)
OH
OH
5
HO
C
H
H
C
OH
H
C
OH
HOH2C
C
H
CH2OH
D-ribose
(b)
From (a) and (b) we can write as
CH2OH
C
OH
O
H
H
C
C
OH
OH
α -D-Ribofuranose
3.3.2
H
CH2OH
C
C
OH
H
O
OH
H
H
C
C
OH
OH
C
H
β -D-Ribofuranose
Ketohexoses
Ketoses also undergo a similar reaction to form hemiacetal. ketohexoses
undergo this reaction between C-2 ketone and C-5 hydroxyl to form furanoses.
68
Biophysical Chemistry
Most stable ring forms are those with 5 or 6 carbon atoms. Both ketohexoses are aldopentoses can form pyranoses when they react with last
−OH group.
3.4
Glycosides
A hemiacetal or a hemiketal can be converted into an acetal or ketal by
adding another alcohol. Both acetal and ketal are stable in neutral solution
but not in acidic solutions.
The hemiacetal-acetal type reaction involving a monosaccharide and
alcohol gives rise to a glycoside, the reaction being
CH2OH
H
C
OH
C
CH2OH
O
H
OH
H
C
C
H
OH
H
OH
C
C
+ CH3OH
H
OH
C
O
H
OH
H
C
C
H
OH
OCH3
C
H
The glycoside ring cannot be opened up. It is neither reducible nor
oxidisable. It cannot form osazones or show mutarotation. It is called α- or
β-glucoside depending on whether the −OH group is down or up.
It is a furanose if it is five membered and pyranoside if it is six membered. If the reaction occurs with an amine instead of alcohol, a compound
called N-glycoside is formed whose structure is given below:
CH2OH
H
C
OH
C
O
H
OH
H
C
C
H
OH
H
C
NHCH3
Carbohydrates, their Reactions, Thermochemistry and Energetics
69
With reagents like acetic anhydride or dimethyl sulfate, the free −OH
groups can be methylated to form
CH2OCH3
H
C
H3C
C
O
H
OCH3
H
C
C
H
OCH3
H
C
OCH3
The carbonyl group of a monosaccharide can be reduced to an alcohol
a sugar alcohol. Oxidation of monosaccharides results in the formation of
sugar acids. Three types of reactions are possible:
(i) In which only the aldehyde is oxidized:
COOH
(CHOH)n
Example:
CH2OH
known as aldonic acid.
(ii) Both aldehyde and primary alcohol are oxidized
COOH
(CHOH)n
Example:
COOH
referred as aldaric acid.
(iii) Only primary alcohol is oxidised:
Example:
known as uronic acid.
A phosphate group can also be incorporated in a sugar giving rise to
glucose-1-phosphate which has the structure
CH2OH
H
C
OH
C
O
H
OH
H
C
C
H
OH
H
C
OPO32–
70
Biophysical Chemistry
Introduction of an amino group by a suitable reaction gives
CHO
H
C
NH2
HO
C
H
H
C
OH
H
C
OH
CH2OH
known as glucosamine. In some sugars, −OH group is not present. For
example: 2-deoxyribose having the structure
CHO
CH2
H
C
OH
H
C
OH
CH2OH
3.5
Oligosaccharides
They contain 2 to 10 monosaccharides joined together. Among the most
important are disaccharides. The bond that holds the monosaccharides
together is known as glycosidic bond. The bond is stable in neutral and
basic media but broken in presence of acid.
There are two important characteristics of any disaccharide: (i) identity
of monosaccharide components, (ii) nature of glycosidic bond, i.e., which
carbons of monosaccharides are linked and by the orientation of bond (αor β-).
In the above figure, the 1st sugar is glucose and the 2nd sugar is mannose. This disaccharide has a 1, 4 bond. Since the aldehyde group of glucose is in the acetal linkage, there is no mutarotation or typical aldehyde
Carbohydrates, their Reactions, Thermochemistry and Energetics
71
reaction in the glucose unit. But mannose has a hemiacetal (potential aldehyde) so it can mutarotate and there will be two forms of disaccharide
(anomeric −OH up or down) which will interconvert in solution.
This disaccharide is a reducing sugar because of the −CHO group in
mannose part of the molecule. Other examples of disaccharides are maltose (C12 H22 O11 ), cellulose, lactose. Maltose is a reducing sugar because of
its free −CHO group. It is broken down by the enzyme maltose which is
specific for α-bond.
The enzyme specific for β-bonds is emulsin. Maltose exists in two
forms i.e., α- (with a specific rotation of 168◦ ) and a β-form with a specific
rotation of 112◦ .
Cellobiose is formed from the break down of cellulose. On hydrolyzing with acids, it forms glucose. It is a reducing sugar that can mutarotate
between α- and β-forms.
Lactose found in milk, form D-glucose and D-galactose on acid hydrolysis. Lactose breaks the disaccharide showing the bond to be β.
Sucrose is formed by plants. On acid hydrolysis it yields glucose and
fructose. It is non-reducing, so both anomeric carbons are in the linkage
(C-l of glucose and C-2 of fructose). It has the structure
CH2OH
H
C
OH
C
O
H
OH
H
C
H
CH2OH
H
C
C
O
CH2OH
H
OH
C
C
C
OH
OH
H
O
C
H
An oligosaccharide is a saccharide polymer containing a small number of monosaccharides (3 to 10). They are generally present as glycans.
Oligosaccharide chains linked to lipids or amino acid side chains in proteins by N- or O-glycosidic bonds.
It is not necessary that all natural oligosaccharides occur as components of glycolipids or glycoproteins. Some occur as raffinose in plants
Microbial break down of larger polysaccharides like starch or cellulose
result in the formation of maltodextrin or cellodextrin disaccharides. It
may be mentioned that glycoproteins have distinct oligosaccharide structures and are important as cell-surface receptors, cell adhesion molecules,
tumor antigens. Glycolipids are lipid molecules bound to oligosaccharides
presents in lipid bilayer. They are important for cell recognition and for
modulating the function of membrane proteins which act as receptors. An
important source of oligosaccharides is the fibre from plant tissue.
72
3.6
Biophysical Chemistry
Polysaccharides
These are also known as glycans and have large molar masses. Among
them, homopolysaccharides contain one type of monosaccharide unit while
heteropolysaccharides contain two or more types of monomers. Any polysaccharide is characterized by its monomer types, types of glycosidic bonds
present and the extent of branching in the carbon chain. The importance
of polysaccharides is two fold: (i) they serve as stores of metabolism fuel,
(ii) they act as structural or support elements of organisms. Three important polysaccharides are—(a) Starch, (b) Glycogen, and (c) Cellulose. Some
details of these components are given below.
3.6.1
Starch
Plants store this compound as granules in cells in heavily hydrated form.
There are two components in starch i.e., α-amylose making up 20% of starch
and the rest amylopectin i.e., 80% starch, α-amylose contains only D-glucose
as an unbranched chain with units linked by 1, 4 glycosidic bonds. It has
molecular masses ranging from 15 × 104 to 60 × 104 (≈ 1000 − 4000 glucose
units). It is non-reducing (because all anomeric carbons are in glycoside
bonds). It can be hydrolyzed by α-amylase to give glucose and maltose.
The amylopectin has a molar mass in the range 100 million (100 × 106 ).
It contains glucose in a branched structure. The structures of α-amylose
and amylopectin are:
The branching point in amylopectin occurs at every 24–30 residues. Amylopectin can be hydrolysed by α-amylase to give glucose, maltose and dextran.
Carbohydrates, their Reactions, Thermochemistry and Energetics
3.6.2
73
Glycogen
It is the storage polysaccharide of animals. It is similar to amylopectin but
even more branched. Enzymes can degrade it to give glucose.
3.6.3
Cellulose
It is found in cell walls of plants. It consists mostly of glucose in linear
chains of 10,000 to 15,000 units. Cellulose contains β1, 4 linkages which
make it different from other polysaccharides. The structure of a polysaccharide depends on the type of glycosidic bond, when the bonds are α, the
shape of the molecule is curved into a coil which provides compact shape
for storage. The coil is stabilized by H–bonds. Many cellulose chains lying
next to each other form a net work of bonds providing strong, stable and
straight fibers. The structure of cellulose may be represented as
OH
O
O
O
OH
HO
OH
O–
OH
OH
O
α -bonds
OH
OH
OH
O
O
O
O
O
OH
OH
HO
β -bonds
It is a homopolysaccharide of N-acetyl-D-glucosamine joined by β 1, 4
linkages. It forms strong extended fibers. It is found in lobsters, insects etc.
Its structure is
CH2OH
H
C
OH
C
O
O
C
H
OH
H
C
C
H
NH
O
C
H
CH3
74
3.7
Biophysical Chemistry
Cell Walls of Bacteria
Their cell walls contain long parallel polysaccharide chains linked together
by short peptides. It is a big cage like molecule called peptidoglycan. Their
polysaccharide chains consist of heteropolymers of alternating N-acetyl
glucosamine (NAG) and N-acetyl muramic acid (NAM) linked by glycosidic bonds. The structure of this polysaccharide unit may be given as
3.8
Thermochemistry of Carbohydrates
Thermochemical data gives heat energy changes associated with physical changes and chemical reactions. Many reactions are associated with
absorption or release of energy. Carbohydrates are polyhydroxy aldehydes
or ketones and they serve as storehouses of energy. One may cite cellulose,
a major structural component of plant cell walls. Other carbohydrates such
as starch and glycogen are also important in that they are produced and
consumed depending on the needs of the cell. The following table contains some thermochemical data such as enthalpies of formation (ΔH ◦f )
and enthalpies of combustion (ΔHc◦ ) of some carbohydrates.
Carbohydrates, their Reactions, Thermochemistry and Energetics
75
Table 3.1 Enthalpy of formation (ΔH ◦f ) and ΔHc◦ at 298K and S◦ .
Compound
D-ribose
α-D-glucose
β-D-glucose
β-D-fructose
α-D-xylose
D-arabinose
D-galactose
α-lactose
β-lactose
α-maltose
β-maltose
Sucrose
D-xylulose
ΔH ◦f /
kcal mol−1
−249.8
−303.2
−302.0
−301.3
−251.1
−251.9
−218.6
−529.0
−532.5
−422.4
ΔHc◦ /
kcal mol−1
−558.9
−667.9
−668.6
−669.5
−557.7
−556.7
−667.5
−1344.0
−1344.8
−1344.2
−528.8
−245.8
−1343.1
S◦
(cal mol−1 K−1 )
50.5
54.3
50.5
34.2
48.9
91.9
96.0
95.3
89.6
Table 3.2 Gibbs energy of formation (ΔG ◦f ) of carbohydrates.
Compound
D-ribose
D-glucose
D-fructose
α-D-xylose
D-arabinose
D-galactose
α-lactose
β-lactose
Sucrose
ΔG ◦f /kcal mol−1
−157.8
−218.1
−195.4
−178.7
−157.8
−189.0
−373.0
−317.7
−314.3
Table 3.3 Thermodynamic data for anomeric conversions at 298 K.
Reaction
ΔG ◦ /kcal mol−1
ΔH ◦ /kcal mol−1
α-glucose to β-glucose
−0.295
−0.274
α-xylose to β-xylose
−0.333
−0.533
α-galactose to β-galactose
−0.452
−0.309
α-mannose to β-mannose
0.405
0.440
Reaction
α-glucose to β-glucose
α-xylose to β-xylose
α-galactose to β-galactose
α-mannose to β-mannose
ΔS◦ /cal mol−1 k−1
0.174
−0.667
0.476
0.120
ΔCP◦ /cal mol−1 k−1
−2.14
-
76
Biophysical Chemistry
3.9
Gibbs Free Energy Changes for Reactions
3.9.1
Glycolysis
It is the metabolic pathway that converts glucose into pyruvate (CH3 COCOO− ) and H+ . It refers to the production of 2 moles of lactic acid from one
mole of glucose i.e.
C6H12O6
2CH3CHOHCOOH
ΔG° = –195.8 kJ mol –1
The free energy released in the process is used to form the high energy
molecules ATP and NADH. Glycolysis (also called cellular respiration) is
a sequence of ten enzyme catalysed reactions. The overall reactions occur
in the cytoplasm and is given by
1. For the oxidation of glucose by O2 according to the reaction
C6H12O6 + 2NAD+ + ADP + 2P
2CH3COCOOH + 2ATP + 2NADH+ + 2
(Pyruvic acid)
ΔG = ?
2.
3. For the reaction glucose-1-phosphate glucose-b-phosphate ΔG = 7.3
kJ (in presence of enzyme, phosphoglucomutas)
4.
ATP + H2O
2–
ADP + HPO4 + H+
ΔG = –30.5 kJ (at pH = 7.0)
5.
Carbohydrates, their Reactions, Thermochemistry and Energetics
77
6.
O–
Adenine-ribose
O
P
O–
O– + H2O
O–
(AMP)
Adenine-ribose
(adenosine)
(at pH = 7.0)
OH + HO
P
O–
O
ΔG = –9.2 kJ
7.
ATP + AMP
ADP + ADP
ΔG = 0
8.
3.10
Enolic Phosphate
The important compound to be considered in the conversion of glucose
to pyruvate (that provides for the generation of ATP from ADP) phosphoenolpyruvic acid (PEP). Its hydrolysis can be given in two stages with the
ΔG of hydrolysis of each step as
78
Biophysical Chemistry
The large negative ΔG of hydrolysis is due to the conversion of the
unstable enol form to the stable keto form. Thus the tautomerization makes
PEP one of the most important energy rich phosphate compound.
3.11
Guanidinium Phosphates
These compounds have an important role in energy transfer and energy
storage. They are known as phosphagens and are formed by the phosphorylation of creatine or arginine with ATP in presence of a suitable enzyme.
They are found in vertebrates and invertebrates.
Creatine + ATP
Creatine
kinase
(enzyme)
Phosphocreatine + ADP
ΔG′ = –12.6 kJ
at pH = 7.0
When the concentration of ATP is high, the above reaction proceeds
from left to right and phosphate is stored as energy rich phosphocreatine.
The hydrolysis of guanidinium phosphate is given by
The hydrolysis products are more stable than the guanidinium phosphate because of their resonance stabilization.
3.11.1
Coupling of Reactions
A coupling reaction is one in which the energy released in an exergonic
reaction is utilized to drive a related endergonic reaction and thereby is
made to work. For example, we consider the reaction:
H
OH
O
C
H
C
OH
+ H 2O
CH2OPO3H2
D-glyceraldehyde
3-phosphate (D-gly-3-phosphate)
O
H
C
OH
H
C
OH
CH2OPO3H2
H
C
OH
C
OH
+ 2H+
CH2OPO3H2
3-phosphoglycerate
ΔG′ = –50.2 kJ
Carbohydrates, their Reactions, Thermochemistry and Energetics
79
Since the reaction has a large −ve ΔG value, the reaction is practically
occurring to the right and is not reversible. Living cells have a way of using
(D-gly-3-phosphate) to generate ATP by coupling this reaction with NAD+
and H3 PO4 to give 1, 3-diphosphoglyceric acid which in turn reacts with
ADP to ATP according to
The acyl phosphate in a subsequent reaction is utilized to convert ADP
to ATP.
O
H
O
C
OPO3H2
C
OH
CH2OPO3H2
+ ADP
H
C
OH
C
OH
+ ATP
CH2OPO3H2
ΔG′ = –18.8 kJ
The above two reactions may be summed up to give the coupled reaction.
From the above data of redox potentials, it is possible to calculate ΔG of any reaction. For example, in the case of the reaction
Acetaldehyde + NADH + H+ −→ NAD+ + Ethanol
where
E0 = −0.16 − (−0.32) = +0.16 V
ΔG = −0.16 × 96, 500 × 2 = −30.88 kJ
7.
8.
9.
10.
11.
12
13.
14.
1.
2.
3.
4.
5.
6.
+56.00
+61.76
+79.13
+82.99
+7.72
−48.25
−7.72
+90.71
+30.88
ΔG (kJ mol−1 )
36.70
19.30
−5.80
−11.60
+38.60
Table 3.4 Redox reactions and their E◦ and ΔG values.
Reaction
E0 (at pH = 7.0)/V
Pyruvate + 2H+ + 2e −→ Lactate
−0.19
Oxalacetate + 2H+ + 2e −→ Malate
−0.10
Fumarate + 2H+ + 2e −→ Succinate
0.03
+
Dehydroascorbic acid + 2H + 2e −→ Ascorbic acid
0.06
Riboflavin + 2H+ + 2e −→ reduced form of riboflavin
−0.20
1, 3 diphosphoglyceric acid + 2H+ + 2e
−→ Glyceraldehyde-3-phosphate + phosphate ion
−0.29
NAD + 2H+ + 2e −→ NADH + H+
−0.32
Acetyl-COA + 2H+ + 2e −→ Acetaldehyde +COA-SH
−0.41
3
+
2
+
Ferredoxin-Fe + e −→ Ferredoxin-Fe
−0.43
Cytochrome-b-Fe3+ + e −→ Cytochrome-b-Fe2+
−0.04
Cytochrome-c-Fe3+ + e −→ Cytochrome-c-Fe2+
0.25
+
−
Oxidized glutathione + 2H + 2 e = reduced glutathione
0.04
Acetate + 2H+ + 2e −→ Acetaldehyde + H2 O
−0.47
Acetaldehyde + 2H+ + 2e −→ Ethanol
−0.16
80
Biophysical Chemistry
Carbohydrates, their Reactions, Thermochemistry and Energetics
81
Consider another common reaction occurring in living tissues
1
NADH + H+ + O2 −→ NAD+ + H2 O
2
where
E0 = +0.82 − (−0.32) = 1.14 V
−96, 500 × 2 × 1.14
ΔG =
= −22 kJ
1000
It is to be emphasized that although ΔG has a large negative value, it has
no bearing on the rate of the reaction (thermodynamic feasibility versus
kinetic occurrence). In fact, NADH is stable in presence of O2 and will
react only in the presence of suitable enzymes.
3.12
Carbohydrates and Microbial Fuel Cells
It is well known that microorganisms can function either as electron donors
or as electron acceptors. This uniqueness indicates that no redox mediators are necessary for their functioning in electrochemical systems. Consequently, this field has now provided new avenues in technological applications viz.: (i) energy conversion and storage devices i.e., fuel cells and
supercapacitors, (ii) removal of pollutants from drinking water and (iii)
selective detection of analytes. Here, a brief overview of microbial fuel
cells is provided with special emphasis on glucose.
The thermochemical data provided earlier indicates that various types
of carbohydrates can be employed in fuel cells. As a typical example,
consider the oxidation of glucose in alkaline media which may be represented as
Anodic reaction: Oxidation of glucose
C6 H12 O6 + 36 OH− − 24e− = 6 CO23− + 24 H2 O
(E0 = 0.93 V)
Cathodic reaction: Reduction of oxygen
6 O2 + 24e− + 12 H2 O = 6 CO23− + 24 OH−
(E0 = 0.52 V)
The combined fuel cell reaction is then
C6 H12 O6 + 12 OH− + 6 O2 = 6 CO23− + 12 H2 O
(E0 = 0.45 V)
82
Biophysical Chemistry
The standard Gibbs free energy change ΔG0 = −3358.2 kJ mol−1 if the
number of electrons transferred equals 24. However, in reality, the oxidation
of glucose yields gluconic acid, with number of electrons involved being 2.
Thus, the standard Gibbs free energy change is considerably lower. The
thermodynamic efficiency is often defined as
Thermodynamic efficiency =
ΔG
ΔH
and ΔH denotes the enthalpy change of the reaction. However, the overall efficiency of any fuel cell depends upon other factors such as voltage
efficiency, fuel utilization, ohmic loss etc.
The voltage efficiency =
Voltage obtained
Theoretically attainable voltage
Fuel employed in the actual operation
Fuel provided
Assuming that the different efficiencies can be considered as independent
events, the overall efficiency is given by the product of the individual
efficiencies.
The thermochemical data of carbohydrates plays a crucial role in dictating the thermodynamic or coulombic efficiency. On the other hand, the
fuel utilization as well as the voltage efficiency is dependent upon: (i) catalysts employed, (ii) nature of the electrolytes, (iii) solvent and (iii) design
considerations. The catalysts employed are often noble metals for cathodic
and anodic processes, thus making the fuel cells, unaffordable in general.
The activation overpotential (ηactivation ) is one of the quantitative parameters to deduce the catalytic efficiency. The nature of the electrolyte and solvent dictate the magnitude of ohmic overpotentials (ηohmic ). It is desirable
to minimise all the overpotentials for obtaining the maximum efficiency of
a fuel cell (or any electrochemical energy storage device).
It is to be mentioned that one method of obviating the use of noble metals for the catalysis of electrode processes consists in employing microorganisms as catalysts. The microbial fuel cells have significant advantages
such as: (i) low operating temperature; (ii) specificity and (iii) enhanced
efficiency. The field of microbial fuel cells is a frontier area of research
in energy storage devices. In addition to the usual components of fuel
cells viz.: anode, cathode, membrane, electrolyte and separators, microbial fuel cells contain microbes such as Geobacter metallireducens, Geobacter
sulphureducens, Schwenella Oneidensis, R. Ferrireducens, Saccharomyces cerevisiae, Pseudomonas aeruginosa etc. These microbes upon attachment to the
electrodes effectively catalyse the desired oxidation of compounds.
Fuel utilization efficiency =
Carbohydrates, their Reactions, Thermochemistry and Energetics
83
Lest one may neglect the importance of cathodic reaction (reduction
of oxygen), it is essential to point out that the reduction of oxygen may
involve either a two-electron or four-electron transfer. The mechanism
pertaining to the electrochemical reduction of oxygen has been a fascinating topic of research for the past few decades so as to replace the noble
metal catalysts with less expensive metals such as Ni, Co etc. Alternately,
chemically modified electrodes (in particular using conducting polymers)
or diverse nanostructured materials appear to be promising in this context.
Here too, several attempts are ongoing to catalyse the reduction of oxygen
using a combination of microbes such as Sphingobacterium, Acinetobacter
and Acidovorax sp, with inexpensive carbon cathodes.
Questions
(1) Which of the following is true regarding monosaccharides? They cannot be further
(a) oxidized
(c) hydrated
(b) reduced
(d) hydrolysed
(2) How many reducing ends are present in Amylopectin?
(a) 0
(c) 2
(b) 1
(d) 5
(3) Sucrose is an invert sugar since the hydrolysis of sucrose with hot
dilute acids leads to
(a) an equimolar combination of D-glucose and D-fructose
(b) only Fructose
(c) only Dextrose
(d) just Glucose
(4) Along with starch, polysaccharides possess nutritional value due to
(a) cellulose
(c) polygen
(b) glucose
(d) glycogen
(5) Furanose is a
(a) Deoxy ribose
(b) 7-methyl sugar
(c) 4 - methyl sugar
(d) Deoxy glucose
84
Biophysical Chemistry
(6) The formation of the red precipitate upon reacting glucose with Benedict’s reagent is
(a) Iron oxide
(c) Cuprous oxide
(b) Cupric oxide
(d) ZnO
(7) Upon heating with dilute acid, lactose undergoes
(a) Enantiomeric inversion
(c) Crystalline transformation
(b) First order phase transition
(d) Isomorphic transition
(8) Mannose and glucose are
(a) Enantiomers
(b) Isomers
(c) Epimers
(d) None of the above
(9) Calculate the entropy change, ΔS◦f , associated with the formation of
5 moles of arabinose (from its elements) given that ΔG ◦f = −662.9
kJmol−1 and ΔH f = −1057.9 kJmol−1 at 298 K.
Solution: Using the relation
ΔG f = ΔH ◦f − TΔS◦f −
we can write,
TΔS◦f = ΔH ◦f − ΔG ◦f −
or
ΔS◦f
=
ΔH ◦f − ΔG ◦f
T
−1057.9 − (−662.9)
=
2.98
−395.0
=
= −1.326 kJ mol−1
2.98
For 5 moles, ΔS◦f = −5 × 1.326 = −6.63 kJ.
(10) Calculate the enthalpy change of the reaction α-glucose to β-glucose
at 35◦ , given that ΔC ◦p of the reaction is −9.0 JK−1 mol−1 and ΔH ◦ at
298 K s −1.15 kJ mol−1 .
Solution: Assuming ΔC p to be independent of temperature, we can
write
ΔH2◦ − ΔH1◦ = ΔC p ( T2 − T1 )
Carbohydrates, their Reactions, Thermochemistry and Energetics
85
ΔH2 = ΔH1◦ + ΔC p ( T2 − T1 )
= −1.15 − 9 × 10−3 (308 − 298)
= −1.15 − 9 × 10−3 × 10
= −1.15 − 0.09
= −1.24 kJ
(11) For the reaction
C6 H12 O6 FGGGB
GGG 2CH3 CHOHCOOH(l)
the standard Gibbs energy is −196 kJ. Given ΔG ◦f (glucose) = −916.0
kJ mol−1 . Calculate ΔG ◦f of pyruvic acid.
Solution:
−196 = 2 × ΔG ◦f (pyruvic acid) − ΔG ◦f C6 H12 O6
= 2 × ΔG ◦f (pyruvic acid) − (−916)
or
2 × (ΔG ◦f (pyruvic acid) = −196 − 916 = −1112 kJ
or
ΔG ◦f (pyruvic acid) =
−1112
= −556 kJ mol−1
2
4
Lipids
4.1
Introduction
Lipids are solid fatty compounds, but there are also oily compounds. They
are sparingly solute in water but possess good solubility in organic solvents. Lipids function as energy storage molecules and they are structural
components of membranes.
The nomenclature ‘Lipids’ refers to diverse set of organic molecules viz
fats, oil, hormones, membranes which have extensive hydrophobic property. Among various lipids, triglycerides comprise of fats present in adipose cells which function as energy storage devices. On the other hand,
steroid hormones are often messengers between different constituents of
the cell. Another class of lipids present in the membranes of the cells and
the organelles are designated as phospholipids and are vital for the origin
of life on account of their specific role in cell functioning.
While most lipids are hydrophobic, amphipathic lipids are less common. Amphipathic lipids possess both hydrophilic and hydrophobic parts,
with the latter being more pre-dominant. In water, amphipathic lipids
yield self-aggregating structures with hydrophilic and hydrophobic ends
being inside and outside respectively.
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_4
87
88
4.2
Biophysical Chemistry
Properties of Lipids
Fatty acids form crystals consisting of stacked layers of molecules, with
each layer having the thickness of two extended molecules. The molecules
within a layer are arranged such that the hydrocarbon chains comprise the
interior while the hydrophilic –COOH groups occupy the two faces. Interestingly, the molecular packing of lipids lead to different crystal forms,
designated as polymorphs.
The melting temperatures of saturated fatty acids of biological interest
are > 27◦ C and increase with the hydrocarbon chain lengths. It is well
known that (mono and poly) unsaturated molecules melt at much lower
temperatures in comparison with their saturated analogues. As anticipated, the lowest melting temperatures occur when the C==C bonds are
located near the centre of the hydrocarbon chain. Hence these molecules
form viscous liquids, even at room temperature. The hydrophobic character of the hydrocarbon chain of fatty acids is more prominent than the
hydrophilic nature of the –COOH group, resulting in lower solubility of
these molecules in water. For example, at 25◦ C, the solubility of fatty acids
∼ μg per gram of water. However, this solubility exhibits an exponential decrease with the addition of each carbon atom to the hydrocarbon
chain. From thermodynamic considerations, this behaviour is a manifestation of Gibbs free energy changes involved in the transfer of molecules
from the organic solvent to water. In other words, upon incorporation of
each CH2 group, more energy is required to orient water molecules around
the hydrocarbon chain of the fatty acid. In aqueous solutions, the dissociation occurs as given by the equation
R − COOH −→ RCOO− + H+ ,
with R representing the hydrocarbon chain; the degree of dissociation is
1). The negatively charged –COO− ions are more polar
quite small (
than the undissociated acid. The dissociation equilibrium can however
be controlled by systematic addition of a base such as NaOH. This leads
to the replacement of H+ by Na+ ions so as to yield the salt of the fatty
acid (soap). The detergent property of soaps arises since RCOO− anions
in spontaneously form micelles. The interior of these structures essentially
consisting of hydrocarbon chains, serves as an efficient medium wherein
grease etc., can be sequestered. The diameter of each spherical micelle is
nearly double the length of the extended fatty acid. The dispersions of
micelles in water are stable. The formation of bubbles, foams on the surface
of these soap dispersions is the consequent outcome of the adsorption of
Lipids 89
RCOO− ions at air-water interfaces. It is hence no wonder that the entire
arsenal of thermodynamic, spectroscopic, electrochemical and microscopic
tools becomes available to unravel the nature of air-water interfaces.
4.3
Bonding in Lipids
The reactive acidic –COOH group of RCOOH form esters (RCOOR’) with
alcohols R’OH, the ester bond being the primarily covalent in nature. A
less frequent occurrence is the ether bond (R’-O–R) in lipids.
All cell membranes consist of lipid bilayers. Each lipid bilayer contains two layers of fat cells which encompass two sheets. The boundaries
of all the cells are dictated by the barrier provided by the lipid bilayer components and hence comprehending the structure and functioning of lipid
bilayers is of paramount importance. The interfacial tension varies from
0.2 to 6 dynes cm−1 while the thickness ranges from 4 to 7 nm. In view of
the lipid bilayers possessing capacitance (∼ 1μFcm−2 ), dielectric constant
(< 4) and conductance (∼ 10−8 mho m−2 ) electrochemical techniques are
especially suited to probe their properties.
4.4
Classification
They may be classified as: (a) fatty acids, (b) acylglycerides, (c) waxes,
(d) phospholipids (or phosphoglyceride), (e) sphingolipids, (f) glycolipids,
and (g) terpenoids lipids. A brief account of the above classes is given
below.
4.4.1
Fatty Acids
They are carboxylic acids with hydrocarbons (carbon atoms ranging from
4 to 30) in straight chain mostly with 16 to 18 number of carbon atoms. The
chain is generally linear but may be branched also. The compounds may
contain double bonds from one to six always (nearly) with a cis configuration. For compounds with one double bond, the usual position is C-9
and C-10 and for compounds with many double bonds the most common
positions are C-9, C-12 and C-15 (mostly unconjugated). The saturated
fatty acids are waxy solids while the unsaturated ones are oily. A few fatty
acids starting from lauric acid are listed below.
90
Biophysical Chemistry
Acid
Lauric
Myristic
Palmitic
Stearic
Oleic
Linoleic
Table 4.1 Examples of a few fatty acids.
Structure
Number of
carbons and
double bonds
CH3 (CH2 )10 COOH
12 : 0
CH3 (CH2 )12 COOH
14 : 0
CH3 (CH2 )14 COOH
16 : 0
CH3 (CH2 )16 COOH
18 : 0
CH3 (CH2 )7 CH = CH(CH2 )7 COOH
18 : 1 (position
of = is 9)
CH3 (CH2 )4 (CHCHCH2 )2 (CH2 )6 COOH 18 : 2 (position
of = is 6)
If a trans configuration occurs in a structure, it is started as for example 18: 3(6-C, 9-C, 12-C) 6, 9, 12 being positions of double bonds. Animal fatty acids are mostly straight chain compounds containing upto six
double bonds. Bacterial fatty acids may be saturated, branched chain compounds while plant fatty acids have a more complex structure. Naturally
occurring fatty acids having 1 to 8 carbon atoms are liquid, while those
with more carbon atoms are solid. Addition of a double bond to fatty acid
lowers the melting point.
4.5
Reactions
4.5.1 Fatty Acids
They react with base to form salts, known as soaps. For example,
CH3 (CH2 )14 COOH + NaOH →CH3 (CH2 )14 COONa + H2 O
(4.1)
Sodium palmitate (soap)
Figure 4.1 Schematic representation of hydrophobic and polar groups in
soaps.
Lipids 91
Such salts have a polar head (carboxyl group) and a non-polar tail (hydro
carbon). Soaps form micelles where the hydrophobic tail points away from
water while the polar heads point towards water as shown in Figure 4.1.
A major portion of acids are found as esters in the cells and the ester
bonds undergo hydrolysis in presence of acids or bases. The acid hydrolysis is a reversible reaction, but the base hydrolysis is not. For example, the
base hydrolysis of triacylglycerol is as follows:
(4.2)
The pKa ’s of most fatty acids range between 4.8 to 5.0.
4.5.2
Geometric Isomerism
When a double bond exists in the hydrocarbon chain, geometric isomerism
occurs. For example
(4.3)
Oleic acid is a bent molecule for the following reasons: (i) It has a cis 9,
10 double bond; (ii) The combination of a cis form and the presence of
sigma and pi bonds in the double bond; (iii) The bent structure may be
represented as:
(4.4)
92
Biophysical Chemistry
Another bent form of oleic acid (CH3 (CH2 )7 CH = CH(CH2 )7 COOH). In
linoleic acid, which has two double bonds in the hydrocarbon chain, it
is more severely bent. It may be mentioned that animal and plant cell
membranes are rich in polyunsaturated acids.
Naturally occurring polyunsaturated fatty acids may contain a nonconjugated double bond like
−CH2 − CH = CH − CH2 − CH = CH − CH2 (non-conjugated)
but also
−CH2 − CH = CH − CH = CH − CH = CH − CH2 (conjugated)
An important poly unsaturated fatty acid, alpha-elaeostearic acid, which
is the principal acid in tung oil, has a conjugated triene group given by
(CH3 )(CH3 )3 CH = CH − CH = CH − CH = CH(CH2 )7 COOH
The two types of double bonded systems have important differences in
their reactivity. The conjugated double bonded systems are more reactive
than their non-conjugated double bounded systems because of the delocalization of pi-electrons. Fatty acids with conjugated double bonds undergo
extensive polymerization, which is of great value in paint industry. Such
systems, which are biochemically important, are retinol and carotenes and
they have important role in visual processes of retina.
4.6
Analysis of Lipids
Gas liquid chromatography and thin layer chromatography are some methods used in analysis of lipids.
4.7
Nomenclature of Phospholipids
Let us consider glycerophosphoric acid as an example.
(4.5)
Lipids 93
(a) The numbers 1 and 3 cannot be interchangeably used for the same primary alcoholic group. (a) As can be seen, the second hydroxyl group (in I)
is shown to the left of C-2 in the Fischer projection and the carbon above
C-2 is referred as C-1 and the one below as C-3. This stereo specific numbering is indicated as “Sn” as prefix before the name of the compound.
Thus glycerol is labeled as
On this basis, compound I (in equation 4.5) is Sn-glycerol-3-phosphoric
acid. Its special antipode is
A mixture of both is called racemic glycerol-3-phosphoric acid. There
are several phospholipids, which contain glycerol, fatty acids and a nitrogeneous base and are considered derivatives of phosphatidic acid whose
structure is
(4.6)
A few representative phospholipids containing different bases are given
below.
Bacteria, animal and plant tissues contain phospholipids. Cell membranes contain choline. They are amphipathic since they contain polar and
non-polar groups.
Polar Base
component
Choline
Amino-ethanol
Non-polar
Component fatty acid
Stearic or palmitic (R1 )
polyunsaturated (R2 )
Stearic or palmitic ( R1 )
polyunsaturated (R2 )
Lecithin (3-Sn-phosphatedyl)
choline
Cephalin
(3-Sn-phosphatidyl)
amino-ethanol
Structure
Name of Phospholipid
94
Biophysical Chemistry
Amino-ethanol
Myo-Inositol
Unsaturated ether (α),
Linoleic (α)
Palmitic (R1 )
Arachidonic (R2 )
Plasmalogen
(3-Sn-Phosphital
amino-ethanol
Inositol-phosphor-lipid
(3-Phosphotidylinositol)
Polar Base
component
Non-polar
Component fatty acid
Name of Phospholipid
Structure
Lipids 95
96
Biophysical Chemistry
There are three other classes of lipids of importance, which must be
mentioned: (a) sphingolipids, (b) glycolipids, and (c) terpenoids. They are
briefly discussed below:
(a) Sphingolipids: The main compound is known as 4-sphnigenine which
has the structure
Two important products formed from 4-sphingosine are
(b) Glycolipids: They are derivatives of carbohydrates and glycerol and
do not contain phosphate. They are present in chloroplasts; galacto
Lipids 97
and sulpholipids are also found in chloroplasts. Two important compounds of this class are:
(c) Terpenoids: Terpenoids are an important group of compounds made
of a simple repeating unit, the isoprenoid unit. This unit by appropriate condensation with other molecules gives rise to compounds like
rubber, carotenoids, steroids and terpenes. The active biological
(counter part of isoprene is isopentyl pyrophosphate which is formed
by a series of enzymatically steps from squalene. Squalene condenses
with itself to form cholesterol. The structures of squalene (C30 H50 O)
and isopentyl pyrophosphate are:
98
Biophysical Chemistry
Another important terpenoid product is beta-carotene which has the
structure as shown earlier.
β-Carotene belongs to carotenoid family and is a constituent of colouring pigment for deep yellow and orange fruits and vegetables. It occurs
in natural form in strawberries, cantalopes, broccoli, carrots etc. It is
a powerful antioxidant and boosts immune system. Other important
members of beta-carotenoid family are lycopene, alpha-carotene etc.
4.8
Lipoproteins
These are classes of bio molecules in which the lipid components are triacylglycerol, phospholipid and cholesterol or its esters. The protein components have relatively high proportion of non-polar amino acid residues
which can participate in binding of lipids. The main binding force between
the protein and lipid is the hydrophobic interaction between the apoproteins and lipids.
The hydrophobic interaction implies the tendency of the hydrophobic
components to associate with each other in aqueous medium. Lipoproteins
are found in membranes of mitochondria endoplasmic reticuli and nuclei.
The electron transport system is mitochondria contains large amounts
of lipoproteins. Lamellar lipoproteins occur in chloroplasts and membranes
of bacteria.
4.9
Role of Lipids in Cell Function
Lipids serve as storage forms of energy and they are also part of the structure of cell membranes.
They participate in many metabolic activities such as
(i) a major source of energy in animals and birds,
(ii) activators of enzymes such as glucose-6-phosphatase,
Lipids 99
(iii) enabling electron transport in mitochondria.
(iv) For example, the isoprenoid compound, undecaprenyl phosphate acts
as a lipophilic carrier of a glycosyl moiety in the synthesis of bacterial
wall lipopolysaccharides and peptidoglycan,
(v) Phosphotidylserine is decarboxylated by a specific decarboxylase to
Phosphatidylethanolamine and in the process CO2 is released.
4.10
Distribution of Lipids
The composition of lipid differs in prokaryotic and eukaryotic cells. In
prokaryotic cells, a bacterial cell wall has over 95% of its total lipid associated with cell membrane. The remaining 5% is distributed between its
cytoplasm and cell wall. Bacteria are limited in their capacity to synthesize conventional polyunsaturated acids. They produce only saturated or
branched chain fatty acids.
Plants: Seeds of higher plants have a fixed composition of fatty acids.
The maturing seed synthesizes its different fatty acid, at different rates and
at different periods during maturation. The exotic fatty acids are found
as triacylglycerols in the mature seed and are not found in chloroplast.
Higher plants synthesize a wide range of polyunsaturated fatty acids.
Animals: Lipids of animal cells are complex and their composition
is characteristic of a particular cell. For example, a nerve cell is rich in
sphingolipids, glyceryl ethers and phospholipids. An adipose cell consists
mainly of triacylglycerols. A special feature, which is unique to lower and
higher forms of animal life, is their inability to form polyunsaturated fatty
acids. Animal cells introduce “cis” double bonds into hydrocarbon chain
towards carboxyl end where as plant cells always introduce double bonds
towards the methyl end.
100
4.11
Biophysical Chemistry
Physicochemical Data on Lipids
Interfacial tension data of some single component bi-layer membranes with
different lipids.
β-carotene belongs to carotenoid family and is a constituent of colouring pigment for deep yellow and orange fruits and vegetables. It occurs in
natural form in straw berries, cantaloupes, broccoli, carrots etc. It is a powerful anti-oxidant and boosts immune system. Other important members
of this family are lycopene, alpha-carotena etc.
Lipids 4.11.1
101
Physicochemical Data on Lipids
I. Interfacial tension data of lipid-cholestrol (1 : 1) complex in bi-layer
lipid membranes.
Complex
Interfacial tension
(Nm−1 )
Pc - Ch
2.17 × 10−3
PE - Ch
2.38 × 10−3
Cer - Ch.SM - Ch
2.33 × 10−3
4.48 × 10−3
II. Interfacial tension data of lipid-lipid (1 : 1) in bi-layer lipid membrane.
Complex
Interfacial tension (Nm−1 )
Pc - PE
2.58 × 10−3
SM - Cer
1.62 × 10−3
III. Interfacial tension data for lipid-fatty acid and lipid-amine complexes
(1 : 1) in bi-layer lipid membranes.
Complex
Pc - SA
Interfacial 7.16 × 10−3
tension
(Nm−1 )
PC - DA
7.25 × 10−3
PC - ST
6.04 × 10−3
PC - DE
3.63 × 10−3
IV. Interfacial tension data for lipid-amino acid complexes (1 : 1) in bilayer lipid membranes.
Complex
Pc - Tyr
Interfacial 1.75 × 10−3
tension
(Nm−1 )
PC - Ile
1.91 × 10−3
PC - Val
2.04 × 10−3
PC - Phe
3.69 × 10−3
V. Interfacial tension data of some single component bi-layer membranes
with different lipids.
Interfacial tension
(Nm−1 )
Phosphatidylcholine (PC)
1.62 × 10−3
Phenylalanine (Phe)
5.30 × 10−3
Ceramide (CER)
1.29 × 10−3
Cholesterol (Ch)
4.72 × 10−3
Phosphatidylethanolamine (Pe)
3.34 × 10−3
Decanoic acid (DA)
−4.2 × 10−3
Decylamine (DE)
−7.5 × 10−3
Valine (Val)
7.0 × 10−4
Sphingomyelin (SM)
1.72 × 10−3
Tyrosine (Tyr)
−3.5 × 10−3
Stearic acid (SA)
−1.54 × 10−3
Stearylamine (ST)
4.40 × 10−3
Isoleucine (Ile)
−2.7 × 10−3
Name of lipid
Interfacial tension
(Nm−1 )
Name of lipid
Interfacial tension
(Nm−1 )
Name of lipid
Interfacial tension
(Nm−1 )
Name of lipid
102
Biophysical Chemistry
Lipids 103
VI. Physico-chemical parameters of one component bi-layer lipid membranes.
Component
pKa
pKb
Isoelectric
point
PC
PE
PS
SM
2.58
2.42
2.58
2.59
5.69
5.98
9.55
5.31
4.12
4.18
3.80
4.01
Interfacial Tension
at Isoelectric Point
(Nm−1 )
3.53
4.06
2.94
4.42
VII. Conductivity of phospholipid (used as lecithin) cholesterol
membranes as a function of pH. Concentration of phospholipid =
5 × 10−6 mol ml−1 = 5 × 10−3 mol liter−1 .
Lipid: PE
X (Mole fraction of cholesterol)
0.20
0.80
Conductance (mho cm−2 )
pH = 5.0
pH = 6.0
−
5
2 × 10
8 × 10−6
−
5
5 × 10
1 × 10−5
Lipid: PC
X (Mole fraction of cholesterol)
0.20
0.80
Conductance (mho cm−2 )
pH = 5.0
pH = 6.0
−
5
1 × 10
0.2 × 10−5
−
5
6 × 10
4.0 × 10−5
VIII. Dependence of membrane conductance on carrier concentration.
Concentration of Valinomycin = 10−8 M
Lipid
Conductance (mho cm−2 )
PG
10−4
PE
10−7
DC
10−7
PG = Phosphatidyl glycerol;
PE = Phosphatidyl ethanolamine;
DC = 7-dehydro-cholestrol
IX. Critical micelle concentration of lipids.
The CMC of a surfactant (in this case, a lipid) is the concentration at
which surfactant micelles form. Below CMC, a surfactant exists as
monomers in solution but above CMC micelles are present.
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Biophysical Chemistry
Lipid = PC
5:0
10 : 0
16 : 0
Lipid = PG
10 : 0
14 : 0
CMC
90 mM
0.005 mM
0.46 nM
CMC
14.0 mM
0.011 mM
Lipid = PS
8:0
10 : 0
CMC
2.28 mM
0.096 mM
X. Temperature dependence of CMC.
Lipid : PC
10 : 0
14 : 0
Temp (20◦ C)
8 × 10−3 moles liter−1
7 × 10−5 moles liter−1
Temp (40◦ C)
7 × 10−3 moles liter−1
8.5 × 10−3 moles liter−1
Spontaneous self-assembly of phospholipids in water is essential to
the stability of biological membranes and to life itself. It is controlled
by thermodynamics, specifically the tendency of hydrophobic lipid
chains to segregate from contact with water coupled with the structural requirement to form a thin, flexible membrane. The membrane
forms a tight permeability barrier to polar solutes in aqueous environment. This requires a strong interplay of lipid polar head-groups
and their hydrocarbon acyl chains, which lead to the formation of
lipid bi-layer membranes.
◦ , ΔH ◦ , ΔS◦ data for some saturated acyl lysophosphatidyl
XI. ΔGtr
tr
tr
cholines (n : 0) and diacetyl phosphatidyl cholines (n : 0)2 of different chain lengths, n, from their monomeric state in water to their
respective micellar states at different temperatures.
Lipid studied
(10 : 0) LPC
(14 : 0) LPC
◦ /kJmol−1
ΔGtr
20◦ C
40◦ C
−21.5 −22.5
−35.0 −36.5
◦ /kJmol−1 ΔS◦ /Jk−1 mol−1
ΔHtr
tr
10◦ C 30◦ C
10◦ C
30◦ C
9.0
2.5
150.0
100.0
5.0
−7.0 140.0
90.0
◦ for 1, 2 diacetyl phosphatidyl cholines ( n : 0)
XII. Dependence of ΔGtr
2
from water to micellar state at 25◦ C.
Chain length
8
10
12
ΔGt◦ /Jmol−1
−30.0
−40.0
−52.0
Formula of the compound
C42 H80 NO8 P
Lipids 105
XIII. Surface charge density (σ ) of various lipids.
Compound
1, 2 dipalmitoyl-3-trimethyl
ammonium propane (DPTAP)
1, 2 dipalmitoyl phosphatidyl
ethanolamine (DPPE)
1, 2 dipalmitoyl phosphatidyl glycerol
(DPPG)
4.11.2
σ
15.1 ± 1−2 mCm−2
5.3 ± 5 mCm−2
Δσ = −44.0 ± 9 mCm−2
Waxes
Waxes are esters of fatty acid, with long chain monohydric alcohol group.
Natural waxes are mixtures of such esters and may also contain hydrocarbons. The formulae of three well-known waxes are given below, the under
lined part being the carboxylic acid moiety.
Compound
Spermaceti
Bees wax
Carnauba wax
Formula
CH3 (CH2 )14 COO − (CH2 )15 CH3
CH3 (CH2 )24 COO − (CH2 )29 CH3
CH3 (CH2 )30 COO − (CH2 )33 CH3
The leaves and fruits of many plants have waxy coatings which protect
them from dehydration and small insects. The feathers of some birds and
furs of some animals have similar coatings which serve as water repellents.
Waxes are highly insoluble in water and have no double bonds in their
chains.
4.12
Lipid Bi-layers
The lipid bi-layer is established as the basis for cell membrane structure.
The bi-layer structure is attributable to the special properties of lipid
molecules which cause them to assemble spontaneously into bi-layers.
Lipid molecules constitute about 50% of the mass of most animal cell membranes, the remainder being protein. There are approximately 5 × 106 lipid
molecules in a (one μm)2 i.e., 10−12 m2 area of a bi-layer. All of lipid
molecules in cell membranes are amphiphilic (or amphipathic) i.e., they
have hydrophilic or polar end and a hydrophobic or non-polar end.
Cylinder shaped phospholipid molecules form bi-layers. Lipid
molecules form any of the above structure in H2 O depending on their
shape. In this energetically most favourable arrangement, the hydrophilic
106
Biophysical Chemistry
Figure 4.2 Schematic depiction of lipid bilayers.
heads face water and the hydrohobic tails are shielded from water inside.
A small tear in a bi-layer creates a free wedge with water. This being energetically unfavourable, the lipids spontaneously rearrange to eliminate the
wedge. In such a case, they form a sealed compartment by closing on themselves, which is fundamental to the creation of a living cell.
Figure 4.3 Energetically favourable structures of bilayers.
Different spectroscopic techniques such as ESR, NMR have been used
to measure the motion of individual lipid molecules. Lipid molecules
Figure 4.4 Structure of cholesterol in free state and fluid region.
Lipids 107
rapidly exchange places with their neighbours within a monolayer (107
times per second). This gives rise to lateral diffusion with a diffusion coefficient of 10−8 cm2 sec−1 which means that the average lipid molecule diffuses the length of a large bacterial cell (2μ m) in about one second.
The lipid bi-layer of many cell membranes contains not only phospholipids but also often cholesterol and glycolipids. Eucaryotic plasma
membranes contain large amounts of cholesterol i.e., upto one molecule of
cholesterol for every phospholipid molecule.
The lipid composition of two different cell plasma membranes is given
below.
Lipid
PE
PS
PC
Cholesterol
Liver cell plasma
membrane
7
4
24
17
Red blood cell
plasma membrane
18
7
17
23
In the above, PS carries a net negative charge while the others are electrically neutral at physiological pH carrying one +ve and one −ve charge.
Asymmetry of the Lipid Bi-layer
The liquid compositions of the two-lipid bi-layer in many membranes are
strikingly different.
4.13
Glycolipids on the Surface of all Membranes
The glycolipids with the most extreme asymmetry in the distribution of
membranes are the sugar containing lipid molecules called glycolipids.
They are found exclusively in the non-cytosolic monolayer of the lipid
bi-layer. The glycolipids tend to self-associate partly through H-bonds
between their sugars and partly through Van der Waals forces between
their long saturated hydrocarbon chains. Glycolipids occur in all animal
cell plasma membranes where they generally constitute about 5% of lipid
molecules in the outer monolayer. They are also found in some intracellular membranes.
In voltammetric studies, glassy carbon electrodes are modified by
deposition of a hydrophobic coating of a lipid. For example, the lipid
asolectin, which is negatively charged, modifies the glassy carbon electrode by selectively accumulating organic molecules depending on their
charge and the hydrophobic-hydrophilic balance.
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Biophysical Chemistry
Dipalmitoyl Phosphatidylcholine
Structure of dipalmitoyl phosphatidylcholine
C40 H80 NO8 P;
Mol. Weight = 734.05g/mol;
CMC = 4.6 × 10−10 M
1, 2 diacyl phosphatidylcholine
or 1, 2-diacyl-Sn-glycero-3-phosphocholine
Formula-C42 H80 NO8 P
4.14
Interfacial Studies of Lipid Bilayers
The cyclic voltammetric and impedance analysis are especially powerful
in this context since they can provide all the system parameters pertaining
Lipids 109
to electron and ion conduction. However, it is essential to choose systems
which mimic the structure and components of lipid bilayers.
As mentioned earlier, in view of the phosphate ions and lipid
molecules together forming a bi-layered system, the nomenclature bilayer
lipid membrane is invoked. Such bilayer lipid membranes are the scene
of action for many important processes e.g., transport of species, charge
transfer. The interior of membranes is non-polar and hence the transfer of
hydrophilic substances is energetically un-favourable. However hydrophobic molecules (and ions such as tetra alkyl ammonium and tetraphenyl
borate) can pass though the membrane, in a facile manner. Comprehending the mechanism of such transport is of paramount importance in so far
as the extent of the drug delivery of the species is crucially dependent on
energetic considerations. The electric-field assisted transport consists of (i)
adsorption of the species and (ii) transfer across the interface. For each of
these steps, the Gibbs free energies can be formulated and interpreted. The
construction of suitable thermochemical cycles can be of immense help in
estimating the overall Gibbs free energy changes involved.
In view of the adsorption step (i) above, it is essential to compute
the surface coverage of the lipids using appropriate adsorption isotherms
such as
Henry isotherm represented as
θ = BCads
Langmuir isotherm viz
θ
= BCads
1−θ
Frumkin isotherm viz
θezaθ
= BCads
1−θ
In the above equations, θ is the surface coverage while B denotes the
adsorption equilibrium constant, Cads being the bulk concentration of the
adsorbate. The parameter ‘a’ known as the Frumkin interaction constant
is solely dependent upon the nearest neighbour interaction energies and
its value if > 2 indicates the onset of phase transitions. It is well known
that the Henry and Langmuir isotherms will be unable to predict the phase
transitions since these do not incorporate interaction energies. For adsorption of phloretin onto the bilayer lipid membranes, the most suitable
110
Biophysical Chemistry
adsorption isotherm remains un-settled. This limitation arises because of
the inability to quantitatively incorporate dipole-dipole, non-electrostatic
and van der Waals type interactions.
Among several studies in this context, adsorption of phloretin on phosphatidylcholine (PC) and phosphatidylethanolamine (PE) deserves mention in view of the unusual dependence of the surface coverage of phloretin
on the bulk concentration. Furthermore, adsorption also induces dipole
potentials whose magnitude ranges from 100 to 300 millivolts.
Analogously, the analysis of the step (ii) involving ion transfer across
dissimilar interfaces is a non-trivial task. In this case, the most effective
experimental strategy consists in constructing cyclic voltammograms. Here,
the hydrophilic ions of BLM yield distinct voltammetric features, wherein
the peak height is a quantitative measure of the amount of the ionic species
while the peak potential is a qualitative measure of the nature of the species
involved. On account of the difficulties involved in mimicking the structure and properties of natural membranes, it has been customary to model
a simpler system such as water/organic solvent interface wherein the ionic
species can be dissolved in the aqueous solvent, with the hydrophobic
species being present in the organic phase. Hence the entire paraphernalia of charge transfer processes across liquid/liquid interfaces can be
employed to unravel all the events of relevance pertaining to biological
processes.
Questions
(1) Which of the following is correct regarding palmitic acid?
(a) saturated
(b) unsaturated
(c) linear
(d) branched
(2) Identify the correct statement regarding Linoleic acid and Linolenic
acid. Both are
(a) Essential fatty acids
(b) Micelles
(c) Inorganic compounds
(d) Cholesterol
Lipids 111
(3) Identify the following structure:
(a) phospholipid
(b) glycolipid
(c) sphingolipid
(d) glycerophospholipid
(4) For the oxidation of the fatty acid, palmitic acid, CH3 (CH2 )14 COOH
give by
CH3 (CH2 )14 COOH + 23O2 16CO2 (g) + 16H2 O(l)
ΔG = −856 kJ mol−1 . Given that ΔG ◦f (CO2 ) and ΔG ◦f (H2 O, l) =
−394.4 kJ mol−1 and −237.2 kJ mol−1 respectively, what is ΔG ◦f of the
acid?
Solution:
−856 = ΔG ◦f (CO2 ) × 16 + ΔG ◦f (H2 O, l)
× 16 × −237.2 − ΔG ◦f (palmitic acid) − 0
or
ΔG ◦f (palmitic acid) = −394.4 × 16 + 16 × −237.2 + 856
= 16(−394.4 + (−237.2) + 856)
ΔG ◦f (palmitic acid) = 16 × −631.6 + 856 = −10105.7 + 856
= −9249.6 kJ mol−1
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Biophysical Chemistry
(5) Explain briefly, giving reasons, as to why the interfacial tension of
phosphatidyl ethanolamine (PE = 3.34 × 10−3 Nm−1 ) is much higher
than phosphatidyl choline.
Solution: The head group in phosphatidyl ethanolamine is more polar
than that in phosphatidyl choline. Further the lipid membrane of PE
is also much more viscous than in phosphatidyl choline. These differences may be largely responsible for the difference in interfacial
tension between the two lipids.
(6) (a) Assuming phosphatidyl choline to be bronsted base, represent its
dissociation equilibrium in aqueous solution.
(b) Given its pKb =5.76, calculate the Gibbs energy change of the above
reaction at 300K.
Solution:
ΔG ◦ = − RT ln Kb = −8.314 × 10−3 × 300 × 2.303 × log Kb
= +8.314 × 10−3 × 300 × 2.303 × 5.76
= 1421.7 kJ
(7) For a spherical micelle aggregate, estimate the approximate radius if
its volume is 100 cm3 , the area inside the aggregate being 0.1 cm2 .
Solution:
V = 100 cm3 ; q = 0.1 cm2
Rsphere = 3 V/a
Rsphere = 3 × 100 cm3 /0.1 cm2
Rsphere = 3000 cm
(8) A electrochemical cell containing a lipid bilayer membrane (BLM) was
formulated as follows:
SCE− (in gel)|BLM|Ag+ (1M)|Ag
Its voltage was found to be 0.660 V. Given (ESCE = +0.22V) and
(E Ag,Ag+ ( M) = +0.800V). What is the potential difference across thelipid dilayer.
Solution:
0.660 = ER − EL + EBLM
= 0.800 − 0.220 + EBLM
EBLM = 0.660 − 0.800 + 0.220
= 0.08V
Lipids 113
(9) Among the following, which possesses higher stacking characteristic?
(10) Fill up the blanks with suitable phrases:
(a) 1,2-Diacyl-sn-Glycero-3-Phosphocholine is a ...................................
(b) 1-Acyl-2-Acyl-sn-Glycero-3-Phosphocholine is an example of
...................................
(c) Linoleic acid is an ...................................
(11) Describe the intermolecular forces present in the lipid bilayer membranes.
(12) Write the structure of a simple triglyceride.
(13) Give a schematic diagram of a cell and indicate its components which
serve to transfer information across the cell.
5
Amino Acids
5.1
Introduction
Amino acids are organic compounds containing an amino group(–NH2 )
and a carboxyl group(–COOH) along with a side chain (–R) specific to
each amino acid. Among the many known naturally occurring amino acids
about 20 are important because they occur are coded genetically. They can
be classified according to the core (structural) functional groups locations
such as α−, β−, γ−, δ–amino acids. The general formula of a naturally
occurring amino acid may be represented as
Figure 5.1 Fischer projection and ball-stick model.
Amino acids are soluble in water but are insoluble in non-polar organic
solvents such as chloroform or ether. They have high melting points.
5.2
Classification of Amino Acids
One way of classifying amino acids is based on the polarity of the R-group.
They may be classified into four groups.
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_5
115
116
Biophysical Chemistry
Table 5.1 Classification of amino acids based on polarity.
Group-I
Group-II
Group-III
Group-IV
Non-polar
Polar but uncharged
Acidic amino
Basic amino
amino acids
amino acids
acids
acids
Group
Examples
Alanine
Glycine
Non-polar
amino acids
H
Leucine
Valine
H
H
H
H2N — C — COOH H2N — C — COOH H2N — C — COOH H2N — C — COOH
H
CH3
H — C — CH3
CH3
CH2
1
H — C — CH3
CH3
Methionine
Creatine
NH
H2N
O
CH3
It plays a crucial role
in protein biosynthesis
Group
Polar uncharged
amino acids
OH
N
Examples
Serine
Asparagine
Glutamine
H
H
H
H2N — C — COOH
CH2
OH
H2N — C — COOH
H2N — C — COOH
CH2
CH2
CH2
Amino Acids Group
Examples
Tyrosine
Threonine
Polar
uncharged
amino
acid
(Note: pKa of phenolic group in
tyrosine is 10.1)
Group
Examples
Aspartic acid
Glutamic acid
Acidic
amino
acids
(Note: Ghutamate is an
excitory neurotransmitter
in the central nervous
sytesm)
Group
Examples
Lysine
Arginine
Histidine
Basic
amino
acids
(The pKa i.e., acid
dissociation constant
of guanidinium group
is 13.8 ± 0.1)
(It makes up the
active sites of
protein enzymes)
117
118
5.3
Biophysical Chemistry
Chirality of Amino Acids
Except glycine, all other amino acids are chiral. They exist in optically
active enantiomeric forms (D and L) that are mirror images of each other.
It is interesting to note that the amino acids found in proteins are mostly
of L-configuration. Enzymes responsible for protein synthesis utilise only
L-configuration. D-amino acids are found in cell walls of bacteria and in
several antibiotics.
5.4
Acid-base Properties
Amino acids are amphoteric molecules (because they contain both amino
and carboxylic acid groups). For example, an aqueous solution of alanine is
neutral and there is no migration of ions under the influence of an electric
field. Thus, although it is a neutral molecule it is best represented as a
zwitterion.
H
H3C
C
COO
NH3
(zwitterion of alanine)
In the potentiometric titration of an aqueous solution of alanine with
NaOH, the pH vs. NaOH curve shows a typical curve with a pKa = 9.7
upon titrating with it is titrated with HCl, a curve with a pKa = 2.3 is
obtained as shown below.
Figure 5.2 Potentiometric titration of aqueous alanine solution.
at pH = 9.7: a group capable of furnishing protons is half neutralized.
at pH = 2.3: a group of accepting protons is half neutralized.
Amino Acids 119
The reactions occurring may be represented as:
It may be seen from the above scheme that alanine in acid solutions,
migrates to the negative electrode (anodes) in an electric field and to the
positive electrodes in alkaline solutions. Because of the zwitterionic nature,
amino acids, dissolve easily in water.
The Zwitterionic nature of amino acids is also proved by the reaction
with formaldehyde.
The secondary and tertiary amines formed are weaker bases (or
stronger acids). This is shown by the lowering of pKa of the amino group
in presence of formaldehyde.
The pH at which an amino acid behaves predominantly as neutral is
known as isoelectric point (pI). The piso (pH at isoelectric point) may be
approximated as half way between the two points of strongest buffering
capacity and is given by
pI =
1
1
(pK1 + pK2 ) = (2.3 + 9.7) = 6.0
2
2
Table 5.2 Isoelectric point of a few amino acids.
Amino acid
Glycine
Alanine
Valine
Leucine
Methionine
Threonine
Cysteine
pKa1
2.34
2.34
2.32
2.36
2.28
2.09
1.96
pKa2
9.60
9.69
9.62
9.60
9.21
9.10
8.18
pI
5.97
6.00
5.96
5.98
5.74
5.60
5.07
Amino acid
Proline
Phenylalanine
Tryptophan
Asparagine
Glutamine
Serine
Tyrosine
Aspartic acid
Glutamic acid
Lysine
Arginine
Histidine
pKa1
1.99
1.83
2.83
2.02
2.17
2.21
2.20
1.88
2.19
2.18
2.17
1.82
pKa2
10.60
9.13
9.39
8.80
9.13
9.15
9.11
9.60
9.67
8.95
9.04
9.17
pKa3
6.30
5.48
5.89
5.41
5.65
5.68
5.66
3.65
4.25
10.53
12.48
6.00
pKa1 = −ve log10 of 1st –COOH group, pI = pH at isoelectric point.
pKa2 = −ve log10 of NH3+ group.
pKa3 = −ve log10 of second –COOH group.
pI
2.77
3.22
9.74
10.76
7.59
120
Biophysical Chemistry
In amino acids having more than one –COOH group or −NH2 group
there are different pKa values for them. For example, the pKa of alphacarboxyl group of aspartic acid is 1.88 while the pKa of the second carboxyl
group is 3.65 and that of −NH3 group is 9.60. The sulphydril group of
cysteine dissociates with a pKa of 8.18 according to the reaction.
5.5
Reactions of Amino Acids
(a) Reactions of carboxyl group
(i) The carboxyl group may be esterified with alcohols
(ii) Using PCl5 or POCl3 , the –COOH group may be converted to an
acyl chloride
The acyl chloride formed is very reactive and can react with a second amino acid to form a peptide bond.
(iii) The carboxyl group of amino acids may be decarboxylated either
chemically or biologically to yield the amine.
Amino Acids 121
In all the above, for example, histamine is produced from histidine in this way. The biological significance of histamine is that it
stimulates the flow of gastric juice in stomach.
(b) Reactions of amino group
(i) Oxidising agents like HNO2 react with amino acids liberating N2 .
The reaction is stoichiometric and is used for the estimation of
alpha-amino group in the amino acid.
(ii) Other milder oxidising agents like ninhydrin may be used to oxidise the amino group.
This reaction is characteristic of amino acids having alpha-amino
group. The reaction can be used to quantitatively determine amino
acids.
(iii) Reaction with 1–fluoro –2, 4 dinitrobenzene
122
Biophysical Chemistry
By this method, one can identify the terminal amino acid in a
polypeptide chain.
(c) Reaction with isothiocyanates: Isothiocyanates are used to degrade
polypeptide chains and to identify the −NH2 terminal amino acid in
the polypeptide chain. For example, the reaction of an amino acid with
phenyl isothiocyanate is given by
5.6
Biochemical Importance of Amino Acids
Our body needs twenty different amino acids to maintain good health.
Among them, people have to obtain nine essential amino acids through
foods such as eggs, tofu, soyabean, meat, quinoa and dairy products. The
body itself makes the remaining eleven amino acids.
Amino acids perform diverse functions in the body such as: (i) build
muscles, (ii) cause chemical reactions in the body, (iii) transport nutrients,
(iv) prevent illness. Deficiency of amino acids results in decreased immunity, digestive problems, low mental alertness, depression etc.
Each amino acid has a different role in the body. They are enumerated
below:
(i) Lysine: It plays important role in building muscle, maintain bone
strength, fast recovery from injury or surgery, regulates hormones
and enzymes. This amino acid is present in meat, eggs, soyabean,
black beans, quinoa and pumpkin seeds.
(ii) Histidine: It promotes growth, creation of blood cells and tissue repair.
Our body metabolises histidine into histamine, which is very important for immunity build up, digestion and reproductive health. Its
deficiency can cause anaemia and low blood levels in the body and
kidney disease. Meat, fish, nuts, seeds and whole grains are good
sources of histidine.
Amino Acids 123
(iii) Threonine: It is necessary for healthy skin and teeth. It helps in
metabolising fat and digestion. It helps in controlling depression.
Cottage cheese and wheat germ are good sources of threonine.
(iv) Methionine and cysteine: They play a role in the health of skin and
hair. They also aid in the proper absorption of selenium and zinc.
This amino acid i.e., methionine is present in eggs, grains, nuts and
seeds.
(v) Valine: It is essential for muscle co-ordination, mental focus and emotional stability. It is present in soyabeans, cheese, peanuts, mushroom, vegetables and whole grains.
(vi) Leucine: It helps regulate blood sugar levels and aids growth and
repair of muscle and bone. It is also necessary for wound healing.
Its deficiency leads to skin rashes, hair loss and fatigue. Dairy, soybeans, legumes, beans are good sources of this compound.
(vii) Isoleucine: It is useful in faster wound healing, immunity, blood sugar
regulation and production of hormones. It is present in muscle tissue.
Meat, fish, poultry, eggs, cheese, lentils are plentiful in this amino
acid.
(viii) Phenylalanine: It is important in the body for effective use of proteins and enzymes. It is converted in the body to tyrosine, which
is necessary for specific brain functions. Its deficiency causes eczema,
fatigue and memory loss in adults. It is used in the artificial sweetener, Aspartame. People with the genetic disorder, phenyl ketonuria
are unable to metabolise this amino acid. It is present in dairy, meat,
fish, soyabeans and beans.
(ix) Tryptophan: Infants need it for good growth. It is a precursor of seritonin and melatonin. The former is a neurotransmitter, regulates
appetite and pain. Melatonin is a sleep aid and hence used as a sedative. It improves mental energy and emotional well being in women.
Deficiency of this amino acid causes a condition known as pellagra,
which can lead to dementia.
It also causes skin rashes and digestive problems. It is present in most
high-energy foods such as cottage cheese and wheat germ.
Foods with Essential Amino Acids
Meat, eggs, tofu, dairy products contain all essential amino acids. However, people who eat vegetarian or vegan diets can also get their essential
amino acids from plant foods such as rice and beans.
124
5.7
Biophysical Chemistry
Electrochemical Studies of Amino Acids
The conversion of amino acids into peptides has been studied extensively
for the past few decades, on account of its importance in evolution of
life. The diversity in the products arising from the anodic oxidation, can
be ascribed to the nature of amino acids, the molar concentrations and
pH dependence. The products may vary from aldehyde and ammonia to
carbon dioxide. In contrast to conventional homogeneous chemical oxidations, electrochemical oxidations are versatile and the product formation can be controlled by (i) nature of electrodes; (ii) choice of electrolytes
and solvents; (iii) potential window; (iv) bulk concentration; (v) choice
of experimental technique and (vi) electrode geometry. Among various
amino acids for which the complete equilibrium and kinetic data are available, the following deserve mention: alanine, glycine, guanine, L-arginine,
L-lysine, asparagine, etc. The electrode potentials are often be expressed
either using half wave potentials or formal potentials. The half wave potentials are approximately equal to the standard electrode potentials since the
diffusion coefficients of oxidised and reduced species are nearly the same.
However, the formal potentials and standard potentials are substantially
different due to the effect of pH and activity coefficients. Thus, it is customary to report the formal potentials of compounds having biological
significance.
The mechanistic elucidation for comprehending such studies requires
not only elaborate experimental techniques but also density functional theories of diverse genre. The theoretical calculations are required in order to
rationalise the experimentally observed products. The oxidations can be
studied using noble metals, carbon paste and glassy carbon electrodes.
In addition, various nanoparticles or polymer-coated electrodes can be
employed. It is of interest to note that for oxidation studies, carbon-based
electrodes are preferable since metals lead to oxide formation in the same
potential range in which amino acids undergo oxidation. In cyclic voltammetric studies, the absence of electron transfer peak is an indication of
adsorption of amino acids (with or without polymerization). A collateral advantage consists in selective and sensitive detection of amino acids
using different steady state and transient electrochemical experiments. The
differential pulse voltammetry and amperometry provide an estimate of
sensitivity, linear calibration range and lowest detection limits pertaining to different biosensors. These aspects have been briefly discussed in
Chapter 12.
Amino Acids 125
Glycine being the simples amino acid, its behaviour at electrode surfaces has been extensively investigated. On noble metals, the oxidation
products include CO, CN− and [O=C=N]− ions. The identification of
products can not solely be identified from electrochemical studies; instead
several in situ spectroscopic techniques become essential. The adsorption
of amino acids such a tryptophan on surfaces may also form self-assembly
which can then be exploited for several applications. An important aspect
in this context is the enantiomeric separation of amino acids which can be
accomplished by chiral electrodes.
Questions
(1) Which of the following is the aprotic and aliphatic amino acid?
(a) tryptophan
(c) Lysine
(b) alanine
(d) cysteine
(2) Which of the following is a neutral pH amino acid
(a) Glutamate
(c) Methionine
(b) Arginine
(d) Valine
(3) Proline is known as
(a) α-helix terminator
(b) α-helix initiator
(c) α-helix responder
(d) α-helix sensor
(4) Methionine is a
(a) Sulphur-containing amino acid
(b) Nitrogen-containing amino acid
(c) Imino acid
(d) Polypeptide
(5) Arginine is
(a) Positively charged
(b) Negatively charged
(c) Neutral
(d) Non-polarisable
(6) Aromatic amino acids with aromatic nature can be identified at a wave
length of
(a) 320 nm
(c) 280 nm
(b) 440 nm
(d) 380 nm
126
Biophysical Chemistry
(7) An amino acid which is both glucogenic and ketogenic are
(a) Lysine
(c) Homocystine
(b) Isoleucine
(d) Cysteine
(8) Amino acid involved in carbon skeleton catabolism releases
(a) Acetoacetyl CoA
(c) Acetoacetyl CoB
(b) Acetone
(d) Acetic acid
(9) Glycine and proline are the most abundant amino acids in
(a) Hemoglobin
(c) Insulin
(b) Myoglobin
(d) Collagen
(10) Lysine has one carboxylic and two amino groups whose dissociation
constants are 6.61 × 10−3 , 1.12 × 10−9 , 2.95 × 10−11 (the latter two are
for NH3+ group). The PI value of the molecule is
(a) 5.56
(b) 6.35
(c) 9.74
(d) 7.42
(11) The PI values of valine (I), cysteine (II) and histidine (III) are 5.96, 5.07
and 7.59 respectively. The increasing order of their migration in an
electrophoresis experiment towards cathode at pH = 7.0 is
(a) I < II < III
(b) III < I < II
(c) II < II < III
(d) I < III < II
(12) Given Ka(1) and Ka(2) of cysteine as 1.1 × 10−2 and 6.61 × 10−9 , calculate its isoelectric point.
Solution: pKa = 1.96, pKb = 8.18
Isoelectric point =
pKa + pKb
1.96 + 8.18
10.14
=
=
= 5.07
2
2
2
(13) Calculate the pH of a 0.05 M solution of glycine given its pKa (of –
COOH group) is 2.34 at 298K.
(14) A solution of 25 ml of 0.1 M alanine is titrated with 25 ml of 0.1 M
NaOH. The potentiometric titration yields a curve with pKa = 9.7
when 12.5 ml of NaOH are added. When the same solution is titrated
with 0.1 M HCl the curve with pKa = 2.3 was deduced. Draw the
potentiometric curve obtained in the two cases as a pH vs. volume of
acid and volume of base. What are the reactions in the two cases?
Amino Acids 127
pKa = 9.7
pH
pKa = 2.3
12.5
0
0.1 M
HCl
CH3 CH COOH
H+
0.1 M
NaOH
CH3 CH COOH
NH3
12.5
OH
NH2
CH3 CH COO H2O
NH2
(15) Classify the following amino acids into various categories: Arginine,
Glutamine, Threonine, Valine and Creatine.
Answer:
Arginine: Basic amino acid
Glutamine: Polar uncharged amino acid
Threonine: Polar uncharged amino acid
Valine: Non-polar amino acid
Creatine: Non-polar amino acid
(16) Draw the structure of the tetrapeptide valine-glycine-serine-alanine
in neutral solution.
6
Peptides
6.1
Introduction
Peptides are short chains of amino acids linked by peptide (or amide)
bonds. They are classified as di, tri, tetra and poly peptides. A polypeptide is a long, continuous, unbranched peptide chain. Peptides are different from proteins on the basis of their size and they contain upto 50
amino acids in their chain. Amino acids that are incorporated into peptides are termed as “residues”. All peptides except cyclic peptides have
a N-terminal (amino group) and a C-terminal (Carboxyl group) residue at
the end of the peptide.
Example:
Figure 6.1 Structure of tetrapeptide containing Val-gly-Serine-Ala with LValine being at left end and L-Alanine at the right end.
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_6
129
130
6.2
Biophysical Chemistry
Classes of Peptides
Peptides are classified on the basis of their source and function. Some
groups of peptides include plant, bacterial, fungal, invertebrate, venom,
cancer/anticancer, vaccine, endocrine, ingestive and gastrointestinal, cardiovascular, renal, respiratory, opiatic, blood-brain peptides. Some organisms produce peptides as antibiotics.
Peptides undergo reactions such as phosphorylation, hydroxylation,
sulfonation, glycosylation and disulfide formation.
Non-ribosomal peptides (NRP’s) are a kind of peptide secondary
metabolites which are synthesized by multidomain mega enzymes named
non-ribosomal peptide synthetases f (NRP’s) without the need for the cell
ribosomal machinery and messenger RNA’s. They are naturally synthesized by microorganisms such as bacteria and fungi. An example is glutathione, which is a component of anti-oxidant defenses of many aerobic
organisms, plants and fungi.
6.2.1
Different Classes of Peptides
Classification of peptides
Antimicro bial
peptides
(Example:
Magainin
family)
Tachykinin
Vaso
Pancreatic
Opioid
family’
active
peptides
and
(such as
intestinal
related
such as
neuropeptides
prodypeptides
kinin A) (such as VIP (such as
norphin
peptide PHI27 NPY, PPY) peptides
Secretin)
Self
assembled
peptides,
such as
amphiphilic
biomimetic
peptide
The abbreviations are explained as follows: VIP = Vasoactive intestinal
peptide; PHI = Peptide histidine isoleucine 27; NPY = Neuropeptide Y;
PPY = Pancreatic polypeptide.
Other peptides include lactotripeptides, natriuretic peptide,
Lactotripeptides are two naturally occurring milk peptides; IsoleucineProline-Proline (IPP) and Valine-Proline-Proline (VPP). They are derived
from casein, which is a milk protein found in dairy products.
6.2.2
Natriuretic Peptides (NP’s)
There are four different groups of NP’s identified as: (i) atrial natriuretic
peptide (ANP), (ii) B-type natriuretic peptide (BNP), (iii) C-type natriuretic
Peptides 131
peptide (CNP) and a D-type natriuretic peptide (DNP) each with its own
characteristic functions. They are hormones which are mainly secreted
from heart.
A polypeptide is a single linear chain of many amino acids held
together by amide bonds. An oligopeptide consists of two to forty amino
acids. Oligopeptides are classified according to molecular structure as
aeruginosins cyanopeptolins, microcystins, microviridins, microginins,
anabaenopeptins, and cyclamides.
6.3
Functions of Peptides
The function that a peptide carries out depends on the types of amino acids
involved in the chain and their sequence as well as the specific shape of
the peptide. Peptides often act as hormones and thus constitute biological messengers carrying information from one tissue to another through
blood.
There are two classes of hormones known as peptide hormones and
steroid hormones. The former are produced in glands and other tissues
such as stomach, intestines, and brain. Examples of peptide hormones are
those involved in blood regulation, including insulin, Glucose like Peptide1 (GLP-1) and those regulating appetite including Ghretin. Ghretin is produced and released by the small intestine and by stomach, pancreas and
brain.
Peptides are found in every cell of human body and perform a wide
range of functions mentioned above. Maintenance of appropriate concentration levels of peptides is necessary to achieve homeostasis and maintain
health. A few common natural peptides are given in Table 6.1.
6.4
Acid-Base Properties of Peptides
All peptides have, at least, two ionisable groups, the carboxyl group of Cterminal residue and the amino group of N-terminal group. Further, there
may be ionisable groups inside chains of some amino acids forming part
of the peptide.
It is important to predict the charge carried by a peptide since this
helps to know how it travels during an electrophoresis experiment. The
charge on a peptide depends on pH due the acid groups present in amino
acid part.
Hypothalamic neurohormone. It is
involved in the regulation of the
hypothalamic pituitary thyroid
axis
Table 6.1 Residues, their sources and amino acid sequences.
Source or function
Amino acid sequence
Living cells, they stimulate tissue
NH3 CH(COO− )CH2 CH2 CONHCH
growth
(CH2 SH)CONHCH2 COOH
TRH(3) peptide i.e., Thyrotropin
releasing hormone (tripeptide)
(C16 H22 N6 O4 )
Name and residues
Glutathione (3)
132
Biophysical Chemistry
Name and residues
Glucagon: It is 29
amino acid peptide
hormone secreted
from the alpha cells
of the pancreas
Insulin
Amino acid sequence
Its structure in humans is NH2 -His-Ser-Gln-Gly-Thr-PheThr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-AlaGln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-COOH.
It has molar mass of 3485 daltons.
It is composed of two peptide chains referred as A chain
and B chain. These chains are linked together by two disulfide bonds, and an additional disulfide is formed within
the A chain. In most species, the A chain consists of 21
amino acids and the B chain of 30 amino acids. The structure is given below.
Source or function
It is generated from the
cleavage of proglucon by
protein convertase 2 in
pancreatic islet α cells
It helps control blood glucose
levels by signalling the liver
and muscle and fat cells to take
in glucose from the blood.
Thus, it helps cells to take in
glucose to be used for energy. It
is a pancreatic hormone.
Peptides 133
134
Biophysical Chemistry
To find the overall charge on a peptide, one has to identify—(i) all ionisable groups, (ii) charge on each group at a given pH. Let us consider, for
example, the compound (Val-Ile-Leu-Met).
O
O
O
H2N CH C NH CH C NH CH C OH
CHCH3
CH2
CH2
CH3
CH
CH2
H3C
CH3
S
CH3
The ionisable groups in the above compound are the −NH2 group
(pKa of NH3 groups 9.0) and the carboxyl group (pKa = 3.0) at each end
of the compound. The net charge on the peptide at three different pH’s is
distributed as follows:
pH
Charge on functional group
C-terminus
N-terminus
Overall charge
2.0
7.0
12.0
0
−1
−1
+1
0
+1
0
−1
Table 6.2 Critical Aggregation Concentration (CAC) of a few peptides and
their diffusion coefficients.
Name of the
compound
EAK 16-II
Alzheimer’s
beta amyloid
oligo peptide
Human Serum
albumin
Bovine Serum
albumin
CAC
gram liter−1
0.1
moles liter−1
6.035 × 10−5
2.5 × 10−5
0.05
7 × 10−7
0.05
7 × 10−7
Diffusion coefficient
(m2 sec−1 )
D.C. of A
Beta 42 = 1.35 × 10−10
CAC of Ab 42 = 9.2 ×
10−8 M
Peptides 135
Among the many peptides, EAM16-II (molar mass = 1656.8 g) is an
important 16-residues peptide (Ala-Glu-Ala-Glu-Ala-Cys-Ala-Cys)2 . It has
a characteristic β-sheet circular dichroism spectrum in water. Upon addition of salts, the peptide spontaneously assembles to from a macroscopic
membrane.
The critical aggregation constants and diffusion coefficients of EAK-II
and a few other peptides are given in Table 6.2.
Based on the amino acid sequence of bovine serum albumin, its molar
mass is 66430.3 daltons which is equivalent of 66430.3g mol−1 . Albumin
found in human blood. It is the most abundant protein in human blood
plasma. It has a molar mass of 66.5 k Da i.e., 66500 daltons or 66,500 a.m.u
Name of compound
λTAPP (human islet amyloid
polypeptide) a 37 amino acid
peptide hormone. Its
structure is: Lys, Cys, Asn,
Thr, Ala, Thr, Cys, Ala, Tur,
Gln, Arg, Leu, Ala, Asn, Phe,
Leu, Val, His, Ser, Ser, Abn,
Asn, Phe, Gly, Als, Ile, Leu,
Ser, Ser, Thr, Asn, Val, Gly,
Ser, Asn, Thr, Thr
6.4.1
CMC
3.25 × 10−6 (by
conductance and
fluorescence
measurements)
ΔG associated with the
incorporation of protein
into membrane is determined by the hydrophobic effect and is about
−63 kJ mol−1
Expansion of Symbols
Lys = lysine (K), Cys = Cysteine (C), Asn = Asparagine (N), Thr = Threonine (T), Ala = Alanine (A), Gln = Glutamine (a), Arg = Arginine (R), Leu =
Leucine (L), Phe = Phenylalanine (F), Val = Valine (V), His = Histidine (H),
Ser = Serine (S), Gly = Glycine (G), Ala = Alanine (A), Ile = Isoleucine (I).
The single letters in brackets are one letter abbreviations. It is secreted
by beta cells and functions to control hyperglycemia.
6.5
Peptides as Biosensors
Peptides also find applications as biosensors upon immobilisation of certain amide sequences on electrode surfaces, such as Au, Ag etc.
136
Biophysical Chemistry
Structure of the ionic complimentary peptide EAK 16-II. It contains
alternating hydrophobic (alanine, A) and hydrophilic (glutamic acid, E and
lysine, K) residues producing amphiphilic structure that is hydrophobic on
one side and hydrophilic on the other. The charged residues are arranged
as type-II − − + + − − ++, where pairs of −vely (E) and +vely charge (K)
residues alternate. Formula of EAK 16-II: C70 H121 N21 O25 . Mol.Wt = 1657 g.
Peptides 137
Structure of MA-1
Structure of TA-1
Using voltammetric studies, the time-dependent variation of the peak
current for various compositions of TA-1 and MA-1 was studied and the
results are shown in Table 6.3. The TA-1 probe was taken in a buffer of pH
= 7.4 and then Trypsin was added.
6.6
Current-Voltage Characteristics
of Peptide Films
Using cyclic voltammetric studies, the current-potential response of F-mocLL and F-moc-YL was studied. The increase in current with potential was
observed (see Table 6.3). The specific conductances of the peptide are also
shown in Table 6.5.
138
Biophysical Chemistry
Table 6.3 Variation of the MB peak current with time.
Time (min) % change in signal using
TA-1
MA-1
0.0
0.0
0.0
10.0
−12.0
−30.0
20.0
−28.0
−43.0
30.0
−38.0
−48.0
40.0
−46.0
−50.0
58.0
−50.0
−52.0
78.0
−53.0
−54.0
96.0
−60.0
−58.0
Table 6.4 Rate constant data for the effective cleavage rate of MB in the
peptides TA-1 and MA-1.
Compound Rate constant (min−1 )
TA-1
7.7 × 10−2
MA-1
3.8 × 10−2
Table 6.5 Current potential response of peptides from cyclic voltammetry
at a scan rate of 500 mv sec−1 at 25◦ C.
Peptide
F-moc-LL
F-moc-YL
Potential
(V)
−2.0
−1.0
+0.5
+1.0
+2.0
−2.0
−1.0
−0.5
+1.0
+2.0
Current
(μA)
−0.25
−0.10
0.00
0.00
0.10
−0.75
−0.50
−0.30
+0.10
+0.45
Electrode
length/space
(in mm)
Specific
conductance
(mho cm−1 )
12/3
4.2 × 10−5
12/3
2.2 × 10−4
F-moc-LL: N-(fluorenyl - 9 methoxy carbonyl)-leucine-leucine
F-moc-YL: N-(fluorenyl - 9 methoxy carbonyl)-tyrosine-leucine-leucine
Peptides 139
It is well known that peptides possess the ability to form self-assembled
monolayers on suitable electrode surfaces. These self-assembled monolayers (SAMs) are the vital source of information for the functional behaviour
of peptides. Furthermore, it is possible to tailor-make peptide sequences of
desired characteristics. In this context, Au electrodes modified using peptides with sulphide moiety offer the most desirable surfaces. The investigations pertaining to the structure and dynamics of SAMs on surfaces
have several facets viz. (i) applicability as sensors; (ii) directional charge
transport; (iii) opto-electronic devices and (iv) biomedical applications.
(i) In the case of electrochemical biosensors, various types of peptides
serve as bio recognition elements for detection of antibodies and proteins. A pre-requisite for employing these peptide-based biosensors
is the correct choice of peptide sequences that possess unique ability of excellent affinity and satisfactory selectivity towards the target
(analyte) protein. Since the peptides themselves are electro-inactive,
modified electrodes of diverse types (SAMs, carbon nanotubes,
graphene, conducting polymers etc.) have been employed.
(ii) A particularly innovative approach consists in employing negatively
(e.g., glutamic acid) and positively charged amino acids (e.g., histidene) on Au. This arrangement upon suitable anchoring is shown to
yield secondary structures-amenable for fundamental and medicinal
applications. In the case of directional charge transport, the system
donor-peptide-acceptor is chosen wherein the electron transfer mechanism designated as ‘super exchange’ operates. The corresponding
operator is known as ‘super exchange operator’. An alternate mechanism of electron transfer deduced using current-potential response
of molecular junctions is electron hopping which occurs in the selfassembly of cyclic peptide nanotubes. In the electron hopping mechanism, the mobility is significantly higher than the conventional electron transfer processes.
(iii) In order to function as opto-electronic devices, electron or photon
responses of the system are essential. In view of the self-assembly
properties as well as chemical and electrochemical activity of the peptides, these constitute potential opto-electronic devices. Under suitable conditions, the peptides can serve as spacers between the donor
and acceptor moieties. Due to the availability of diverse peptide functional groups viz oligothiophene, oligopyrrole, oligoaniline etc., these
systems function as light emitting diodes, electrochromic device, solar
cells etc.
140
Biophysical Chemistry
(iv) Peptides function as biorecognition elements in diverse amperometric and voltammetric biosensors on account of their selectivity and
satisfactory adhesion to electrodes. Among several analytes employed
in this context, mention may be made of proteins, nucleic acids, antibodies. The most commonly employed electrochemical technique in
this context is differential pulse voltammetry, in view of its sensitivity
and low detection limits.
Questions
(1) Which of the following is true regarding the nature of the peptide
bonds?
(a) Covalent
(c) Metallic
(b) Ionic
(d) Three centre-two electron
(2) Which of the following is true regarding complete proteins? They are
(a) conjugated
(c) saturated
(b) unconjugated
(d) unsaturated
(3) When a protein denatures, it undergoes
(a) folding
(c) oxidation
(b) un-folding
(d) reduction
(4) For a peptide given by the sequence Glu-Cys-Asn-Met-Lys Met-GluThr-Arg-Trp Ile-Tyr, the reagent for specific cleavage is
(a) Trypsin
(b) Chymotrypsin
(c) Pepsin
(d) Renin
(5) Myoglobin has a
(a) Primary structure
(b) Secondary structure
(c) Tertiary structure
(d) Quaternary structure
(6) Cysteine has
(a) Azo bond
(b) Disulphide bond
(c) Thiol bond
(d) Hydrogen bond
(7) The rate constant for the cleavage of methylene blue in the peptide
TA-1 is 7.7 × 10−2 min−1 . Calculate the time required for 90 percent
cleavage of the same.
(8) Draw the structure of a tetrapeptide and indicate the end groups in it.
Peptides 141
(9) Give the chain of amino acids linked in EAK 16-II. What are the special
features of the peptide.
(10) Calculate the percent aggregation of EAK 16-II after 1200 sec given its
aggregation rate constant is 1.14 × 10−4 min−1 .
(11) With illustrative examples, explain the application of peptides as
biosensors.
7
Proteins
7.1
Introduction
Proteins are essentially linear polymers built of monomer units of amino
acids. The function of a protein is dependent on its three-dimensional
structure. Proteins fold up spontaneously into three dimensional structures that are determined by the sequence of amino acids in its chain.
They are versatile macro molecules in living systems and have crucial functions in all biological processes. They act as catalysts, transport and store
molecules like O2 and provide immune protection.
7.2
Composition of Proteins
Proteins are built from around twenty amino acids. The amino acids are
linked by peptide bonds to poly peptide chains in a primary structure and
subsequently these chains fold into other structures like α-helix, beta sheets
and loops. Proteins contain a wide range of functional groups and they
may be alcohols, thiols, carboxylic acids and also a variety of basic groups.
This array of functional groups, when combined in various sequences
accounts for a broad spectrum of protein function. The chemical reactivity
associated with these groups is essential to the function of enzymes.
7.3
Some Characteristics of Proteins
Proteins can interact with one another and with other biological macromolecules to form complex assemblies. Proteins within these assemblies
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_7
143
(iii) They are softer than fibrous
proteins.
(iv) They are soluble in water.
(v) They form antibodies, enzymes
and hormones.
(vi) Examples: Collagen, myosin, silk,
keratin.
(vi) Examples: Insulin,
Haemoglobin, DNA, and RNA
polymerase.
(iv) They are blood clotting proteins.
(v) Examples: Fibrinogen.
(iii) They are soluble in water.
(ii) Tertiary structure is very
important in them.
(ii) For this class of proteins,
secondary structure is a most
important functional structure.
(iii) They are tough and strong.
(iv) They are insoluble in water.
(v) They form long fibres or sheaths.
Intermediate proteins
(i) Their structures intermediate
between linear and globular
proteins.
(ii) They are short and linear shaped.
Globular proteins
(i) They are spherical in shape and are
tightly folded into this shape.
Fibrous proteins
(i) They are linear (long fibrous) in
shape
Table 7.1 Classification of proteins based on structure.
144
Biophysical Chemistry
Proteins 145
can act (in synergy) to generate capabilities not afforded by individual
components. For example, human insulin is a protein hormone crucial for
maintaining blood sugar levels at proper levels. Its structure with chains
of amino acids is a specific sequence defines its function. The protein lactoferrin undergoes conformational changes on binding with iron that allow
other molecules to distinguish between iron free and iron bound forms.
7.4
Classification of Proteins
Proteins are classified on the basis of the following three criteria: (i) based
on the structure of proteins, (ii) based on composition of the proteins and
(iii) based on functions of the protein.
Classification Based on Composition
(a) Simple proteins composed only of amino acids: They possess relatively simple structure. They may be fibrous or globular. Example: Collagen, Myosin, Insulin, Keratin.
(b) Conjugated proteins: They are complex. They contain some nonamino acid components. The protein part is tightly or loosely bound to
one or more non-protein parts which are called prosthetic groups. The
prosthetic groups may be metal ions, carbohydrates, lipids, phosphoric
acids, nucleic acids. These groups are essential for the biological function of the proteins. Conjugated proteins are globular in shape and are
water soluble. Most enzymes belong to this class.
Based on the nature of prosthetic groups, conjugated proteins are further classified as follows:
– Phosphoprotein: In this case, the prosthetic group is phosphoric
acid.
– Chromoproteins: In this case, the prosthetic group is pigment or
chrome. Example: Hemoglobin, Phytochrome, Cytochrome.
– Lipoproteins: Prosthetic group is lipids. Example: Membrane proteins.
– Flavoproteins: Prosthetic group is FAD (i.e., Flavin adenine dinucleotide). Example: Proteins of electron transport system.
– Metalloproteins: Prosthetic group is metal ions. Example: Nitrate
reductase.
146
Biophysical Chemistry
Classification Based on Functions
(a) Structural proteins: Most of these proteins are fibrous proteins insoluble in water. Example: Collagen, Keratin, Elastin. Collagen and Elastin
are found in connective tissues such as tendons and ligaments. Keratin
is found in hair, feathers and beaks. Collagen is recognized as the most
abundant protein in mammals. It has a triple helix structure and is
not α-helix. It contains specific amino acids: glycine, proline, hydroxyl
proline and arginine.
(b) Enzymes: They are biological catalysts. They speed up reactions by
reducing the activation energy of reactions. Many of them are globular
conjugated proteins. Example: DNA polymerase, Lipase, Nitrogenase.
(c) Hormones: These include proteinaceous hormones in cells. Example:
Insulin, Glucagon, ACH.
(d) Respiratory pigments: They are coloured and are conjugated proteins
containing pigments as prosthetic groups. Example: Haemoglobin,
Myoglobin.
(e) Transport proteins: They transport material in cells, form channels in
plasma membranes. They form one of the components of blood and
lymph in animals. Example: Serum albumin.
(f) Contractile proteins: They are force generators in muscles. They can
contract with expense of energy from ATP molecules. Example: Actin,
Myosin.
(g) Storage proteins: They store metal ions and amino acids in cells. They
are found in seeds, egg, milk and pulses (legume seeds). Example: Ferritin which stores iron, casein, ovalbumin, gluten of wheat.
(h) Toxins: They are toxic proteins. Example: Snake venom.
7.5
Nature of Bonds in Protein Structure
The polypeptide bonds present in proteins fold into specific structures so
as to get a proper conformation. Many types of bonds support and stabilize the specific shapes and conformations. Important types of bonds that
contribute to the conformation are peptide bonds, ionic bonds disulphide
bonds, H-bonds and bydrophobic interactions. The various bonds are discussed below.
Proteins 147
(1) Peptide bond: It is a covalent bond formed between the amino group
of one amino acid and carboxyl group of another amino acid. The bond
has high dissociation energy and is also planar since the electrons are
delocalised to give double bond character to the C–N bond.
(a) During protein synthesis, this bond is formed.
(b) The resulting compound after the
formation is a di-peptide.
(c) Several amino acids combine to form polypeptides having many
peptide bonds
(d) Peptide bond formation is favoured by the enzyme peptidyl transferase. Two amino acids react according to
(2) Ionic bonds: The ionic bonds are formed between the ionized acidic
(−COO− ) or ionised basic Ō
⎛ +
⎞
⎜ − N H3 − ⎟
⎝
⎠
groups of amino acids. The R-groups of some amino acids, may contain
additional
148
Biophysical Chemistry
The ionic bonds may break down by a change of pH. Example: Denaturation of protein. Tertiary or quaternary structures of proteins are
stabilized by ionic bonds.
(3) Disulphide bonds: This bond is covalent and hence quite strong. The
covalent bond formed between two thiol groups of two cysteine residing in a protein is an example. Disulphide bonds stabilize the tertiary
structure of protein.
(4) H-bonds: It is formed by electrostatic attractions between a H atom
which is covalently linked to atoms such as O or N. Example: H : O : H.
The H atoms carry a partial +ve charge due to attraction of highly electro negative oxygen atom. The same is true with nitrogen. Example:
NH3 .
The H-bonds give regular shape to the dipeptide chain such as α-helix.
H-bond is not very strong. These bonds stabilize the secondary and
tertiary structure of proteins.
(5) Hydrophobic interactions: The non-polar R-groups in some amino
acids are hydrophobic and they repel water. Example: alanine, valine,
leucine, methionine. In an aqueous environment (inside a cell) the linear polypeptide chain will fold in such a way that the hydrophobic
groups come together and exclude water due to hydrophobicity. The
hydrophilic groups form a shell over the hydrophobic moieties and
point towards water in the interior of the cell.
7.6
Structure of Proteins
Research on protein structure resulted in the conclusion that a specific configuration, the right handed a α-helix, is favourable for its stability. The
α-helixes are stabilised due to H-bonding between an −NH group in the
helix and the C=O group of the form the amino acid down the chain.
The α-helix, a common structural motif of proteins consists of a right
handed helix with a repeat length of 3.6 amino acid residues per helical
turn. The α-helix is stabilised by H-bonds between an amide hydrogen
of one amino acid and a carbonyl hydrogen four amino acids away. The
side chains of the amino acids, R, protrude from the surface of the helix
core, allowing interaction of these functional groups with other proteins
Proteins 149
Figure 7.1 Structure of α-helix.
structural motifs or with H-bonding sites in the major or minor grooves of
the DNA.
The amino acids, glycine, proline, serine, glutamine, threonine and
asparagines are α-helix breakers because they have Nα atom in a rigid ring
structure.
7.7
Role of Amino Acids in Proteins
(1) Cysteine: It can cross link with another cysteine sulfhydril group in
the same or on different polypeptide chains by oxidation to a covalent disulphide bond. The structure of insulin is a good example of the
importance of disulphide bonds. Thioether bridges occur in cytochrome
C between the iron protoporphyrin groups and two cysteine residues
of the protein.
(2) Histidine: The lone pair of electrons in the ring nitrogen may serve as a
potential ligand as in the iron containing proteins in haemoglobin and
cytochrome C.
(3) Lysine: It is involved in binding pyridoxal phosphate, lipoic acid and
biotin. Like in serine and histidine, it may serve in making up the active
site of an enzyme like muscle aldolase.
150
Biophysical Chemistry
(4) Serine: It serves as a nucleophile in a number of proteolytic enzymes.
Along with histidine residue, a specific serine residue serves as a component of the active site of chymotrypsin. It may be mentioned that
chymotrypsin is a digestive enzyme which breaks down proteins in
the small intestine (it is secreted by pancreas and converted into an
active form by trypsin). Serine residues also serve as active sites of
phosphoryl groups which modify the activity of a number of enzymes.
(5) Proline: It forces a bend in polypeptide chain and disrupts α-helicity
due to its rigid rang. Polar amino acids such as glutamic acid, aspartic
acid, arginine, lysine are ionised over a wide pH range and can form
ionic bonds in protein structure. Protein, being a complicated macro
molecule, is defined in terms of four basic structural levels.
(a) Primary structure: It is a linear sequence of amino acid residues
making up its polypeptide chain.
(b) Secondary structure: It refers to the structure of a polypeptide or a
protein may possess from H-bond inter actions between amino acid
residues which are fairly close to one another in primary structure.
(c) Tertiary structure: This structure refers to the tendency of the
polypeptide chain to undergo extensive coiling or folding to produce a complex rigid structure. Folding occurs between amino acid
residues relatively far apart in the sequence.
(d) Quaternary structure: This defines the structure resulting from
interactions between separate polypeptide units of a protein containing more than one sub unit. Thus the enzyme phosphorylase
“a” contains two identical sub units that alone are catalytically inactive but when joined as a dimer form the active enzyme.
Figure 7.2 Quaternary structure of a complex globular protein (dimer).
Proteins 7.8
151
Examples of Proteins
(1) Blood proteins: About 60 proteins have been identified and characterized in blood plasma. They are divided into carbohydrate free proteins
and glycoproteins.
Among the predominant carbohydrate free proteins is albumin
which constitutes 50% of total serum protein. It serves as a carrier
protein because of high concentrations of fatty acids and anions in it.
Serum albumin controls the osmotic pressure of blood as well as maintaining the buffering capacity of blood pH. It is a globular protein with
a molar mass of 69,000.
(2) Glyco proteins: They occur widely in nature. In these proteins, a covalent link exists between carbohydrate polymer and N-or acylglyconyl
linkage. They are classified into three categories based on carbohydrate
composition, linkage of carbohydrate to protein.
(i) Plasma glycoproteins: They are found in blood sera.
After NH-C and vertical double bonded O.
(ii) Mucin glycoprotein protein
152
Biophysical Chemistry
(iii) Mucopolysaccharide
7.9
Hemoglobin
It is a respiratory protein in all vertebrates and is localized in erythrocytes.
It reacts reversibly with O2 and transports O2 from lungs to all parts of
body. It is a conjugated heterogeneous tetrameric protein composed of
different subunits (α and β). Each monomeric unit (Molar mass = 16,000)
contains a heme group linked to protein via the imidazole nitrogen of the
histidine residues in the monomer of protein. There are four iron atoms in
each molecule of hemoglobin which can bind four oxygen atoms.
Globin contains two linked pairs of polypeptide chains.
Figure 7.3 Structure of hemoglobin.
Proteins 7.10
153
Antibodies
A large group of glycoproteins, classified as immunoglobulins are found
in blood plasma. Some of these proteins are produced in the spleen and
lymphatic cells in response to a foreign substance called antigen. The
newly formed protein is called “antibody”. Each immunoglobulin is made
of two pairs of polypeptide chains, a pair of short and a pair of long chains.
7.11
Hormones
These polypeptides are small proteins found in low concentrations in animal tissues. Among these, the important ones are pituitary hormones (oxytocin), large protein (glucagon) and insulin.
7.11.1
Nutrient Proteins
The essential amino acids (in proteins) are readily synthesized by plant but
must be acquired by humans.
7.12
Denaturation of Proteins
Proteins are maintained in their native state (i.e., their natural 3D configuration) by stable secondary, tertiary and quaternary structures. Denaturation takes place when the folded native structures break down because
of extreme temperatures, pH changes which disrupt the stabilizing structures. Under these conditions, the structure becomes random and disorganised. Most proteins are biologically active over a temperature range of
0 to 40◦ C only. Heat is often used to kill organisms and deactivate their toxins. Heat denaturisation is used to prepare vaccines against some diseases.
Denaturation can also be caused by α-helices packed on one side.
Other methods such as treatment with organic solvents, strong acids
or bases, detergents and heavy metal ions like Hg2+ , Pb2+ , Ag+ .
Table 7.2 Equilibrium constants, Kh−c for helix-coil transformation.
Amino acid residue
Glycine
L-serine
L-alanine
L-leucine
Kh−c
0.62
0.79
1.06
1.14
154
Biophysical Chemistry
Equilibrium constants, Kh−c for helix-coil transformation in some proteins having amino acid residues at 30◦ C.
7.13
Helix-Coil Transitions in Proteins
The helix form dominates at low temperatures. As temperature is raised,
the random form dominates. More often, the transition occurs over a narrow range of temperatures indicating that the helix unwinds suddenly
rather than gradually. A plot of fractional helicity (in terms of optical rotation) as a function of temperature for protein chains of different length (N)
is depicted in Figure 7.4.
Figure 7.4 Schematic depiction of helix-coil transitions in proteins.
Note: Tc is the temperature in the midpoint of the transition when half of
the total number of chains are helical.
7.14
Kinetics of Helix-Coil Transformation
Using small synthetic polypeptides rich in alanine, temperature jump studies were carried out and the mechanism was deduced as consisting of two
steps: (i) a very fast step in which amino acids at either end of the helical
segments undergo transitions to coil regions and (ii) a slower (rate determining step) that corresponds to the co-operative melting of the rest of the
chain and loss of helical content. Using X and Y to denote an amino acid
Proteins 155
residue as belonging to a helical and coil region the mechanism may be
written as
The relaxation time of the slow step, measured by laser T-Jump techniques for an alanine rich polypeptide chain (containing 21 amino acids)
around 290 K was found to be 160 ns (or 160 × 10−9 sec).
Table 7.3 Electrophoretic mobility data of a few typical proteins.
Protein
pH Electrophoretic mobility
(10−4 cm2 v−1 sec−1 )
Ribonuclease A
7.0
0.55
Lysozyme at an ionic strength of 0.2 9.6
−0.24
7.15
Membrane Proteins
Integral membrane proteins, also called intrinsic proteins, have one or
more segments that are embedded in the phospholipid bilayer. Many integral proteins contain residues with hydrophobic side chains that can interact with fatty groups of the membrane phospholipids thus anchoring the
proteins to the membrane. Membrane proteins perform several functions
vital to the survival of organisms. They relay signals between the cells
internal and external environments.
Effect of micellar structure, electrostatic effects and activation barrier
on detergent mediated unfolding of 101-residue monomeric mixed α-helix
S6 protein.
Figure 7.5 Shapes of proteins under different coordinates.
156
Biophysical Chemistry
Ribosomal protein S6 (sub unit 6) is a small α/β protein consisting of
101 amino acids with a molar mass of about 12 kDa. S6 is constructed
from 4β-sheets and two α-helices. S6 has two different conformations; it is
either folded or unfolded with no intermediate states. It follows two-state
folding event from the denatured state (D) to the native state (N) with the
energy diagram shown in Figure 7.6.
Its 3D structure consists of 4 stranded antiparallel β-sheets with two.
Figure 7.6 An energy diagram of a two-state folding event.
The protein crosses an energy barrier, the transition state, to pass from
denatured state to the native state without populating any intermediate
states. The energy difference between the “D” state and “N” state corresponds to the difference in ΔGD− N and the reaction co-ordinate is the total
difference in exposed surface area, Δm D− N (divided from m f and mu ). The
reaction rate from the denatured state to TS is seen as the folding rate (k f )
and the opposite direction, from the native state to TS as the unfolding
rate (k u ). The detergents used in these studies are sodium dodecyl sulfate
(SDS), lauryl trimethyl ammonium bromide (LTAB) and lauryl trimethyl
ammonium chloride (LTAC). Two schemes were proposed for the unfolding of S6
Proteins 157
Table 7.4 Kinetic parameters for unfolding of S6 in SDS.
Concentration
of NaCl (M)
−1
kmodel
un f ( s )
K1 ( M − 1 )
−1
kSDS
0.5m ( s )
CMC (mM)
0.0
0.02
0.06
0.15
0.30
0.40
19.5
12.3
4.34
2.49
-
364
303
445
177
-
31.3
43.8
67.6
88.9
125.2
162.0
2.0
1.7
1.0
0.5
-
Conditions: Temperature 25◦ C, pH = 7.0. K1 = association constant
between micelles (of surfactants) and free protein. CMC values (of surfactants) in presence of S6 at the given NaCl concentration and 25 mM Tris
HCl, pH = 8.0. The kinetic runs were made using stopped flow technique.
7.16
Modelling of Tertiary Structure of Proteins
As mentioned earlier, the sequence of amino acids dictates the primary
structure of proteins. The secondary structures often comprise α-helices
and β-sheets, while the three-dimensional arrangement of peptides constitutes the tertiary structure. Interestingly, diverse types of long-range and
short-range interactions predominantly influence the tertiary structures.
In fact, it is this presence of electrostatic and non-electrostatic along with
van der Waals type interactions that poses a major challenge in the prediction of the structures. Consequently, it is imperative to employ minimalistic approaches for comprehending the structures from the constituent
amino acids sequences. The mysterious connection between the native
structures of proteins and their most stable sequences vis a vis the mechanism underlying the energy landscape remains formidable even today.
It is hence essential to formulate the fundamental rules governing the
native protein structures, in a hierarchical manner. The lowest-energy state
is identified as the native state of the protein; among different conformations available for a given sequence, the folded state is often referred to as
the most compact structure.
The Hydrophobic-Polar (HP) model for a chain of N amino acid
residues is a simple but powerful approach to analyze the conformations
of proteins. The energies and degeneracies of 2N states need to be computed
for identifying the native states. Since exact enumeration is feasible only
for small values of N (< 16), Monte Carlo simulations as well as graph
158
Biophysical Chemistry
Table 7.5 Thermodynamic data of polypeptides in proteins relating to
helix-coil transformation in aqueous solutions.
theoretical procedures have been employed for larger values of N (∼ 100).
The tertiary structures of proteins is dictated solely by the topological
arrangement of the H-P sequences. The H-P lattice models are analyzed
in both two and three dimensions, for deducing the designable protein
sequences vis a vis conformations. Furthermore, these models correctly
predict the temperature dependence of the coil to globule transition, thus
Proteins 159
Table 7.6 Equilibrium constants (Khel −nonhel ) for some natural amino
acids in nine globular proteins.
Amino acid
Glu
Ala
His
Met
Lys
Phe
N
62
165
54
20
106
56
helix/N
0.63
0.57
0.56
0.50
6.44
0.38
ΔG ◦ /JK−1 mol−1
−611.7
−236.2
−324.7
-
exp(−ΔG ◦ /RT)
1.28
1.10
> 1.0
> 1.0
1.14
> 1.28
Note: N = number of residues of the amino acid in 9 globular proteins
(myoglobin, α and β chains of haemoglobin, lysozyme, ribonuclease, αchymotrypsin, papain, subtilisin, carboxypeptidase)
demonstrating their remarkable validity. The input in these lattice models
is the pair-wise interaction energies between H-H, P-P and H-P contacts.
There are several versions of H-P models, depending upon the energies ( E) assigned for H-H, H-P and P-P contacts. In the original HP model
due to Dill et al., in 1989, the interaction energy between H-H contacts
(EH − H ) was assumed to be negative (i.e., favorable) while the H-P (EH − P )
and P-P (EP− P ) energies were assigned zero.
Subsequently, other
parametrization schemes for the interaction energies were envisaged e.g.,
negative values for both EH − H and EH − P with EP− P = 0.
Typically, for two-dimensional H-P lattice models of N amino acid
chains, containing hydrophobic and polar residues, the total number of
sequences is 2N . In an amino acid chain of length ‘N’, let ‘p’ be the number
of hydrophobic residues and q be the number of hydrophobic-polar contacts. The enumeration of A( p, q) indicating the total number of ways in
which the H-P contacts can arise for a given composition of proteins is a
non-trivial task even for N = 16 since the total number of states allowed
is 216 . In the parlance of graph theory, this enumeration is known as the
counting of black-white edges.
Table 7.7 provides the values of A( p, q), for a square lattice of 16 sites.
It is easy to verify that the addition of all the entries of Table 7.7 yields
216 (65,536). This enumeration becomes tedious when the chain length of
amino acids increases.
As an illustrative example, consider a sequence HHHPHHPHHHHPHHP with the chain length N = 16, with p = 4 (black circles denoting
the polar part). It has been demonstrated that the structures (i) and (ii) are
the native and compact structures for this sequence.
Note: ‘p’ denotes the number of polar residues and ‘q’ denotes the number of polar-hydrophobic contacts.
Table 7.7 A( p, q) values which arise from the counting of ‘hydrophobic-polar’ contacts for a square lattice of 16 sites,
assuming periodic boundary conditions.
q
0 1
2
3
4
5
6
7
8
9
10
11
12
13 14 15 16
p
0
1
1
2
4
16
16
6
32
32
8
88 96
24
8
24
96 88
10
256 256 192
96
64
0
64
96
192 256 256
12
208 736 688
704
624
768
624
704
688 736 208
14
576 1664 1824 1920 1600 1920 1824 1664 576
16
228 1248 2928 3680 4356 3680 2928 1248 228
18
448 1568 3136 3264 3136 1568 448
20
128
768 1392 2112 1392 768
128
22
64
512
576
512
64
24
56
96
120
96
56
26
0
64
0
28
16
0
16
30
0
32
2
160
Biophysical Chemistry
Proteins 161
Figure 7.7 (i) Native and (ii) a compact structure of amino acid chain
lengths 16 within the two-dimensional HP lattice model framework.
7.17
Levinthal Paradox
The process by which a given amino acid composition chain acquires a
three-dimensional shape is customarily designated as ‘protein folding’.
The folding of any protein into its native structure remains a challenging
problem, and the complexity can be comprehended with a typical example. For a chain length N = 100 undergoing three-dimensional random
walk, the total number of conformations is ∼ 1077 . Given these astronomically large numbers, ferreting out the native protein structure by any algorithmic methodologies will consume billions of years even with the fastest
computers. On the other hand, the protein folding often occurs within
the time scale of a few seconds, implying that the protein folding does
not occur by any systematic search among all the conformations. This is
known as Levinthal paradox.
One of the strategies by which this paradox can be resolved consists
in assigning first order kinetic rate constants to the process by which the
native state arises. This clever method entirely eliminates the enumeration
protocols.
7.18
Proteins in Nutrition
Protein is found in plants and animals. Two kinds of amino acids must
be considered for the body. Essential amino acids are required for normal
body function but they can not be directly made by the body and must be
obtained from food. Nine amino acids out of 20 are considered essential.
The non-essential amino acids can be made by the body from the essential
amino acids consumed in food. Proteins are found in a variety of foods
such as beans, peas, eggs, nuts and seeds, meat, sea-food and soy products.
Proteins provide energy for the body. They are a component of every
cell in the body and are necessary for proper growth and development.
Protein helps the body build and repairs cell and body tissue.
162
Biophysical Chemistry
Protein foods are important sources of vitamins and minerals, such as
B-vitamin, choline, iron, phosphorus and selenium. They are also a sources
of vitamins D, E and zinc. The daily value for protein is 50 gms per day
based on 2000 calories diet.
Questions
(1) If the electrophoretic mobility of ribonuclease A is 5.5 × 10−5 cm2 v−1
sec−1 , how much distance will it travel under a potential gradient of 20
V cm−1 in 60 min.
Solution:
Distance travelled in 1 sec = 5.5 × 10−5 cm sec−1 × 20V cm−1
= 11.0 × 10−4 cm
In 60 minutes, distance travelled = 11.0 × 10−4 cm × 60 × 60
= 11.0 × 36 × 102 × 10−4
= 396 × 10−2 = 3.96 cm
(2) The rate constant for the unfolding of S6 proteins in 0.5 M sodium
dodecyl sulfate (SDS) is 31.3 sec−1 . Given that the equilibrium constant for the reaction
kfold
S6 + SDS FGGGGGGGGB
GGGGGGGG S6(SDS)
kunfold
is 364 sec−1 , calculate the rate constant for the folding of S6.
Solution:
K = 364 =
kfold
kunfold
kfold = 364 × 31.3 = 41410.5 liter M−1 sec−1
(3) For the polypeptide, poly-L-Lysine, the enthalpy and entropy changes
ΔH ◦ and ΔS◦ associated with the helix-coil transformation are −3.4 kJ
mol−1 and −10.04 J deg−1 mol−1 respectively. Calculate the equilibrium constant at 300K.
Proteins 163
Solution:
ΔG ◦ = − RT ln K
ΔG ◦ = ΔH ◦ − TΔS◦ = −3400 − (300 × −10.04)
= −3400 + 3012 = −388 J
−388 = −8.314 × 300 × 2.303 log K
388
log K =
= 0.0675
8.314 × 300 × 2.303
K = 1.17
(4) Classify proteins on the basis of their structure and describe their properties.
(5) Discuss briefly the various types of bonds observed in proteins.
(6) Write the structure of haemoglobin and explain its role in human body.
(7) Describe briefly (a) denaturation of proteins and (b) helix-coil transition.
(8) Discuss the effect of temperature on helix-coil transformation in
proteins.
(9) Consider the square lattice model for a protein of 16 amino acids, with
equal number of hydrophobic and polar parts. Show that the total
number of hydrophobic-polar contacts is 4356.
8
Nucleosides and Nucleotides
8.1
Introduction
A nucleoside consists of a nitrogenous base covalently linked to a 5-carbon
sugar (ribose or deoxyribose) but having no phosphate group. A nucleotide
consists of a nitrogenous base sugar (ribose or deoxyribose) and one to
three phosphate groups. A comparison of the two is given in Table 8.1.
Table 8.1 Comparison of nucleosides and nucleotides.
Chemical
Medical
Examples
composition
significance
Nucleoside Nucleoside sugar
Many
Cytidine, uridine,
+ base. When the
nucleoside
adenosine,
phosphate group
analogues are
guanosine,
of a nucleotide is
used as
thymidine
removed by
antiviral or
and inosine
hydrolysis, the
anti-cancer
structure of
agents
nucleoside remains
Nucleotide
Sugar + base +
The
All names as
phosphate
malfunctioning
given above and
nucleotides is
with inclusion of
one of the main
phosphate. For
causes of all
example 5’-uridine
cancers
monophosphate
Compound
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_8
165
166
8.2
Biophysical Chemistry
Biological Function (DNA & RNA)
Nucleotides are building blocks of nucleosides. A nucleic acid contains a
chain of nucleotides linked together with covalent bonds to form a sugar
phosphate backbone with protruding nitrogenous bases. For example:
DNA contains two chains spiraling round each other in the shape of a double helix. The two chains in the double helix are held together along their
length by H-bonds that form between the base, on one chain and the bases
on another.
8.3
Examples of Nucleotides
Nucleic acids are made of monomers known as nucleotides. A nucleotide
has three parts as mentioned earlier.
I. A 5-carbon sugar ring: For example, deoxyribose in DNA: ribose in
RNA; ribose in ATP.
II. Phosphate group: In RNA and DNA the phosphate group binds to
another nucleotides carbon forming the backbone of the molecule.
In ATP, there are three phosphate groups that store energy.
III. A nitrogenous base: The bases in DNA, are adenine, guanine, cytosine,
and thymine.
The bases in RNA are adenine, guanine, cytosine and uracil. The base in
ATP is always adenine.
Figure 8.1 Structure of nucleoside, nucleotide, nucleoside di and tri phosphates.
Nucleosides and Nucleotides 167
Figure 8.2 Structures of purines.
Figure 8.3 Structures of pyrimidines.
Figure 8.4 The structural elements of the nucelosides and the phosphate
bearing nucleotides.
(i) The reduction of adenine involves a primary potential controlling
reduction of the N(1) = C(6) double bond to give 1, 6-dihydro-6
amino purine. It is reduced again is a 2ē − 2H + process to give
1, 2, 3, 6-tetrahydro-6-amino purine. The scheme of reduction is
given in Table 8.2.
168
Biophysical Chemistry
Table 8.2 Typical polarographic data for the
sine and guanine.
Compound Nature of polarographic
wave
Adenine
Single diffusion-controlled
wave
Cytosine
Single diffusion-controlled
wave
reduction of adenine, cytoHalf wave potential (V)
−0.975 to 0.084 in the pH
range 1 to 6
−1.125 to 0.073 in the pH
range 4 to 6
Figure 8.5 Scheme depicting electrochemical reduction of adenine.
(ii) The basic reaction pattern for cytosine involves a rapid protonation at the N(3) position to form the electro active species A 2electron reduction of the N(3) = C(4) bond leads to the carbanion. The protonation of the latter followed by de-amination, regenerates the N(3) = C(4) bond giving 2-oxypyrimidine. The protonation and subsequent one-electron reduction of 2-oxypyrimidine
gives a free radical which then dimerizes to give 6-6’ bis (3, 6)
dihydropyrimidone-2. In the case of guanine, the mean potential
for oxidation is 0.99 V and the scheme of oxidation is shown below.
Figure 8.6 Electro-oxidation of guanine.
Nucleosides and Nucleotides 8.4
169
Naming of Nucleosides and Nucleotides
Nucleotides are commonly abbreviated with 3 letters (4 or 5 in case of
deoxy or dideoxy-nucleotides). The first letter indicates the identity of the
nitrogenous base (for example: A for adenine, G for guanine), the second
letter indicates the number of phosphates (mono, di, tri) and the third letter “P” denotes phosphate. Nucleoside triphosphates that contain ribose
as the sugar are abbreviated as NTPS, while nucleoside triphosphates containing deoxyribose as the sugar are abbreviated as DNTPs (example: dATP
refers to deoxyribose adenine triphosphate) NTPs are the building blocks
of RNA and dNTPs are the building blocks of DNA.
The carbons of the sugar in a nucleoside triphosphate are numbered
around the carbon atom starting from the original carbonyl of the sugar.
By convention, the carbon numbers in a sugar are followed by the prime
symbol ( ) to distinguish them from the carbons of the nitrogenous base.
The nitrogenous base is linked to the 1 carbon through a glycosidic bond
and the phosphate groups are covalently linked to the 5 carbons. The 1st
phosphate linked to the sugar is termed as α-phosphate, the second is βphosphate and the third is the γ-phosphate.
Figure 8.7 Helical structure of DNA.
Compound
Adenosine
triphosphate
Importance
It is a most important energy form for all organisms
and is the cells energy currency
Structure
Table 8.3 Functional importance of a few nucleotides.
170
Biophysical Chemistry
Compound
Ribonucleic
acid (RNA)
Table 8.3 (Continued)
Importance
It is a single stranded molecule composed of building
blocks called ribonucleotides. A ribonucleotide is composed of 3 parts (i) a ribose i.e., ringed 5 carbon (ii)
sugar nitrogeneous base and (iii) a phosphate group.
In RNA, the bases present are adenine, guanine, cytosine and uracil. There are three functionally different RNA’s; messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA) mRNA carries information about a protein sequence to ribosomes, the protein
synthetic factories in the cell. t-RNA is a small RNA
chain of 80 nucleotides that transfers a specific amino
acid to a growing polypeptide chain. rRNA is the catalytic component of ribo somes.
Structure
Nucleosides and Nucleotides 171
Compound
Deoxyribonucleic acid (DNA)
Table 8.3 (Continued)
Importance
DNA is made up of molecules called nucleotides.
Each nucleotide contains a phosphate group, a
sugar group and a nitrogen group. The four
types of nitrogen bases are Adenine (A),
Thymine (T), Guanine (G) and Cytosine (C). The
order of these bases is what determines DNA’s
instructions, or genetic code. Each nucleic acid
strand contains nucleotides that appear in a
certain order within the strand, called its base
sequence. The base sequence of DNA is
responsible for carrying and retaining the
hereditary information in a cell.
Structure
172
Biophysical Chemistry
Nucleosides and Nucleotides 173
Table 8.4 Sites of protonation and pKa ’s of four nucleobases and the
ribose-phosphate backbone.
Reaction
pKa1 pKa2
1.
3.50
—
2.
1.60
9.20
3.
4.20
—
4.
9.2
—
174
Biophysical Chemistry
pKa and ΔG ◦ (kJmol−1 /at 298 K of the nucleotides 3’-ethyl phosphate
[d | rN Pet] (1a-5a and 1b-5b, scheme-1) and 3’, 5’-bisethyl phosphate [Etp
(d | rN) pEt] (6a-10a and 6b-10b), scheme-1 in both 2’-deoxy (dN) and ribo
(rN) series using NMR technique
1a : B = A, R = H
2a : B = C, R = H
3a : B = T, R = H
4a : B = U, R = H
5a : B = G, R = H
1b : B = A, R = OH
2b : B = C, R = OH
3b : B = T, R = OH
4b : B = U, R = OH
5b : B = G, R = OH
Explanation of symbols:
6a : B = A, R = H
7a : B = G, R = H
8a : B = U, R = H
9a : B = T, R = H
10a : B = C, R = H
6b : B = A, R = OH
7b : B = G, R = OH
8b : B = U, R = OH
9b : B = T, R = OH
10b : B = C, R = OH
Nucleosides and Nucleotides 175
Table 8.5 pKa data and ΔG ◦ values of nucleotides.
Nucleotide pKa (nucleo base) ΔG ◦ (kJmol−1 )
1a
3.35
19.1
2a
4.12
23.5
3a
9.92
56.6
4a
9.35
53.3
5a
9.40
53.6
6a
3.82
21.8
7a
9.59
54.7
8a
9.58
54.6
9a
10.12
57.7
10a
4.34
24.8
1b
2b
3b
4b
5b
6b
7b
8b
9b
10b
3.11
3.84
9.66
9.21
9.23
3.72
9.29
9.26
9.78
4.25
17.1
21.8
55.1
52.5
52.7
21.2
53.0
52.9
55.8
24.2
Examples of symbols in explanation:
Table 8.6 Conductivity data of nucleotides and nucleotides at 400 K.
Nucleosides
Nucleotides
−
11
−
13
−
1
−
7
10
to 10
mho cm
10 to 10−12 mho cm−1
176
Biophysical Chemistry
Solid State Conductivity of Dry DNA
It shows a small semi conductivity based on the equation
x = x0 e−Δε/RT
where Δε = 2.42 electron volts,
log10 ( x0 , mho cm−1 ) = 3.4
DNA Duplex and the Thermodynamic Parameters
Associated With It
Duplex DNA is another name for double stranded DNA. This means that
the nucleotides of two DNA sequences have bonded together and then
coiled to form a double helix. This double stranded structure facilitates the
stable duplication of genetic material, a requirement for cell division.
A strand of DNA is composed of nucleotides each of which consists of
a nitrogen base bonded to a sugar and triphosphate group. When two
of these strands are antiparallel, meaning lagging end of one strand is
aligned with the other strands leading end, hydrogen bonds form between
complementary nitrogen base pairs of adenine and thymine (A–T) or guanine and cytosine (G–C). This allows two separate DNA segments to fuse
together to create a ladder-like structure, where the sugar and the phosphate group are the vertical sides and the complementary nitrogen base
pairs serve as the ladder’s rungs. The DNA molecule is technically classified as a biopolymer, which means that it contains two polymer chains that
link up to form the larger molecule. Each of these polymer chains is composed of a DNA monomer, or nucleotide whose structure is formed from
a phosphate group, a deoxyribose sugar and a nitrogen containing base.
Areas mentioned below: (i) Better crops (drought and heat resistant),
(ii) recombinant vaccines (i.e., hepatitis B), (iii) prevention of sickle cell
anaemia, (iv) prevention of cystic fibrosis, (v) production of clotting factors, (vi) production of insulin, (vii) plants that produce their own insecticides.
More Explanatory Information on (ITS)1 and (ITS)2
Internal transcribed spacer (ITS) is the spacer DNA situated between
the small sub-unit ribosomal RNA (rRNA) and large subunit rRNA genes
in the chromosome or the corresponding transcribed region in the polycistronic rRNA processor transcript.
Table 8.7 Thermodynamic parameters for DNA duplex formation in 4 MNaCl and 4 M choline dihydrogen phosphate
(Choline dhP).
Medium
Nucleotide
ΔG ◦ /kJ mol−1 ΔH ◦ /kJ mol−1 TΔS◦ /kJ mol−1 Tm (◦ C )
4 M NaCl
Oligonucleotide with 9 chains
−35.1
−195.0
−159.8
38.6
(0 DN 9)
4 M NaCl
Oligonucleotide with 10 chains
−53.4
−228.8
−175.7
63.0
(0 DN 10)
Choline DhP 0 DN 9
−42.2
−279.5
−237.2
43.6
Choline dhP 0 DN 10
−35.1
−200.0
−164.8
38.2
Nucleosides and Nucleotides 177
178
Biophysical Chemistry
Details of ITS-1 and ITS-2 Terms
In bacteria and archaea, there is a single ITS located between the 16S and
23S rRNA (ribosomal RNA, S = Svedberg unit = 10−13 sec). In contrast
there are two ITS’s in eukaryotes: ITSI is located between 18S and 5.8S
rRNA genes, while ITS2 is between 5.8S and 28S rRNA genes. ITSI corresponds to the ITS in bacteria and archaea while ITS2 originated as an
insertion that interrupted the ancestral 23S rRNA gene.
Table 8.8 Stability constant data of the complexes formed between the
nucleotides and polyamines in aqueous solution at 25◦ C.
Nucleotide
AMP
Polyamine (en)
(r = 2)
2.61
log (Kr)
CdV
ptr
(r = 2) (r = 2)
2.22
2.24
Spd
(r = 2)
(r = 3)
2.52
3.04
UMP
(r = 2)
2.64
(r = 2)
2.04
(r = 2)
2.1
(r = 2)
(r = 3)
3.13
3.17
IMP
(r = 2)
2.73
(r = 2)
2.31
(r = 2)
2.3
(r = 2)
(r = 3)
3.54
3.51
GMP
(r = 2)
2.65
(r = 2)
1.95
(r = 2)
1.61
(r = 2)
(r = 3)
3.09
3.24
Sper
(r = 2)
(r = 3)
(r = 4)
2.63
3.44
4.09
(r = 2)
(r = 3)
(r = 4)
2.67
3.19
3.76
(r = 2)
(r = 3)
(r = 4)
3.19
3.21
4.04
(r = 2)
(r = 3)
(r = 4)
2.70
3.01
4.04
Nucleosides and Nucleotides 179
Questions
(1) Which of the following statements is true of Duplex DNA
(a) It has no relation to the genetic material.
(b) It does not convey any information about the bonding of the
nucleotides in DNA.
(c) It is another name for double stranded DNA.
(d) Cell division does not require participation of DNA.
(2) Indicate the correct answer:
(a) Ribonucleic acid contains the nitrogenous bases adenine, guanine
cytosine and uracil.
(b) Ribonucleic acid contains adenine, six membered sugar, thymine
and uracil.
(c) Ribonucleic and deoxyribonucleic acids are nucleosides.
(d) There is no relation between the order of presence of nitrogenous
bases and genetic code.
(3) The pKa ’s of adenosine, guanosine and cytosine are 3.50, 1.60 and 4.20
respectively. Hence, their acidic strength lies in the order
(a)
(b)
(c)
(d)
adenosine > guanosine > cytosine
guanosine > adenosine > cytosine
cytosine > guanosine > adenosine
cytosine > adenosine > guanosine
(4) What are the essential components of ribonucleic acid (RNA). Give the
structure of a RNA fragment?
(5) Draw the helical structure of DNA and indicate the positions of amines
and the sugar phosphate backbone.
(6) State the compositions of a nucleoside and nucleotide and indicate
their medical significance.
(7) Write down the dissociation of adenosine and guanosine acids. If their
pKa ’s are 3.50 and 1.60, what are their percent dissociations values at
0.1 M.
180
Biophysical Chemistry
(8) Estimate the vertical electron affinity of adenine using the following
data:
(a) Standard enthalpy of formation of the neutral form = 1.0042 eV
(b) Standard enthalpy of formation of the anion radical = 1.5607 eV
(c) Total tautomeric energy = 1.3034 eV
9
Enzymes
9.1
Introduction
Enzymes are mostly proteins that accelerate the reaction rates. They are
vital for life and execute diverse functions in the body such as: (i) aiding
the digestion and (ii) assist metabolic processes. Enzymes are made from
amino acids. An enzyme is formed by stringing together about 100 to 1000
amino acids in a specific and unique order. This chain of amino acids folds
into a unique shape.
9.2
Different Types of Enzymes
Three different kinds of enzymes are known
(i) Amylase and carbohydrase enzymes: They break down starch into
sugar.
(ii) Protease enzymes: They break down proteins into amino acids.
(iii) Lipase enzymes: They break down lipids into fatty acids and
glycerol.
9.3
Nature of Enzyme Action
Some enzymes break large molecules into smaller ones which are more
easily absorbed by the body. Other enzymes bind two molecules together
to form a new molecule. Enzymes are very selective catalysts. They react
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_9
181
182
Biophysical Chemistry
Figure 9.1 Schematic depiction of enzyme - substrate complex.
with compounds known as substrates. The substrates bind to a region on
the enzyme referred as active site. Figure 9.1 depicts the enzyme-substrate
complex, schematically. The energy diagram of the reaction may be represented as shown below:
Figure 9.2 Energy diagram with and without the presence of enzymes.
It is seen from Figure 9.2 that the activation energy of the reaction gets
lowered from ΔE to ΔE thereby enhancing the reaction rate.
9.3.1
Examples of Digestive Enzymes
(i) Amylase is produced in the mouth and helps in breaking down starch
into smaller sugar molecules. (ii) Pepsin is produced in the stomach and
helps to break down proteins in the food. (iii) Trypsin, lipase, DNA and
RNA are produced in pancreas. (iv) Lactose, found in mammalian milk,
Enzymes 183
breaks down the sugar lactose. Nature of enzyme action: Applying chemical kinetic principles, a chemical reaction may be expressed as occurring
according to the scheme.
X+Y
Reactants
. . Y ∗
X . −→
Transition State
Z + Z
Products
The quantitative measurement of the relation between rate and substrate concentration in an enzyme catalyzed reaction is due to Michaelis
and Menten. According to them, an enzyme catalyzed reaction involves
the formation of an enzyme-substrate complex in an equilibrium step which
then decomposes to give the enzyme and products. The reaction scheme
may be represented as
k1
k2
BG ES FGGGGGGGB
E + S FGGGGGG
GGGGG
GGGGGGG E + P
k −1
k −2
(9.1)
where k’s represent the rate constants of the appropriate steps. After the
enzyme and substrate are brought together, the concentration of E-S complex builds up under condition when “S” is in large excess and k1 >> k2
i.e., under steady state conditions. In this case, the rate of decomposition
of E-S complex balances the rate of its formation. Thus,
Rate of formation of [ES] = Rate of decomposition of [ES]
k1 [ E][S] + k −2 [ E][ P] = k −1 [ ES] + k2 [ ES]
(9.2)
(9.3)
From equation (9.3),
[ E]{k1 [S] + k −2 [ P]} = [ ES]{k −1 + k2 }
(9.4)
[ ES]
k [ S ] + k −2 [ P ]
k1 [S]
k [ P]
= 1
=
+ −2
[ E]
k −1 + k 2
k −1 + k 2
k −1 + k 2
(9.5)
To simplify equation (9.5), the initial stage of reaction can be determined
when the concentration of P will be small. In this case, the rate of formation
of ES from E and S will be small. Thus, the second term of equation (9.5)
can be neglected, i.e.,
k −2 [ P ]
=0
(9.6)
k −1 + k 2
Thus
[ ES]
k1 [S]
=
[ E]
k −1 + k 2
(9.7)
184
Biophysical Chemistry
By defining
k −1 + k 2
= Km
k1
(9.8)
[ E]
Km
=
[ ES ]
[S]
(9.9)
eqn. (9.8) may be written as
The total enzyme concentration,
Et = [ E] + [ ES]
[ E] = [ Et ] − [ ES]
or
(9.10)
Substituting equation (9.10) in equation (9.9), we get
[ Et ] − [ ES]
Km
=
[ ES]
[S]
(9.11)
[ Et ]
Km
−1 =
[ ES]
[S]
(9.12)
or
The maximum initial velocity, Vmax , is obtained when the total enzyme, Et ,
is completely complexed with saturating amounts of S or
Vmax = k [ Et ]
(9.13)
where k is a rate constant for this reaction. Also, the initial velocity of the
reaction (V ) is given by
V = k [ ES]
(9.14)
at a given concentration of S. Thus,
[ Et ]
Vmax
=
V
[ ES]
(9.15)
Comparing equations (9.12) and (9.15), it is seen that
Vmax
Km
−1 =
V
[S]
or
Km
Vmax
−1 =
+1
V
[S]
or
V
Km
[S]
+1
=
Vmax
Km +[S]
[S]
=
Vmax [S]
Km + [ S ]
(9.16)
(9.17)
Enzymes 185
Km of equation (9.8) is known as Michaelis–Menten constant. Some conditions may be considered which give interesting results for equation (9.17).
(i) when [S] is very large, Km (in equation 9.17) becomes negligible. Then
V = Vmax
(9.18)
In such a case, it is seen that V is independent of [S] or it becomes a zero
order reaction.
(ii) when V = Vmax /2, equation (9.17) may be written as
Vmax [S]
Vmax
=
2
Km + [ S ]
or
Km + [ S ] =
Vmax [S] × 2
= 2[ S ]
Vmax
or
Km = [ S ]
(9.19)
It is to be noted that under these conditions, the dimensions of Km is mols/
liter. If Km >> [S]
Vmax [S]
V=
(9.20)
Km
Now V depends on concentration of [S] as a first order dependence. Graphically, the above conclusions may be represented as in Figure 9.3.
Figure 9.3 Dependence of the reaction rate on concentrations of (a)
enzyme and (b) substrate.
In the above diagram, it is assumed that the concentration of enzyme
is constant and the concentration of substrate is increasing.
9.3.2
Significance of Km
The rate limiting steps in a biochemical reaction can be predicted by using
Km values which is similar to suggesting rate determining steps of ordinary
chemical reactions.
186
Biophysical Chemistry
For example, consider
In the above scheme, L → P, at low concentrations of L (< 10−4 M
lower), the rate L → M will be very low and controls the overall change
L → P. It may be mentioned that Km may be defined as the equilibrium
constant of an enzyme-catalysed reaction because
k −1
ES FGGGGGGGB
GGGGGGG E + S
k1
or
Ks =
k
[ E][S]
= −1
[ ES]
k1
(9.22)
and
k −1 + k 2
k1
Thus Km >> Ks . It is to be noted that Km depends on ionic strength and
temperature.
Km =
Lineweaver-Burke Plot
Taking reciprocal of equation (9.17), it follows that
1
Km
1
1
=
+
V
Vmax [S]
Vmax
From the slope and intercept values, Km and Vmax can be evaluated.
Figure 9.4 Lineweaver-Burke plot.
(9.23)
Table 9.1 Kinetic data on some reactions catalysed by enzymes (or inhibited in presence of inhibitors).
Substrate
Enzyme
Km (μmol/L) Ki (μmol−1 ) Inhibitor
Ribulose diphosphate
Ribulose diphosphate
1.2 × 10−4
4.2 × 10−3
Phosphate (C)
carboxylase (spinach)
Bicarbonate ion (HCO3− )
Ribulose diphosphate
2.2 × 10−2
9.5 × 10−3
3-phosphoglyceric acid (C)
carboxylase (spinach)
Fructose 1, 6 diphosphate Fructose 1, 6 diphosphate
3 × 10−4
2 × 10−4
L-Sorbose-1 Phosphate (C)
aldolase (yeast)
Ethanol
Alcohol dehydrogenase
1.3 × 10−2
6.7 × 10−4
Acetaldehyde (NC)
(yeast)
Succinate ion
Succinate dehydrogenase
1.3 × 10−3
4.1 × 10−5
Malonate (C)
(bovine heart)
D-glyceraldehyde 3
Triose phosphate
9.0 × 10−3
3.0 × 10−6
1, 3-diphosphoglycerate (C)
phosphate
de-hydrogenase (rabbit
muscle)
Glucose-6-phosphate
Glucose-6-phosphatase (rat
4.2 × 10−4
6 × 10−3
Citrate (C)
liver)
(C) = Competitive inhibitor; (NC) = Non-competitive inhibitor
Enzymes 187
188
9.4
Biophysical Chemistry
Michaelis-Menten Mechanism
Two types of mechanisms have been suggested: (i) ordered and (ii) random. In the ordered type, it is suggested that the substrates must be added
to enzyme before any products are released. The sequence of release of
products is also precise. In the random mechanism, the substrates add
to the enzyme and the products are released randomly. A few reaction
schemes under the above categories are given below.
Ordered reaction mechanism: Consider the reaction
(In equation 9.24, E = Enzyme, L, M = Substrates, X, Y = Products).
The scheme (9.24) states that E first forms a complex with L and only
then M can form the complex ELM. After catalysis, first X and then Y are
released (in that order). The reaction,
CH3 CH2 OH + NAD+
alcohol
+
(9.25)
FGGGGGGGGGGGB
GGGGGGGGGGG CH3 CHO + NADH + H
dehydrogenase
is a typical example of scheme. The kinetic analysis of reaction (9.25) may
be given as
An example of a reaction following random mechanism is
An example of such a mechanism is
Ping-Pong mechanism: A typical reaction sequence of this type is
Enzymes 189
The enzyme complexes formed in this case are EL, FX, FM and EY, with L
being converted to X and then M to Y, F being designated as a modified
enzyme (of X). F combines with M with a subsequent transfer of X to M
so as to form Y, the product with simultaneous regeneration of Y. The
reaction
Acetyl CoA + ATP + HCO3− −→ Malonyl CoA + ADP + Pi
(9.30)
is an example of the above scheme.
9.5
Effect of Temperature on
Enzyme-catalysed Reactions
Consider a chemical reaction with an activation energy of 58.5 kJmol−1 and
an enzyme catalysed reaction with an activation energy of 25.0 kJmol−1 .
By using Arrhenius equation, it is possible to calculate by what factor the
enzyme catalysed reaction proceeds faster vis a vis the chemical reaction.
For the chemical reaction
log k c = log A −
58.5
58.5
= log A −
= log A − 23.6
2.48
8.314 × 10−3 × 298
For the enzyme catalysed reaction,
25.0
8.314 × 0.001 × 298
25.0
= log A −
= log A − 10.1
2.48
log k e − log k c = −10.1 + 23.6 = 13.5
log k c = log A −
or
log
ke
= 13.5 or
kc
ke
= 1013.5
kc
It is seen that the enzyme catalysed reaction is 1013.5 faster than the chemical reaction.
Although it is possible to increase the rate of a reaction by raising
the temperature, this is not favourable in the case of a reaction in the cell
because the protein in the enzyme may undergo denaturation making the
enzyme lose its activity. One must choose an optimum temperature which
takes into consideration the increasing rate with temperature and the
decreasing activity of the enzyme at higher temperature.
190
Biophysical Chemistry
Effect of pH
Since the change in pH profoundly affects the ionic character of amino
and carboxyl groups in proteins, they markedly affect the catalytic sites.
Apart from the ionic effects, low or high pH may cause denaturation and
the enzyme loses its activity. It may also be noted that many substrates
are ionic in nature (NAD+ , amino acids) and the active site of an enzyme
may require specific ionic species for optimal activity. The effect of pH on
enzyme catalysed reactions may be shown in the following diagram.
Figure 9.5 Variation of the rate of enzyme-catalysed reaction with pH.
9.6
Specificity of an Enzyme
An enzyme will select only specific compounds with which it can combine. This is necessitated by the requirements such as the conformation of
the complex protein in the enzyme, the uniqueness of its active site and
the structure of the substrate. An enzyme often exhibits group specificity
in a group of substrates. Another aspect of enzyme specificity is its stereospecificity towards substrates i.e., an enzyme may prefer to react with an
L- or D-isomer. For example,
D-amino acids
O2
α-keto acids + NH3 + H2 O (9.31)
FGGGGGGB
GGGGGG
D-amino acid oxidase
A similar reaction for L-amino acids may be applied using L-amino acid
oxidase.
alanine
BG D-alanine
L-alanine FGGGGGGGGGGGG
(9.32)
GGGGGGGGGGG
racemase
Enzymes 9.7
191
Classification of Enzymes
There are six classes of enzymes: (i) Oxidoreductase (ii) Transferases, (iii)
Hydrolases, (iv) Lyases, (v) Isomerases, and (vi) Ligases. A brief account
of these is given below.
(i) Oxidoreductases: These are concerned with biological oxidation
reduction reactions. Respiration and fermentation processes are thus
included in this category. Also, dehydrogenases, oxidases and peroxydases (which use H2 O2 as oxidant) are part of this category.
Hydrolysates, and oxygenases which introduce −OH groups and
molecular O2 (in place of a double bond) are also included.
(ii) Transferases: These enzymes catalyse the transfer of one carbon
group (example: methyl group), aldehydic or ketonic groups,
P-containing and S-containing groups also.
(iii) Hydrolases: Examples of this class are phosphatases, peptidases and
glycosidases.
(iv) Lyases: This class includes aldolases, dehydratases, decarboxylases.
They remove groups from their substrates leaving or adding double
bonds.
(v) Isomerases: Some enzymes in this class are cis-trans isomerases, racemases, intramolecular oxidoreductases and intra molecular reductases.
(vi) Ligases: These enzymes catalyse the joining of two molecules coupled with the breakdown of a pyrophosphate bond in ATP or other
similar PO34− groups.
Conditions (to be fulfilled) for Specific Interactions
Between Enzyme and Substrate
Three conditions must be fulfilled for a reaction between an enzyme and
substrate: (i) The substrate must be associated with the enzyme in a a
specific orientation at least at three sites. (ii) The reactivities of the three
enzymic sites must be different. (iii) The compound may have two groups
(x1 and x2 ) affected by the enzyme and two different groups (y1 and y2 )
which are associated with a central carbon atom C. For example, in the
synthesis of citrate from acetyl CoA and oxaloacetic acid only x1 is derived
from CoA and x2 , y1 and y2 come from oxaloacetate.
192
Biophysical Chemistry
However, citrate in solution is optically inactive since it has a plane of symmetry. The following diagram shows the positioning of a substrate to its
active site on the surface of the enzyme.
Figure 9.6 Positioning of a substrate to an active site.
9.8
Inhibitors of Enzymes
Inhibitors are compounds which have the ability to combine with some
enzymes reversibly or irreversibly and thereby inhibit the catalysis by that
enzyme. There are a variety of compounds like drugs, antibiotics and
metabolites which can act as inhibitors. An irreversible inhibitor forms
a covalent bond in the enzyme often an amino acid residue which may
be associated with the catalytic activity of the enzyme. Some enzyme
inhibitors physically block the active site.
Scheme depicting non-competitive inhibition
Enzymes 193
In a kinetic sense, the rate of the enzyme-catalyzed reaction is lowered in
proportion to the concentration of the inhibitor. This is known as noncompetitive inhibition.
A specific case is the reaction of the inhibitor, iodoacetate, which reacts
with the sulfhydryl group of an enzyme, triosephosphate dehydrogenase
according to
E − SH + ICH2 COOH → E − S − CH2 COOH + HI
(9.35)
Another type of irreversible inhibition is that a latent inhibitor becomes an
active inhibitor by binding to the active site of the enzyme. This newly
formed inhibitor reacts chemically with the enzyme leading to its irreversible inhibition. For example, the inhibition of D-3-hydroxyl decanoyl
ACP dehydrase (of E-coli) by the latent inhibitor D-3-decenoyl-N-acetyl
cystamine to form an active inhibitor proceeds according to the following
scheme.
(9.36)
9.9
Reversible Inhibition
This type of inhibition involves establishment of equilibrium between the
enzyme and inhibitor, the equilibrium constant, Ki , being a measure of the
inhibitor to the enzyme. There are three types of reversible inhibition: (i)
Competitive inhibition (CI), (ii) Non-competitive inhibition (NC), and (iii)
Uncompetitive inhibition (UC).
In the first category (CI), the inhibitor and the substrate compete for
the same active site on the enzyme which may be represented as
194
Biophysical Chemistry
Scheme showing competitive inhibition
here, complexes ES and EInh are formed but the complex EInh.S. It may
be noted that high concentrations of “S” will overcome the inhibition by
causing the reaction to shift to the right. In non-competitive inhibition
(NC), compounds that bind reversibly with either the enzyme or E-S complex are designated as non-competitive inhibitors. The following scheme
depicts this reaction.
In this case, the inhibitor can combine with ES while S can combine with
E.Inh to form E.Inh.S in both cases, the value of Km is not altered in this
case since the inhibitor binding site is not identical to the active sites nor
does it modify the latter directly.
9.10
Uncompetitive Inhibition
In the case of uncompetitive inhibition (UC) compounds that combine only
with ES complex but not with the free enzyme are called UC inhibitors.
The reaction scheme in this case is
Scheme showing uncompetitive inhibition
Enzymes 195
Comparing schemes (9.38) and (9.39), it is seen that a part of uncompetitive inhibition (UC) is always a component of non-competitive inhibition as E.Inh.S is formed in both cases. The kinetic expressions in all
three cases (CI; NC; UC) may be worked out in a similar way to MichaelisMenten derivation and the results are summarized below.
Table 9.2 Kinetic expressions for different inhibition types.
Inhibition
type
No inhibitor
CI
NC
UC
Equation
V = Vmax [S]/
(Km + [S])
V = Vmax × [S]/
{(Km (1 + 1/Ki ))} + [S]
V = Vmax × [S]/
(Km + [S])(1 + 1/Ki )
V = Vmax × [S]/
(Km ) + [S](1 + 1/Ki )
Vmax
-
Km
-
No change
Increases
Decreases
No change
Decreases
No change
The variation of rate of reaction (V ) with substrate concentration in all
the above cases is shown in Figure 9.7.
Figure 9.7 Graph showing the variation of reaction rate with substrate
concentration.
In the case of NC (curve b), there is no change in Km (see the curves “no
inhibitor and NC”) but Vmax shifts to the top in the case of NC. In the case
but there is no change in V .
of curve for (a), there is a change in Km to Km
m
196
Biophysical Chemistry
Figure 9.8 Graph showing the variation of reaction rate: (i) without
inhibitor; (ii) competitive and (iii) non-competitive inhibitor.
and V
In this case Km decreases to Km
max decreases to Vmax . A typical
reciprocal plot of 1/V vs. 1/[S] in the case of CI is shown below:
Figure 9.9 Variation of the reciprocal velocity with reciprocal substrate
concentration.
Negative Feed Back Inhibition
This inhibition is caused by interaction of a product with enzyme early in
the sequence of its formation. This is similar to the type of competitive
inhibition.
Enzymes 9.11
197
Allosteric Enzymes
They are enzymes that change their conformational isomers upon binding an effector. Every enzyme contains an active site where it catalyses its
specific reactions.
However, allosteric enzymes contain a second site called allosteric site.
This site, through its binding of a non-substrate molecule influences the
activity of the enzyme. Such enzymes play crucial role in many fundamental biological processes like cell signaling and regulation of metabolism.
Kinetic Aspects of Allosteric Enzymes
A normal hyperbolic curve in a velocity [S] plot is obtained when a
molecule of substrate has no effect on the intrinsic dissociation constant
of vacant sites. However, if the binding of one substrate molecule induces
structural changes that result in altered affinities for vacant sites, the velocity curve does not follow.
Michaelis-Menten Kinetics. Allosteric enzymes yield a sigmoid type
of velocity [S] curves. The binding of one substrate facilitates the binding
of he next substrate (or effector) molecule by increasing the affinities of
the vacant binding sites. This phenomenon is called co-operative binding.
The following diagram shows a typical sigmoid curve vis a vis a normal
hyperbolic velocity curve.
Figure 9.10 Dependence of the maximum velocity on the substrate concentration.
198
Biophysical Chemistry
It is to be noted that between [S] = 0 and [S] = 3.0, the hyperbolic
curve decelerates but still rises to 0.75 Vmax . In the same limits, the sigmoid
curve acelerates but reaches only about 0.1 Vmax . However, the sigmoidal
curve increases from 0.1 Vmax to 0.75 Vmax with only an additional 2.3 fold
increase in [S]. But for the same increase the hyperbolic curve requires 27
fold increase in [S].
Cofactors in enzyme catalysis: Many enzymes require an additional
component before it can carry out its catalytic functions. The cofactors
may be divided into three groups: (i) Prosthetic groups, (ii) Co-enzymes,
and (iii) metal activators.
A prosthetic group is a cofactor firmly bound to enzyme protein. For
example, the porphyrin moiety of the hemoprotein peroxidase is a prosthetic group. It may be represented as
A co-enzyme is a small organic molecules stable to heat, which readily
dissociates off an enzyme protein and can be dialysed away from the protein. Examples of Co-enzymes are NAD+ , NADP+ , thiamine phosphate.
The function of a co-enzyme to interact with different co-enzymes is shown
below:
(9.41)
The metal activator group is necessitated by the requirement of a large
number of enzymes for mono or divalent cations like K+ , Mn2+ , Mg2+ ,
Ca2+ and Zn2+ ions. These ions may be loosely or firmly bound to an
enzyme protein by chelation with phenolics phosphoryl or carboxyl
groups.
Enzyme (Zymogen)
Trypsinogen
Chymotrypsinogen A
Pepsinogen
Procarboxypeptidase A
Activating agent
Enterokinase
Chymotrypsin
H+ or pepsin
Trypsin
Active enzyme
Trypsin
α-chymotrypsin
Pepsin
Carboxypeptidase A
Inactive peptide
+ hexapeptide
+ amino acid residues
+ fragments
+ fragments
Table 9.3 Conversion of zymogens to active enzymes.
Enzymes 199
200
Biophysical Chemistry
Enzymes as proteins: There are three groups of this class: (1) The
monomeric enzymes, (2) Oligomeric enzymes, (3) Multi-enzyme complexes.
(1) First category: They are enzymes with only one polypeptide chain in
which the active site resides. Examples of this class include Lysozyme
(Egg white: Molar mass = 14,600; amino acid residues = 129) Trypsin
(Molar mass = 23,800, amino acid residues = 223); Carboxypeptidase A
(Molar mass = 34,600, amino acid residues = 307).
(2) Second category: The enzymes contain 2 to 60 more sub-units firmly
associated to form catalytically active enzyme protein.
(3) Third category: In this case, a number of enzymes engaged in a sequential series of reactions in the conversion of substrate to product are
strongly associated. Attempts to dissociate these enzyme complexes
leads to deactivation. This class contains only a small number of
enzymes and they catalyse hydrolytic reactions. They are highly reactive proteases and cannot be biosynthesized in the active form in the
cell as the cell is damaged in this process. They are, therefore, synthesized in an inactive form known as “Zymogens” and transported out
of the cell into the digestive tract where they are again converted into
the active form. The enzymes, chymotrypsin, trypsin, and elastase are
called serine proteases since their catalytic sites contain highly reactive
serine residue.
9.12
Oligomeric Enzymes
These enzymes include proteins with molar masses ranging from 35,000
to a few millions. They contain a number of polypeptide combinations
and form catalytically active enzymes. Since these enzymes contain multipolypeptide structures, they exhibit properties necessary for proper
functioning of metabolic activities. Some important aspects of the enzymes
are given below:
(a) The dissociation of many oligomeric enzymes into sub-units results in
complete loss of their activity.
(b) The association of many sub-units may yield an active site involving
amino acid residues contributed by different components. Thus, the
widely separated amino acid residues in ribonuclease constitute the
active sites of its monomeric enzyme structure.
(c) The association of two sub-units with different individual enzyme
activity often yields appropriate enzyme reaction (Example: Tryptophan synthetase system).
Enzymes 201
(d) In a number of enzymes, a sub-unit serves as a specific carrier of a
substrate. For example, the two sub-units, biotin carboxylase and transcarboxylase in the enzyme acetyl CoA carboxylase of E.coli catalyse
the following reactions.
(i) Biotin carboxylase carrier protein
(9.42)
(ii) Catalysis using transcarboxylase
(9.43)
(e) Many oligomeric enzymes are regulatory in nature with regulatory
sites and catalytic sites residing on separate sub-units.
(f) An assembly of enzymes would allow highly efficient movement of
intermediates between reactants and products (than individual
enzymes).
9.12.1
Glycolytic Enzymes
These enzymes convert glucose-6-phosphate and nicotinamide adenine
dinucleotides (NAD+ ) to pyruvate and NADH by producing two
Table 9.4 Data on glycolic enzymes.
Enzyme
Phosphorylase A
Phosphofructokinase
Fructose diphosphatase
Glyceraldehyde-3Phosphatase dehydrogenase
Creatine kinase
Lactic dehydrogenase
Pyruvic kinase
Number of
of sub units
4
2
2
2
2
Molar
mass
92,500
78,000
29,000
37,000
72,000
Total
molar mass
3,70,000
1,90,000
1,30,000
2
4
4
40,000
35,000
57,200
80,000
1,50,000
2,37,000
1,40,000
202
Biophysical Chemistry
molecules of ATP according to
C6 H12 O6 + 2NAD+ + 2ADP + 2P → 2CH3 C
= OCOOH (Pyruvic acid) + 2ATP + 2NADH + 2H+
(9.44)
Each enzyme in this group is not a simple monomeric type of protein but
oligomeric type consisting of varying number of sub-units.
9.13
Isoenzymes
An enzyme which has multiple molecular forms in the same organism catalyzing the same reaction is known as isoenzyme. An example of this
class is lactic dehydrogenase (LDH) which occurs in five forms in organs of
many vertebrates. One type predominates in the heart and known as heart
LDH. The other one is characteristic of skeletal muscles and is referred as
muscle LDH. While the heart LDH consists of four identical monomers,
called H sub-units, muscle LDH consists of four identical M units, each
sub-unit being enzymatically inactive. Both types of sub-units have different amino acid compositions and different immunological properties. The
two sub-units are believed to be produced by different genes.
9.14
Bifunctional Oligomeric Enzymes
A typical enzyme in this category is Tryptophan synthetase of E.coli. It
consists of two proteins designated as A and B. The complete tryptophan
synthetase consists of two A proteins and one B protein designated as α2 β 2 .
The α2 β 2 protein catalyses the reaction.
α2 β 2
Indole-3-glycerophosphate + L-SerineGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGA
in presence of pyridoxal phosphate
L-Tryptophan + glyceraldehyde-3-phosphate
(9.45)
Individually, the α and β proteins catalyse the reactions.
α
BG Indole+glyceraldehyde-3-phosphate
Indole-3-glycerophosphate FGGGGG
GGGG
(9.46)
Enzymes β2
Indole + L-SerineGGGGGGGGGGGGGGGGGA L-Tryptophan
pyridoxylsulfate
9.15
203
(9.47)
Multienzyme Complexes
In this case, a number of complexes are described which consist of an organized mosaic of enzymes in which each component enzyme is so located
as to allow effective use of individual reactions catalysed by them. For
example, the E-coli pyruvic acid dehydrogenase complex catalyses the oxidation of pyruvic acid to acetyl CoA and CO2 . The total complex (Mol.
Mass 40 × 106 ) consists of three separate catalytic active components EI, EII
and EIII named pyruvic dehydrogenase, dihydrolipoyl transacetylase and
dihydrolipoyl dehydrogenase. The manner in which this complex enzyme
acts is shown below:
Scheme depicting the catalytic activity of components E1 , E2 and E3
An even more complex multienzyme system is the fatty acid synthetase
complex which occurs as a tightly knit group responsible for the conversion of acetyl CoA and malonyl CoA to palmitic acid. These complexes are
found in animal and yeast cells. In bacteria and plants, these enzymes are
completely separable and are easily purified.
For the same reaction above, in presence of enzyme cytidine deaminase (from E.Coli)
58.6
3.8
−42.2
62.3
56.1
Table 9.5 Activation parameters for the reactions of some enzymes.
ΔG ΔH TΔS
(kJ mol−1 ) (kJ mol−1 ) (kJ mol−1 )
127.1
92.5
−34.7
kcat = 300 s−1
k (rate constant
for hydrolysis)
3 × 10−10 s−1
Reactions
204
Biophysical Chemistry
Table 9.5 (Continued.)
ΔG’
(kJ mol−1 )
ΔH’
(kJ mol−1 )
113.4
ΔG0 (binding) of cytidine with the enzyme cytidine deaminase = −22.0 kJ mo1−1
TΔS◦ = −31.8 kJ mol−1 (at 298 K), K for binding = e−ΔG0 /RT = 9.12 × 103
Reactions
TΔS’
(kJ mol−1 )
−3.4
k (rate const.
for hydrolysis)
Enzymes 205
206
9.16
Biophysical Chemistry
Modification of the Specificity of an
Oligomeric Enzyme
The enzyme lactose synthetase catalyses the synthesis of lactose by the
reaction
UDP-galactose + glucose UDP + Lactose
(9.48)
in the mammary gland. This enzyme (isolated from raw milk) is separable
into proteins A and B. None of them catalyse reaction (9.48). Protein A,
however, catalyses the reaction
UDP galactose + N - acetylchol glucose amine
←→ N-acetyl lactosamine + UDP
(9.49)
The addition of protein B inhibits the reaction (9.49) and in presence of
glucose allows catalysis of reaction (9.48). Thus protein B modifies the
substrate specificity of protein A by combining to form lactose synthetase
complex. Protein B is alpha-lactalbumin found only in mammary glands
while protein A is distributed widely in animal tissues. This leads to the
formation of lactose in mammary glands.
9.17
Measurement of Enzymatic Activity of Lactose
Dehydrogenase (LDH) obtained from
Different Organisms
LDH catalyses the interconversion of pyruvate and lactate with concomitant interconversion of NADH and NAD+ . The reaction scheme is
308
303
303
Cod
Rabbit
Lobster
D-glyceraldehyde-3-phosphate
dehydrogenase
D-glyceraldehyde-3-phosphate
dehydrogenase
Muscle glycogen phosphorylase-b
Muscle glycogen phosphorylase-b
308
308
308
Rabbit
Tuna
Rabbit
Muscle type LDH
Temp (K)
Animal
Enzyme
60
70.8
225
1080
4500
180
Vmax (μ mol
substrate)
88.7
66.5
60.7
Ea
54.8
39.1
79.5
Table 9.6 Enzymatic activity of lactose dehydrogenase.
63.6
63.2
61.9
86.2
64.0
58.1
74.5
3.4
−12.1
ΔS±
−10.5
−56.5
47.3
ΔG ±
52.3
36.8
62.3
ΔH ±
52.3
36.8
77.0
J mol−1 K−1
kJ mol−1
Enzymes 207
208
Biophysical Chemistry
The above data pertains to the following reaction
9.18
Turn Over Rates (T.O.R) of Some Enzymes
T.O.R is defined as the number of substrate molecules that can be converted to the product by a single molecule of enzyme (per unit time).
T.O.R is also given by
T.O.R = k cat =
Vmax
[ ET ]
where ET = total enzyme concentration.
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
Table 9.7 Turn over numbers for selected enzymes.
Enzyme
T.O.R (moles of product sec−1
mol enzyme−1 )
Carbonic anhydrase
6 × 105
Catalase
9.3 × 104
β-galactosidase
2.0 × 102
Chymotrypsin
100.0
Tyrosine
1.0
Staphylococcal nuclease
95.0
Cytidine deaminase
299.0
Triose phosphate isomerase
4300
Cyclophilin
13,000
Enzymes 209
Table 9.7 (Continued)
T.O.R (moles of product sec−1
mol enzyme−1 )
Ketosteroid isomerase
66,000
3-ketosteroid isomerase
2.8 × 105
Acetylcholinesterase
2.5 × 104
Penicillinase
2.0 × 103
Lactate dehydrogenase
1.0 × 103
DNA polymerase I
15
Tryptophan synthetase
2
Lysozyme
0.5
Enzyme
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
9.19
Immobilisation of Enzymes
Immobilised enzyme systems “fix” the enzyme in warm solution of agar,
which on cooling sets to a gel. This gel can be cut off into small pieces
and can be used to catalyse a reaction. The enzyme can be recovered after
the reaction and can be reused. Because of the advantage to recover them
again, they find extensive application in industry. Once a biocatalyst has
been immobilised, it can be put to use in a range of continuous flow reactors enabling a continuous supply of substrate that can be converted to
product.
Table 9.8 Some industrial processes using enzymes.
Industrial process
High fructose col syrup
Lactose hydrolysis
Aspartame production
L-aspartic acid production
Semisynthetic penicillin
production
Acrylamide production
Transesterification of food
oils
Enzyme used
Glucose isomerase
Lactase
Thermolysin
Aspartase
Penicillin acylase
Nitrile hydratase
Lipase
Rate of production
(in tons year−1 )
107
105
104
104
104
105
105
Alkaline proteases
Amino acylase
Amyloglucosidase
β-galactosidase
α-amylase
Glucose isomerase
Penicillin acylase
L-Asparaginase
Urokinase
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
Table 9.9 Illustrative examples of industrial catalysts.
Reaction
Source
Application
Protein digestion
Aspergillus niger
Coagulation of milk in cheese
Kluyveromyces lactis manufacture
Protein digestion
Bacillus species
Detergents and washing
powders
Hydrolysis of acylated
Aspergilus species
Production of L-amino acids
L-amino acids
Dextrin hydrolysis
Aspergillus species
Production of glucose
Lactose hydrolysis
Aspergillus species
Hydrolysis of lactose in milk
or whey
Starch hydrolysis
Bacillus species
Conversion of starch to glucose
or dextran
Conversion of glucose to
Spectromyces species High fructose syrup production
fructose
Penicillin sidechain
E.Coli
6-APA formation for production
cleavage
of semisynthetic pencillins
Enzymes as therapeutic agents
Removal of L-Asparagine
E.Coli
Cancer chemotherapy,
essential for tumor growth
especially for leukemia
Plasminogen activation
Human
Removal of fibrin clots
from blood stream
Name of enzyme
Acid proteases
210
Biophysical Chemistry
Glucose oxidase
Peroxidase
Urease
Luciferase
Lysozyme
Nucleases
DNA polymerases
(11)
(12)
(13)
(14)
(15)
(16)
(17)
Name of enzyme
Hydrolysis of 1,
4-glycosidic bonds
Hydrolysis of
phosphodiester bonds
DNA synthesis
Hydrolysis of urea to CO2
and NH3
Biduminescence
Thermus aquaticus
Various bacteria
Marine bacteria or
firefly
Egg white (Hen)
Jack bean
Table 9.9 (Continued)
Source
Enzymes as Analytical Reagents
Glucose oxidation
Aspergillus niger
Dye oxidation using H2 O2
Horse radish
Reaction
Bioluminescent assay involving
ATP
Disruption of mucopeptide
in bacterial cell walls
Enzymes used in genetic
manipulation to cut DNA
DNA amplification in
polymerase chain reaction
Detection of glucose in blood
Quantification of hormones and
antibodies
Urea quantification in blood
Application
Enzymes 211
212
Biophysical Chemistry
Table 9.10 Michaelis-Menten constants for some enzyme substrate reactions.
Enzyme
Substrate
Km (mM)
(a) Carbonic anhydrase
CO2
12
(b) Hexokinase
Glucose
0.15
Fructose
1.5
(c) β-galactosidase
Lactose
4
(d) Glutamate dehydrogenase NH4+
57
Glutamate
0.12
NAD+
0.025
NADH
0.018
(e) Aspartate amino
Aspartate
0.9
transferase
α-ketoglutarate
0.1
Oxaloacetate
0.04
Glutamate
4
(f)
Threonine deaminase
Threonine
5
(g) Pyruvate carboxylase
HCO3−
1.0
Pyruvate
0.4
ATP
0.06
(h) Penicillinase
Benzyl penicillin
0.05
(i)
Lysozyme
Hexa-N-acetyl glucosamine 0.006
Illustrative Calculation of Km
The estimation of Km can be demonstrated with the following examples:
Reaction of horse radish peroxidase with H2 O2
Keq =
k2
= 2 × 10−8
k1
k1 = 1.2 × 107 lmol−1 sec−1 ; k2 = 2 × 10−8 × 1.2 × 107 = 0.24 sec−1 ; k3 =
5.1 sec−1
Km =
k2 + k3
0.24 + 0.51
5.34
=
=
= 4.45 × 10−7
7
k1
1.2 × 10
1.2 × 107
It is essential to point out that other interpretations of Km also exist such as
the one given by Briggs and Haldane. According to them, Km represents
the ratio between the sum of the two unimolecular rate constants and the
bimolecular rate constant representing the formation of the enzyme substrate complex.
Enzymes 213
Questions
(1) The activation energy of a chemical reaction is 60 kJ mol−1 . An enzyme
employed as a catalyst for the same reaction has an activation energy
of 30 kJ mol−1 . Calculate the ratio of the rate constants. (Assume that
the frequency factor is the same in both cases). (The temperature may
be taken as 27◦ C).
Solution:
k che
Ae− Eche /RT
e− Eche /RT
=
=
k enz
Ae− Eenz /RT
e− Eenz /RT
or
k che
− Eche Eenz
=
+
k enz
RT
RT
−60
30
=
+
−
3
8.313 × 10 × 300 8.313 × 10−3 × 300
1
=
(−60 + 30)
2.494
= +0.4 × −30 = −12
−12
k
log che =
= −5.21
k enz
2.303
k
log che = 6.17 × 10−6
k enz
ln
(2) In an enzyme catalyzed reaction, a substrate was analyzed with time,
its initial concentration being 10−4 M. After 10 min of the reaction, 5 percent of the substrate reacted. Calculate the amount of product formed
after 30 min. (Assume a 1st order decomposition of substrate).
Solution:
a
2.303
log
t
a−x
10−4
2.303
log
=
−4
10
10−4 − 10100×5
k = 364 =
10−4
2.303
log −4
10
10 − 5 × 10−6
10−4
2.303
log
=
−
4
10
1 × 10 − (5 × 10−4 × 10−2 )
=
214
Biophysical Chemistry
10−4
23.3
log
10
(1 − 0.05) × 10−4
1
2.303
2.303
log
=
=
× 0.0223
10
0.95
10
= 0.00513 min.
k=
after 30 min,
10−4
2.303
log
0.00513 =
30
(a − x)
−4 0.00513 × 30
10
log
=
= 0.0668
a−x
2.303
−4 1 × 10−4
10
= 1.166
or ( a − x ) =
a−x
1.166
= 0.8576 × 10−4
amount reacted = 1 × 10−4 − 0.8576 × 10−4
= 0.1424 × 10−4
or product formed = 1.424 × 10−5 M
(3) Write the balanced equation for the combination of oxaloacetate (OAA)
and the acetyl group from acetyl coenzyme A (ACCOA), in the presence of H2 O, to form citrate ions, thiol coenzyme, and hydrogen ions.
Solution:
ACCOA0 + OAA2− + H2 O CIT3− + COAS− + 2H+
(4) Identify all the products in the hydrolysis of ATP given below.
ATP4− + H2 O product
Solution:
ATP4− + H2 O ADP3− + HPO24− + H+
(5) The membrane potential is 50 mV during the transport of K+ ions. If
the equilibrium potential is 59 mV and the current is −0, 5μA cm−2 ,
estimate the ionic conductance in S cm−2 .
VK+ =
RT
ln
F
+
Kout
+
Kin
Enzymes 215
Solution: Current
I = g(Δφ − VK+ )
−0.5 × 10
−6
A cm
−2
= g(0.05 − 0.059) V
Conductance
g = 5.56 × 10−5 S cm−2
(6) Write down the reaction between an enzyme and a substrate leading to
a product. Derive the equation relating the velocity of the reaction to
the Michaelis–Menten constant.
(7) Describe briefly the various modes of inhibition exhibited by certain
compounds on the activity of an enzyme.
(8) What are allosteric enzymes? Discuss their mode of reaction with a
substrate.
(9) Write the equation for the net flux in the following scheme assuming
Michaelis-Menten mechanism:
k +1
k +2
A + E FGGGGGGGB
GGGGGGG C FGGGGGGGB
GGGGGGG B + E
k −1
k −2
10
Co-Enzymes and Vitamins
10.1
Introduction
Co-enzymes are substances that enhance the action of enzymes. They are
small organic non-protein molecules that bind with a protein molecule
(apoenzyme) to form the active enzyme (holoenzyme). They can not by
themselves catalyse a reaction but they can help enzymes to do so. Coenzymes are a type of cofactors that temporarily bind to an enzyme to
change its configuration or shape.
Figure 10.1 Conversion of an apoenzyme to a holoenzyme.
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_10
217
218
Biophysical Chemistry
Once the enzyme is in an active form, it can build upon or break down
reactents to products.
Figure 10.2 Conversion of an active enzyme with substrate to yield an
enzyme with products.
Functioning of Co-enzymes
Co-enzymes bind temporarily to apoenzymes and detach themselves easily after a biochemical reaction takes place. Other types of cofactors,
known as “Prosthetic groups” which work as co-enzymes, are metal ions
bind more tightly to their apoenzymes through covalent bonding and cannot easily detach themselves from the enzyme. Once the cofactor (enzyme
or metal ion prosthetic group) and apoenzyme form the complex, it
becomes a holoenzyme (active form of the enzyme). Co-enzymes help
transfer of compounds between enzymes and also connect compounds to
the active site of enzyme.
10.1.1
Importance and Need for Co-enzymes
Co-enzymes are mainly responsible for the transfer of functional groups,
electrons, hydrogen atoms or ions and energy. Some enzymes won’t function in the absence of vitamins. Vitamin deficiencies arise when some vitamins are converted into co-enzymes. Some combinations of enzyme-coenzyme systems in presence of vitamins for effecting some functions are
given below.
10.1.2
Support for Proper Enzyme Function
Many diseases arise in the body due to nutritional deficiencies. Though
co-enzymes function at molecular level, co-enzymes and their vitamin precursors are very important for health. The vitamin precursors are found in
Table 10.1 Typical combinations of enzyme-co-enzyme systems in presence of vitamins for their effective functioning.
Co-enzyme
Enzyme
Vitamin
Function
Precursor
Methyl cobalamin (B-12)
Methionine synthase
B-12
Transfers methyl group
NAD (nicotinamide adenine
Malate dehydrogenase, pyruvate
B-6
Transfers electrons and H-atoms
dinucleotide)
dehydrogenase
FAD (Flavine adenine
D-lactate dehydrogenase
B-2
Transfers electrons and H-atoms
dinucleotide)
Co-enzyme Q (Ubiquinone)
Cytochrome C-oxido reductase
B-5
Transfers electrons and H-atoms
Biotin
Propionyl-CoA carboxylase
Biotin
Carries carboxyl groups
Co-Enzymes and Vitamins 219
220
Biophysical Chemistry
whole foods, plant foods, nuts, peanuts, beans etc. Considering the correct
cofactor form of vitamin B12 , it is necessary to check for methylcobalamin
as the body uses this enzyme to detoxify the tissues of homo cysteine to
convert into methionine. Adenosyl cobalamine is another active form of
B12 which is vital for the metabolism, proteins and fats. These two cobalamines are important for vascular and brain health.
10.2
Relation between Co-enzymes and Vitamins
Many enzymes are simple proteins consisting of one or more amino acid
chains. Other enzymes contain a non-protein component called a cofactor
that is necessary for the functioning of enzyme. There are two types of
cofactors: (i) inorganic ions like Zn++ , Cu2+ , and (ii) organic compounds
known as co-enzymes. Most co-enzymes are vitamins or derived from
vitamins.
10.3
Vitamins
10.3.1
Definition and Types
They are nutrients needed by the body to function and fight disease; types
of vitamins are: (i) Fat soluble vitamins and (ii) Water soluble vitamins.
The first category are stored in fat cells of the body where as the second
category are not stored in the body. They need to be replaced daily.
Vitamins are not a source of energy but help enzymes which generate
energy from nutrients such as carbohydrates and fats.
10.3.2
Some Specific Functions of Vitamins
1. Vit-A: Production of retinol which is used in the rods and cones of eyes
to sense light and prevent night blindness. Also, it is important for
healthy teeth, bones, skin and immune system.
2. Vit-B: Aid in energy production in the body, making of red blood cells,
making of new DNA cells, needed for healthy nerve and brain function,
and also needed for intestinal and cardiovascular health.
3. Vit-C: Is an antioxidant, helps prevents cell damage and reduces heart
disease and risk of certain cancers. It is a vital ingredient information of
collagen. Also it helps in faster wound healing.
Table 10.2 Water soluble vitamins along with their co-enzymes and their physiological functions.
Vitamin
Co-enzyme
Function of co-enzyme
Deficiency causes
Vit-B1 (Thiamine)
Thiamine Pyrophosphate
Decarboxylation reactions
Beriberi
Vit-B2 (Riboflavin)
Flavin mono nucleotide or Oxidation reduction reactions
—
Flavin adenine dinucleotide involving two H-atoms
Vit-B3 (Niacin)
NAD or NADP
Oxidation-reduction reactions
Pellegra
involving hydride ion
Vit-B6 (Pyridoxin)
Pyridoxyl phosphate
Many reactions including
—
transfer of amino groups
Vit-B12 (Cyano cobalamine) Methyl cobalamin or
Intramolecular rearrangement
Pernicious anaemia
deoxyadenosylcobalamin
reactions
Biotin
Biotin
Carboxylation reactions
—
Folic acid
Tetrahydrofolate
Carrier of one carbon units such Anaemia
as formyl group
Pantothenic acid
Co-enzyme-A
Carrier of acyl groups
—
Vit-C (Ascorbic acid)
None
Antioxidant, formation of
Scurvy
collagen proteins found in
tendons, ligaments and bone
Co-Enzymes and Vitamins 221
Table 10.3 Fat soluble vitamins and their physiological function.
Physiological function
Effect of deficiency
Formation of vision pigments and
Night blindness
differenciation of epithelial cells
Vit-D (Chole calciferol) Improves the body ability to absorb calcium Softening of bones (Osteomalacia), rickets
and phosphorus
in children
Vit-E (Tocopherol)
Fat soluble antioxidant
Damage to cell membranes
Vit-K (Phylloquinone)
Formation of prothrombin, a key enzyme in Longer time required for blood clotting
blood clotting process
Note: Vitamins C and E as well as provitamine β-carotene act as antioxidants in the body. Antioxidants prevent damage from free radicals. It is known that
such radicals form through metabolic reactions involving O2 but they can also form by environmental factors such as pollution and radiation. β-carotene is
known as a provitamin because it is converted to Vitamin-A in the body.
Vitamin
Vit-A (Retinol)
222
Biophysical Chemistry
Co-Enzymes and Vitamins 223
Table 10.4 Typical examples of fat-soluble vitamins and their source.
Vitamins Source of the vitamins
Vit-A
Orange coloured fruits, vegetables, dark leafy greens
like kale
Vit-D
Fortified milk, dairy products, cereal, sunshine is a
good source of this vitamin
Vit-E
Fortified cereals, green leafy vegetables, nuts and seeds
Vit-K
Dark green leafy vegetables, turnip or beet greens
Table 10.5 Water soluble Vitamins: Vitamins B-1 to B-7, B-9, B-12, Vitamin C and their source.
Vit-B-1 (Thiamin)
Whole gains, enriched grains, nuts
Vit-B-2 (Riboflavin)
Whole grains, enriched grains and dairy
products.
Vit-B-3 (Niacin)
Meat, fish, poultry, whole grains
Vit-B-5 (Pantothenic acid)
Meat, poultry, whole grains
Vit-B-6 (Pyridoxine)
Fortified cereals, soy products
Vit-B-7 (Biotin)
Meats and fruits
Vit-B-9 (Folic acid)
Leafy vegetables
Vit-B-12 (Cyanocobalamin) Fish, poultry, meat, dairy products
Vit-C
Citrus fruits like oranges, grape fruits,
red, yellow and green peppers
10.4
Biochemical Functioning of the
B-Vitamin Group
10.4.1
Niacin
The biochemically active form of this vitamin is nicotinamide whose structure is
It is widely distributed in plant and animal tissues. The co-enzyme forms
are nicotinamide nucleotides.
The nicotinamide nucleotides are
co-enzymes for the “dehydrogenases” enzymes that catalyse oxidationreduction reactions. For example, alcohol dehydrogenase, catalyses the
224
Biophysical Chemistry
oxidation of ethanol with simultaneous reduction of NAD+ according to
CH3 CH2 OH + NAD+ CH3 CHO + NADH + H+
(10.1)
The equilibrium constant K of the above reaction is 10−4 at pH = 7.0 and at
pH = 9.0 is 10−2 . The shift in the equilibrium constant towards right is due
to the replacement of H+ ions by OH− at higher values of pH.
The enzymes possess several functional roles. The dehydrogenases
that require NAD+ and NADP+ catalyse the oxidation of alcohols, aldehydes, α-amino acids and α-, β-hydroxy carboxylic acids. Some reactions
catalysed by nicotinamide nucleotides (enzymes) are given in Table 10.6.
The reduction of acetaldehyde to ethanol by yeast (in presence of alcohol dehydrogenase) is linked to the oxidation of glyceraldehyde-3phosphate (in presence of glyceraldehyde-3-phosphate dehydrogenase,
according to the scheme.
(g)
(h)
(f)
(c)
(d)
(e)
(a)
(b)
Table 10.6 List of reactions catalysed by nicotinamide nucleotides.
Enzyme
Co-enzyme
Substrate
Product
Alcohol dehydrogenase
NAD+
Ethanol
Acetaldehyde
Glycerophosphate
NAD+
Sn-glycerol-3Dihydroxyacetone phosphate
dehydrogenase
phosphate
Lactic dehydrogenase
NAD+
Lactate
Pyruvate
Malic enzyme
NADP+
L-malate
Pyruvate + CO2
+
Glyceraldehyde-3-phosphate
NAD
Glyceraldehyde-31, 3-diphospho glyceric acid
dehydrogenase
phosphate + H3 PO4
Glucose-6-phosphate
NADP+
Glucose-6-phosphate
6-Phosphogluconic acid
dehydrogenase
Glutathrone reductase
NADPH
Oxidised glutathione
Reduced glutathione
Quinone reductase
NADH, NADPH p-benzoquinone
Hydroquinone
Co-Enzymes and Vitamins 225
226
Biophysical Chemistry
Another way in which nicotinamide nucleotides function is in the
reduction of flavin co-enzymes. For example, the reduction of oxidized
glutathione (GSSG) by glutathione reductase in presence of NADH. The
reaction is
glutathione
NAD+ + H+ + GSSG GGGGGGGGGGGGGGGGGA
reductase
NADH + 2 GSH (reduced glutathione)
(10.2)
The above reaction is facilitated by the presence FAD due to the large
ΔG required by reaction (10.2) and is broken down into two reactions of
smaller ΔG as shown below:
NADH + H+ + FAD → NAD+ + FAD − H2
FAD − H2 + GSSG → FAD + 2GSH
(10.3)
(10.4)
The nicotinamide nucleotides and their dehydrogenases are important in
the kinetic and mechanistic study of enzyme reactions for two reasons: (i)
These hydrogenases are available in highly pure crystalline forms; and (ii)
Easy distinguishability of reduced nicotinamide nucleotide from its oxidized form because the reduced form absorbs strongly at 340 nm where
the oxidized form does not absorb.
Co-Enzymes and Vitamins 10.4.2
227
Riboflavin B2
It consists of ribitol attached to 7, 8-dimethyl alloxane. Its structure is
The vitamin occurs in nature as a constituent of two flavin prosthetic
groups, flavin mono nucleotide (FMN) and flavine adenine dinucleotide
(FAD). Riboflavin is produced in green plants, fungi and many bacteria.
Table 10.7 Redox potential data on water soluble B-Vitamins.
Reaction
Formal potential (V)
NAD+ + H+ + 2e NADH
−0.32
+
FAD + 2H + 2e FADH2
−0.22
NADH + H+ + 12 O2 NAD+ + H2 O +1.44
The E0 values are against [H+ ] = 10−7 i.e., pH = 7 at 298K and is not
with respect to standard hydrogen electrode (pH = 0).
228
10.4.3
Biophysical Chemistry
Flavin Adenine Dinucleotides (FAD)
Riboflavin functions as a coenzyme because of its ability to undergo redox
reactions.
These enzymes belong to a group of proteins termed flavoproteins.
Some reactions catalysed by these enzymes are listed in Table 10.8.
10.4.4
Metalloflavoproteins
They are characterized by their multicomponent structure. They transfer electrons from substrate to oxygen, NO3− , NO2− , NAD+ and ferricytochrome C. A metalloflavoprotein is a single isolable enzyme moiety. The
iron in metalloflavoproteins is a single isolable enzymemoiety. The iron in
metalloflavoproteins is a single isolated enzyme moiety and is frequently
of the non-heme type found in iron-sulphur proteins known as ferridoxins.
In these molecules the iron atom is bonded to the sulfur atoms of cysteine
residues and mutually linked by S-bridges.
10.4.5
Lipoic Acid
It exists both in oxidized and reduced forms due to the ability of the disulfide linkage to undergo reduction.
Yeast and liver are rich sources of this compound. Lipoic acid is bonded
to the lysyl residues of the protein in lipoyl enzymes as for example in EN-lipoyl.
Note: Many flavoproteins react directly with molecular O2 to produce H2 O2 ; NHI = non hemetype.
Table 10.8 Typical examples of enzymes (belonging to group of flavoproteins) and reactions catalysed by them.
Enzyme
Electron donor
Product
Co-enzyme and other
(substrate)
components
(1)
D-amino acid and L-amino acid
D- and L-amino
α-keto acids + NH3
2 FAD
oxidases
acids
(2)
L-amino acid oxidase (kidney)
L-amino acid
α-keto acids + NH3 O2 → H2 O
FMN
(3)
L(+)-lactate dehydrogenase (yeast)
Lactate
Pyruvate
1 FMN; 1 heme (cyt B5)
(4)
Glycolic acid oxidase
Glycollate
Glyoxalate
FMN
+
(5)
NAD ± cytochrome C-reductase
NADH
NAD
2 FAD, 2 MoNHI
(6)
Aldehyde oxidase (liver)
Aldehydes
Carboxylic acids
FAD, Fe Mo
(7)
α-glycerol phosphate
Sn-glycerol-3
Dihydroxy acetone FAD, Fe
dehydrogenase
phosphate
phosphate
(8)
Succinic dehydrogenase
Succinate
Fumarate
FAD, Fe, NHI
(9)
Acyl-CoA (C6 - C12 ) dehydrogenase
Acyl-CoA
Enoyl CoA
FAD
(10) Xanthine oxidase
Xanthine
Uric acid
FAD, Mo, Fe
(11) Lipoyl dehydrogenase
Reduced lipoic acid
Oxidised lipoic acid
2 FAD
Co-Enzymes and Vitamins 229
230
Biophysical Chemistry
L-lysine whose structure is
Lipoic acid is a cofactor of the multienzyme complexes, pyruvic
dehydrogenase and α-glutaric dehydrogenase. In these complexes, the
lipoyl containing enzymes catalyse the generation and transfer of acyl
groups.
10.4.6
Biotin
It serves as a growth factor for yeast and bacteria. Egg white contains a
basic protein known as Avidin and it has great affinity for biotin or its
simple derivatives.
Biotin + Avidin Product with equilibrium constant ≈ 1015
(10.7)
This large value indicates that the equilibrium is entirely shifted to the
right. The structure of Biotin is
Liver and yeast are excellent sources of Biotin. This vitamin occurs
mainly in the form bound to protein E - N - Lysine moiety.
10.5
Biochemical Function of Biotin
The specific enzyme protein bound to Biotin is associated with many carboxylation reactions. The overall reaction catalysed by Biotin dependent
carboxylase may be represented as
Co-Enzymes and Vitamins 231
The conversion of acetyl CoA to malonyl CoA in E.coli follows the
sequence in which three proteins participate: (i) biotin carboxylase, (ii)
biotin carboxyl carrier protein (BCCP), and (iii) acetyl CoA, malonyl CoA
carboxylase.
10.5.1
Thiamin
The structure of this compound is:
232
Biophysical Chemistry
It occurs in the outer coats of the seeds of many plants including cereal
grains. Unpolished rice and foods made of whole wheat are good sources
of this vitamin. In yeast and animal tissues it occurs as the co-enzyme
thiamine phosphate or co-carboxylase whose structure is
10.5.2
Biochemical Function
Thiamine pyrophosphate acts as a co-enzyme in α-keto acid dehydrogenases, pyruvic decarboxylase, transketolase, phosphoketolase. For example,
Phospho
D-xylulose-5-P+Pi FGGGGGGGGGGGGGGGGGGGGGGGGB
GGGGGGGGGGGGGGGGGGGGGGGG Acetyl-P+glyceraldehyde-P
ketolase, co-carboxylase
(10.11)
10.5.3
Vitamin B-6 Group
The compounds belonging to this group are pyridoxal, pyridoxine and
pyridoxamine whose structures are
Cereal grains are especially rich sources of this group of vitamins. Pyridoxal is converted to pyridoxal phosphate in the liver according to the
scheme.
Co-Enzymes and Vitamins 233
Biochemical function: Pyridoxal phosphate is a versatile derivative
which takes part in the catalysis of several important amino acid
metabolisms known as transmission, decarboxylation and racemisation.
It is inferred from the above data that the reduction of Vitamins D3,
D2, E and α-tocopherol is favourable on the basis of Gibbs free energy considerations.
Table 10.9 Half wave potential data of a few vitamins and related compounds.
Vitamin
Reaction
E1/2 (V) vs. NHE Medium
D group vitamins D3
(Cholecalciferol) D → Dox
+1.45
CH3 CN, CH2 Cl2
E
E → Eox
+1.20
CH3 CN
α-tocopherol
Eox → Eox,2
+0.80
CH3 CN
Vit-K
Kred → K
−0.60
CH3 CN + 50 mM H2 O
Phylloquinone
Kred,2 → Kred , Kred,2 → Kred −1.2, −0.5
CH3 CN + 7.2 M H2 O
234
Biophysical Chemistry
Co-Enzymes and Vitamins 10.6
235
Adsorption of Riboflavin
Using Langmuir isotherm in the form
C
1
C
= +
a
K
as
where C = equilibrium concentration of riboflavin, a = number of moles of
riboflavin adsorbed per gm of clay, K = Langmuir adsorption coefficient.
Thiamine has two ionizable groups viz. (i) amino and (ii) hydroxyl.
The mean dissociation constant at different temperatures is provided in
Table 10.10.
Table 10.10 Average dissociation constant of thiamine at different temperatures.
Temp (K) pKa
288
9.43
293
9.33
298
9.23
303
9.15
308
9.05
313
8.96
Scheme depicting catalytic decarboxylation of pyruvate by thiamine
O OH
O
H 3C
C
COO + Thiamine
H3C
C
C CH3
H
acetoin
+CH3COCOO
b
H
a
CO2 + CH3CHO
O OH
CO2 + H3C C C CH3
COO
(acetoacetate)
Rate of evolution of CO2 = 2.25 × 10−3 mols l−1 hr−1 .
Rate of formation of acetoin = 1.13 × 10−3 mols l−1 hr−1 .
From the above reaction, it is deducted that acetoin decomposes at a faster
rate in comparison with its rate of formation through the reaction between
pyruate and thiamine.
236
Biophysical Chemistry
Table 10.11 Kinetic data on the reaction of hydrated e− with cobalamine.
Rate constant
Reaction
(dm3 mol−1 sec−1 )
−
e (hyd) + cyanocobalamin −→ Vit-B12
3.8 × 1010
·
OH + cyanocobalamin −→
yellow brown compound
6.5 × 109
−·
CO2 + hydroxocobalamine −→
product (carboxyl radical)
1.45 × 109
CO2− + Vitamin B12 r −→ cobalamin (Vit-B12 ’s)
8.2 × 108
−
Note: ehyd
and OH were generated by pulse radiolysis of water.
Redox chemistry of cobalamin and iron—sulphur containing cofactors
(in tetra-chloroethylene reductase) of dehalobactorretus. (dehalobacter is a
halogen removing rod shaped bacterium).
Tetrachloroethene reductase is an enzyme specific for removing halogens from any compound like dehalobacter reductus. This substance contains cobalamine and iron—sulphur cofactors. It has a mass of 60 k Da =
60,000 Daltons = 60,000 a.m.u. Its reduction was studied by optical and
electron spin resonance spectroscopy.
Table 10.12 Redox potential of cobalamines.
Reaction
Redox potentials/V
Co+1 −→ Co2+ + e
−0.350
Co2+ −→ Co3+ + e
0.150
The redox chemistry of iron-sulphur containing cofactors in tetrachloro
ethylene reductase dehalobactorretus can be represented as below:
[4Fe − 4S]1+ −→ [4Fe − 4S]2+ + e − 0.480
The above data is presented in decreasing order of redox potentials. Hence
the Gibbs free energy changes too increase in the same order. In the case
of NAD+ , NADH system, it can be deduced that the Gibbs free energy
change has a large positive value.
Folic acid gives a pair of well defined redox waves at a 2-mercapto benzothiazole self assembled gold electrode. It can bind strongly 2-MBT and
form a closely packed monolayer whose average electron transfer
rate is 0.085 sec−1 with a 2P, 2H+ transfer. The maximum surface coverage is 2.8 × 10−10 mol cm−2 and the adsorption equilibrium constant is
4 × 105 lmol−1 .
Co-Enzymes and Vitamins Table 10.13 Redox potential data against S.H.E. at pH = 7.0.
Redox pair
Eo (V)
O2 /H2 O
+0.82
Vit-E oxidized/reduced
+0.37
Co-enzyme Q-10 oxidised/reduced
+0.10
Vit-C oxidized/reduced
+0.08
Cystine/cysteine
−0.22
Glutathione oxidised/reduced
−0.24
Lipoate oxidized/reduced
−0.29
+
+
NAD , NADH, NADP /NADPH
−0.32
H+ /H2
−0.42
Biotin reduction in aprotic solvents.
−1.6 to −1.8 (V) vs.
−
The e is attached to –COOH group to Ferrocene/Ferricenium
couple
form a carboxylate anion
Irreversible oxidation of pyridoxine in 0.50
AN
237
238
Biophysical Chemistry
Table 10.14 Kinetic data on the oxidation of pyridoxine.
Temp (K)
288 293 298 303 308
k c × 103 (dm3 mol−1 sec−1 ) 2.5 3.0 3.7 4.2 4.8
Kc (dm3 mol−1 )
207 199 190 196 79
Table 10.15 Protonation constants of folic acid and stability constants of
metal complexes.
Metal ion log K1 ( M : 1) log K2 ( M : 2) log K3 ( M : 1)
H+
8.6
5.1
3.63
Al (III)
9.20 (1 : 1)
7.66 (1 : 2)
8.11 (1 : 3)
Pb (II)
14.83 (1 : 2)
13.22 (1 : 3)
Ba (II)
14.37 (1 : 2)
12.97 (1 : 3)
Ca (II)
7.10 (1 : 1)
Cel (II)
12.71
Co (II)
14.81 (1 : 1)
13.93 (1 : 2)
12.24 (1 : 3)
Fe (III)
14.70 (1 : 1)
13.54 (1 : 2)
12.04 (1 : 3)
Li (I)
2.50 (1 : 3)
Mg (II)
14.05 (1 : 1)
12.76 (1 : 2)
10.01 (1 : 3)
Ni (II)
14.37 (1 : 1)
13.60 (1 : 2)
11.85 (1 : 3)
Cr (III)
8.12 (1 : 1)
Sr (II)
14.42 (1 : 2)
12.92 (1 : 3)
Th (IV)
12.92 (1 : 1)
9.53 (1 : 2)
7.14 (1 : 3)
Zn (II)
Cu (II)
14.06 (1 : 1)
11.59 (1 : 2)
8.62 (1 : 3)
Note: On titration with dilute KOH (0.2M), the maximum number of protons that can be released from folic acid is 3 in the pH range 2.8–12.0. Folic
acid functions as a triprotic acid (H3 -FH), the protonic centers being 1st
carboxyl group, 2nd carboxyl group and imino group of glutamic acid.
One proton is released from 1st carboxyl group in the pH range 3.63–4.95,
the second proton is released from 2nd carboxyl group in the pH range
5.10–8.40, the 3rd proton is released from the imuno group in the pH range
8.60–9.20. The equilibria established are
H 3 − FA H 2 FA− + H + ( pH = 3.63 − 4.95)
H 2 − FA− HFA2− + H + ( pH = 5.10 − 8.40)
HFA2− FA3− + H + ( pH = 8.60 − 9.20)
Co-Enzymes and Vitamins 239
Table 10.16 Conductances of the metal complexes formed at two points
of addition in the titration of 25 ml of metal ions (1 × 10−3 M) with folic
acid (0.01 M).
Metal ion
FA added
Conductance
(mho cm−1 ) ×106
Ca (II)
3ml 6ml
Co (II)
3ml 6ml
Cu (II)
3ml 6ml
Fe (III)
3ml 6ml
42
15
10
5
54
48
36
10
The addition of FA increases the specific conductance of the metal ions
while passing from Cd2+ to Fe3+ . This suggests that the ionic character of
the complex is enhanced due to the addition of FA.
Table 10.17 Values of surface tension and conductivity at the inflection
point pertaining to the formation of 1: 1 complex.
Concentration
Concentration
γ/m
K (sp. Cond.)
−
3
−
1
of β-CD/mM
mM
10 Nm
mho m−1
4.81
5.19 nicotinic acid
73.13
5.42 × 10−3
4.94
5.06 ascorbic acid
72.98
10.65 × 10−3
Table 10.18 Association constant (Ka ) and thermodynamic parameters of
different Vit-β-cyclodextrin inclusion complexes.
Temp
ΔH 0
ΔSe
−
3
−
1
−
1
−
1
Vitamin
(K)
Ka × 10 /M
(kJ mol ) (kJ mol−1 )
Nicotinic acid 298.15
1.25
−20.59
−9.96
308.15
0.92
Ascorbic acid 298.15
3.10
−21.67
−5.87
308.15
2.33
Table 10.19 Distribution constants (K) and Gibbs free energy changes
(ΔG ◦ ) of Vitamin E partitioned between dry reversed micelles of surfactants and non-polar organic solvents.
System
K
ΔGt◦ kJmol−1
Vitamin-E/AoT/n-heptane
10.6
−5.8
Vitamin-E/DDAB/n-heptine
130
−12.1
Vitamin-E/Lecithin/cyclohexane 80.2
−10.9
Vitamin-E/C12 E4 /n-heptane
53.00 −9.8
240
Biophysical Chemistry
Note:
1. AoT = Sodium bis (2 ethyl hexyl) sulfosuccinate
2. DDHB = Didodecy dimethyl ammonium bromide
3. Lecithin = L-α-phophatidyl choline
4. C12 E4 = Tetra ethylene glycol monododecyl ether
10.7
Surface Tension Data of Complexes
with Vitamin-A
BLES (Bovine Lipid Extract Surfactant) whose common brand name is
Suvanter is used to treat respiratory distress syndrome, neonatal (prophylaxis and treatment). It is a natural lung surfactant and is a mixture of lipids
and apoproteins. The surfactant reduces the surface tension of pulmonary
fluids and thereby increases lung compliance.
Compound
BLES
BLES + retinylacetate + ethanol
Surface tension, γ /nNm−1
37.5
36.2
Vitamin-KI: It is highly insoluble in water due to its long hydrocarbon
chain. A substantial increase in its solubility was obtained in solutions
of commercial surfactants containing carboxy methyl ethoxylates. Its solubilisation was studied by UV-Vis spectrometry. The CMC of aqueous
Vitamin-A from studies of variation of γ with concentration is estimated
as 6.0 × 10−9 M. The solute permeability (W ) of various permeants in the
presence of liquid membranes generated by Vitamin-A (W1 ) along with
control values (W0 ) when no Vitamin-A was used.
Permeant
Serine
Threonine
Arginine
Histidine
CaCl2
Na(Cl)
KCl
Initial concentration
(mg ml−1 )
0.2
0.2
0.2
0.2
10.0
5.4
10.4
W0 × 105 (mols
sec−1 N−1 )
498.3
219.9
38.2
7.1
10.7
2.4
3.2
W1 × 105 (mols
sec−1 N−1 )
238.2
105.6
90.0
8.2
16.0
2.7
3.8
Co-Enzymes and Vitamins 241
Table 10.20 Optical data of Vit-K2 and Vit D3 in ethanol.
Vitamin γ (Nm) Extinction coefficient ml/μg Cell length (cm)
Vit-K2
320
0.008
1
Vit-D3
270
0.0444
1
Table 10.21 Zeta potential data of loaded liposomes.
Concentration
Zeta potential Zeta potential Zeta potential
of chitosan
in the absence
(mV) of
(mV) of
(% V/v)
of Vitamins
solution-1
solution-2
0.0
−35.4
−38.4
−36.2
0.0025 (2.5 × 10−3 )
−22.4
−20.9
−31.9
−
3
6.25 × 10
−18.7
−19.8
7.5 × 10−3
−21.7
−20.3
1.0 × 10−2
−17.6
−20.2
Concentration of Vit-K2 used < 0.005% (V/V);
Concentration of Vit-D3 = 32.4 mg in 5 ml of ethanol.
Various forms of Vit-K: Natural forms: K1 and K2
Synthetic forms: K3 , K4 , K5 . Their structures are
As one of the four lipophilic vitamins, Vit-K is a group of vitamins structurally similar molecules that are essential cofactors for γ-glutamyl carboxylase, an enzyme that catalyses the carboxylation of glutamic acid
residues in a number of proteins that are involved in blood coagulation.
Vit-K also plays a role in bone metabolism and the regulation of blood calcium levels. In healthy individuals, like for any lipid compound, orally
administered Vit-K is emulsified in the intestine by bile salts produced by
Diffusion coefficient of Vit-K
in phosphate buffer μm2 sec−1
124
98
Zeta potential (mv)
−23.1
−4.6
Fluorosein
DSPE + PEG
DSPE + PEG
Formulation
Non-PEGylated (Egg phosphotidyl Choline
(EPs)) + Glycocholate + Vit-K
PEGylated EPC, DSPE + PEG Glycocholate
and Vit-K DSPE = distearoyl phosphatidyl
ethanolamine
242
Biophysical Chemistry
Co-Enzymes and Vitamins 243
liver to form mixed micelles with phospholipid to facilitate the absorption
of Vit-K by enterocytes. However, in some cases, like new born patients
with cholestasis showed that the absorption of Vit-K is low.
Permeability coefficients for the transport of Vit-K loaded in mixed
micelles (with and without PEG coating) through CaCo-2 cell
Monolayers
For non-PEGylated micells of Vit-K
For PEGylaled micelles of Vit-K
Permeability coefficient
3.2 × 10−7 cm sec−1
1.1 × 10−7 cm sec−1
Note: The above results imply that the PEGylation reduced the transport
of Vit-K loaded in mixed micelles through CaCo-2 cell monolayers. The
cells have characteristics that resemble intestinal epithelial cells such as the
formation of a polarised monolayer well defined brush order on the apical
surface and intercellular junctions.
Zeta potential of different Vit-K loaded mixed micelles (with and without PEG coating).
Questions
(1) Explain the terms (i) Apoenzyme (ii) Holoenzyme.
account of the functioning of co-enzymes.
Give a brief
(2) Calculate the Gibbs energy change for the reaction
NAD+ + FADH2 NADH + FADH+
given E0 values for the reactions
NAD+ + H+ + 2e NADH
and
FAD + 2H+ + 2e FADH2
are −0.32 V and −0.22 V respectively. Is the above reaction spontaneous under the conditions? (Note that the E0 values are at pH = 7.0)
while E0 of hydrogen electrode is −0.421 V. What will be the E0 values
against S.H.E, that is when [ H + ] = 1.0 molar.
(3) Calculate the pH of a 0.02 M solution of Nicotinic acid given its pKa =
4.75.
244
Biophysical Chemistry
(4) Calculate the degree of dissociation and the dissociation constant of
ascorbic acid at 298 K form the following data.
Ascorbic acid (mol dm−3
∧ (mho cm−2 ) eq −1
5.8 × 10−3
37.00
2.1 × 10−2
20.22
8.0 × 10−2
10.04
∧0 of the acid = 380.4 mho cm−2 eq−1 .
(5) Calculate the molar mass of Nicotinic acid from the following data
of the freezing points of its aqueous solutions K f (H2 O) = 1.86 K kg
mol−1 .
Concentration of acid/mol kg−1
ΔT f
0.0122
0.024
0.0239
0.046
0.0638
0.121
(6) Calculate the partial pressure of a 0.01 M solution of Nicotinic acid in
water at 298 K, given the Henry’s law constant for the acid as 2.90 ×
10−12 atm m3 mol−1 .
(7) Explain the term “cofactor”. Give examples of co-enzymes and their
combinations with vitamin B group that result in (i) decarboxylation
of a reaction (ii) transfer of amino groups and (iii) carriers of acyl
groups.
(8) Classify vitamins. Give two examples in each category and explain
their function in human metabolism.
(9) Name the vitamins of vitamin B and elaborate on their biochemical
functions.
(10) What are the compounds belonging to vitamin B6 and B12 groups?
Give their structure and explain their function.
(11) Write down the structure of ascorbic acid. Given its pKa1 =4.2, calculate its degree of dissociation at a concentration of 0.1 M and its pH.
11
Bioenergetics
11.1
Introduction
The field of bioenergetics deals with diverse types of energy transformation in cells; in particular, the reactions involving adenosine triphosphate
play a central role. The pathways in which the living organism produce
and consume energies is central to various metabolic processes. Any analysis of bioenergetics will be incomplete without adequate description of
thermodynamic concepts and kinetic principles.
The Gibbs free energy changes in various processes involving ATP predict the thermodynamic feasibility; in this context, it is important to analyse the role of pH. Consequently, the Gibbs free energy changes too vary
depending upon the nature of the electrolyte. Thus, the formal potentials
of cell reactions need to be incorporated in this context rather than the standard electrode potentials-customarily invoked. Furthermore, the reaction
rates pertaining to ATP hydrolysis and related reactions are functions of
temperature.
11.2
Metabolism
Metabolism involves a series of interlinked chemical reactions that begin
with a particular molecule which is converted into some other molecule (or
molecules) in a chosen fashion. There are many such pathways in a cell.
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_11
245
246
Biophysical Chemistry
For example, we consider the glucose metabolism as shown in the following scheme:
Metabolic pathways may be divided into two categories: (1) those that
convert energy into biologically useful forms and (2) those that require
input of energy to proceed. As an example of first category, the scheme
below may be considered
Fuels (Carbohydrates, fats) −→ CO2 + H2 O + useful energy
(11.1)
This class of reactions are described as “Catabolism” (Catabolic reactions).
As examples of reactions of second category, which are referred as anabolic
reactions (or anabolism)
Useful energy + small molecules −→ Complex molecules
11.3
(11.2)
Coupled Reactions
This concept is of great importance in the study of bio-chemical reactions.
This means that a thermodynamically unfavourable reaction (i.e., are
which has a +ve ΔG ◦ ) can be driven by a thermodynamically favourable
reaction (i.e., are which has a more negative ΔG ◦ value) to which it is coupled. The following example illustrates this principle.
(11.3)
On combining the reactions,
(11.4)
The reaction (11.3) is driven by coupling it to the second reaction.
Bioenergetics 11.4
247
ATP as an Energy Source
Most energy requiring processes such as biosynthesis, transport of species
across membranes derive their energy from the free energy donor i.e., ATP.
ATP consists of the units adenine, ribose and three phosphate units. The
active form of ATP is usually a complex of ATP with Mg2+ or Mn2+ . The
role of ATP as an energy barrier lies in its triple phosphate moiety. The
presence of two phospho anhydride bonds in its triphosphate groups contributes to the energy rich character of ATP. Considerable amount of free
energy is liberated when ATP is hydrolysed to ADP or AMP as may be
noted from the following equations
ATP + H2 O −→ ADP + Pi
ATP + H2 O −→ AMP + PPi
ΔG ◦ = −30.5 kJ mol−1
ΔG ◦
= −45.6 kJ
mol−1
(11.5)
(11.6)
where Pi is inorganic phosphate and PPi is inorganic pyrophosphate.
Under the conditions of concentrations prevailing in cells, the ΔG ◦ values for the above hydrolyses are of the order of −50 kJmol−1 . Energyrequiring processes such as muscle contraction use the energy derived
out of ATP hydrolysis. The ATP, ADP cycle is fundamental to the energy
change in biological systems. Other analogous nucleoside phosphates
whose hydrolysis is similar to ATP are guanosine phosphate (GTP), uridine triphosphate (UTP) and cytidine triphosphate (CTP). Enzymes catalyse the transfer of the terminal phosphoryl group from one nucleoside to
the other. While the phosphorylation of nucleoside monophosphates is
catalysed by the enzyme nucleoside monophosphate kinase, the phosphorylation of nucleoside diphosphate is catalysed by nucleoside diphosphate
kinase.
Thermodynamically, ATP acts as a coupling agent. This means that a
thermodynamically unfavourable reaction can be converted into a
favourable one by coupling it to the hydrolysis of sufficient number of
ATP molecules. It may be shown that the hydrolysis of “n” ATP molecules
changes the equilibrium quotient of the overall reaction by a factor of 108n .
11.5
High Phosphoryl Capacity of ATP
It is known that the transfer of phosphoryl group is a common way of
energy coupling and it is extensively used in intracellular transmission of
information. To understand the efficient transfer of phosphoryl group by
ATP, the following two reactions [one of them being reactions] (11.5) and
248
Biophysical Chemistry
(11.6) and the reaction
Glycerol-3-phosphate + H2 O −→ Glycerol + Pi
ΔG ◦ = −9.2 kJmol−1
(11.7)
may be considered.
Since ΔG ◦ for reaction (11.5) is much smaller than that of (11.7), one
may conclude that ATP has a much stronger tendency to transfer its terminal phosphoryl group to water than glycerol-3-phosphate.
Thus ATP has a higher phosphoryl group transfer potential than does
glycerol-3-phosphate. Examination of the structures of ATP, ADP and Pi
will give an explanation for the same. ADP and Pi, especially Pi has greater
resonance stabilization than ATP. Apart from this, other factors like electrostatic repulsion and stabilization due to hydration must be considered.
Further, orthophosphate has a number of resonance forms of nearly the
same energy as shown below:
Another important reason is that water can bind more effectively to
ADP and Pi than to phosphoanhydride part of ATP. This leads to stabilization of ADP and Pi.
11.6
Significance of Phosphoryl Transfer
Potential (PTP)
ΔG ◦ of hydrolysis serves as a measure of comparing PTP of phosphorylated compounds. It is observed that some compounds in biological systems have a higher PTP than ATP. A few of such compounds are phosphoenol pyruvate (PEP); 1, 3-bisphosphoglycerate (1, 3-BPG) and creatine
phosphate whose structures are shown.
Bioenergetics 249
Table 11.1 Standard Gibbs energies of hydrolysis (ΔG ◦ ) of some phosphorylated compounds.
Compound
Phosphoenol pyruvate
1, 3-BPG
Creatine phosphate
ATP → ADP
Glucose-1-phosphate
Glucose-6-phosphate
Glycerol-3-phosphate
ΔG ◦ /kJmol−1
−61.9
−49.4
−43.1
−30.5
−20.9
−13.8
−9.2
To understand the significance of the above data, we consider the reaction.
creatine
C.P. + ADP + H+ FGGGGGGGGGGGGB
GGGGGGGGGGGG ATP + creatine
kinase
(11.8)
C.P. in vertebrate muscle serves as a reservoir of high potential phosphoryl
groups that can be readily transferred to ATP. This reaction occurs every
time we exercise to generate ATP.
The equilibrium constant of the above reaction is 162. When body
muscle is resting, typical concentrations of the constituents in equation
(11.8) are [ATP] = 4 × 10−3 M; [ADP] = 1.3 × 10−5 ; [C.P.] = 25 × 10−3 ;
[Creatine] = 13 × 10−3 . The large amount of C.P. and its high P.T.P. relative
to ATP makes it a highly effective phosphoryl buffer.
11.7
Intracellular Conditions Pertaining to
ATP Hydrolysis
The value of ΔG due to ATP hydrolysis depends upon the activities (in
dilute solutions, may be considered as concentrations) of the various species
in the reaction. For example, in the reaction (11.5).
Keq =
[ADP]eq /[IM] × [Pi]/[IM]
[ATP]eq /[M] × [H2 O]eq /[55M]
(11.9)
ΔG ◦ = − RT ln Keq = −31.0 kJmol−1
Correcting for physiological concentrations
ΔG =
[ADP]Phys /[IM]/[Pi]Phys [IM]
[ATP]Phys
ΔG = ΔG ◦ + RT ln Q ≈ −60 kJmol−1
(11.10)
250
11.7.1
Biophysical Chemistry
Hydrolysis Reaction
O
Phosphoanhydride (acid-base) bond hydrolysis
O P OH
O
+1
OPO
R
Reaction
ΔG ◦ (kJ/mol)
ADP + H2 O → AMP + Pi
− 31.0
ATP + H2 O → AMP + PPi
− 38.0
PPi + H2 O → 2Pi
− 24.0
Table 11.2 Standard Gibbs free energies of phosphoesters.
ΔG ◦
Reaction
(kJ/mol)
3-Phosphoserine + H2 O → Serine + phosphate
−10.0
AMP + H2 O → Adenosine + phosphate
−14.0
Dihydroxyacetonephosphate (DHAP) + H2 O → DHA
+Phosphate
−15.0
Fructose1, 6-biphosphate + H2 O → F6P + Phosphate
−16.0
Glyceroldehyde-3-Phosphate + H2 O → Glyceraldehyde
+Phosphate
−17.0
Threoninephosphate + H2 O → Threonine + Phosphate
−19.0
Fructose-6-phosphate + H2 O → Fructose + Phosphate
−16.0
AcetylCoA + H2 O + Oxaloacetate → Citrate + CoA
−31.4
5-adenonylmethionine + H2 O → Methylthioadenosine
+homoserine
−41.8
When the concentrations are farther from equilibrium values |ΔG | is
larger. In practice, the physiological condition depends on the organism
being studied, the time or the cell compartment under consideration and
the energy demand for metabolic reactions. Thus ΔG values may vary
widely.
Gibbs free energy changes for ATP hydrolysis in various organisms
under different physiological conditions are shown in the following table.
Table 11.3 Gibbs free energy changes for ATP hydrolysis in various organisms under different physiological conditions.
Concentration of
Physiological condition of organism
ATP
ADP
Pi
ΔG/kJ mol−1
Standard conditions
1M
1M
1M
−31.0
E.Coli aerobic exponential growth on glucose
10 mM 0.6 mM 20 mM
−54.0
E.Coli anaerobic exponential growth on glucose
3 mM 0.4 mM 10 mM
−54.0
E.Coli anaerobic exponential growth on glycerol
7 mM 0.7 mM 10 mM
−55.0
S.Cerevisiae aerobic growth on glucose
2 mM 0.3 mM 22 mM
−52.0
Spinach spinacia oleracea chloroplast stroma in light
2 mM 0.8 mM 10 mM
−51.0
Spinach spinacia oleracea cytosol + mitochondria in light 3 mM 0.7 mM 10 mM
−52.0
Homosapiens (resting muscle)
8 mM
9 μM
4 mM
−68.0
Homosapiens (muscle recovery after exercise)
8 mM
7 μM
1 mM
−72.0
Bioenergetics 251
252
Biophysical Chemistry
The calculations of ΔG require accurate measurement of relevant intracellular concentrations. NMR can be used in humans to measure concentrations of 31P in vivo because 31P has magnetic properties. In E. Coli, the
concentrations of ATP can be measured by an ATP bioluminescence assay.
The luciferase enzyme uses ATP in a reaction that produces light which
can be measured using a luminometer and the ATP concentration can be
inferred from signal strength.
11.8
Methods by which ATP Transfers Energy
Cells require chemical energy for three general tasks: (i) To drive metabolic
reactions that would not occur automatically to transport substances
needed across membranes and to do mechanical work such as moving
muscles. ATP is not a storage molecule for chemical energy (it is the job of
carbohydrates, glycogen and fats). (ii) When energy is needed by the cell, it
is converted from storage molecules into ATP. ATP then serves as a shuttle
delivering energy to places within the cell where energy consuming activities are taking place. ATP is a nucleotide that consists of three groups: the
nitrogenous base glucose, adenine, the sugar ribose and a chain of three
phosphate groups bound to ribose. (iii) The actual power source is the
phosphate tail of ATP which the cell taps. When hydrolysis occurs, the
phosphate tail is broken and one phosphate group is broken from ATP to
yield energy i.e.
ATP + H2 O −→ ADP + Pi
(11.11)
ATP is able to power cellular processes by transferring a phosphate group
to another molecule (called phosphorylation). This process is carried out
by special enzymes that couple the release of energy from ATP to cellular activities that require energy. As seen above, cells continuously break
down ATP to obtain energy. ATP also gets synthesized from ADP and Pi
through the process of cell respiration. This is facilitated by the enzyme
ATP synthase which converts ADP and Pi to ATP. The enzyme ATP synthase is located in the membrane of cellular structures called mitochondria,
inplant cells the enzyme is found in chloroplasts.
The three processes of ATP production include glycolysis, oxidative
phosphorylation and tricarboxylic acid cycle. In eukaryotic cells, the
latter two processes, occur with in mitochondria. Electrons that are passed
through the electron transport chain ultimately generate free energy capable of driving the phosphorylation of ADP.
Bioenergetics 11.9
253
Citric Acid Cycle
Citric Acid Cycle is also known as CAC, TCA or Krebs Cycle. It is a series
of chemical reactions used by aerobic organisms to release stored energy
through oxidation of acetyl-CoA derived from carbohydrates, fats and proteins into ATP and CO2 . In addition, the cycle provides precursors of certain amino acids as well as the reducing agent NADH that are used in
numerous other reactions.
CH2 COOH
This metabolic pathways is derived from citric acid HO C
COOH
CH2 COOH
that is consumed and regenerated by this cycle. The cycle consumes acetate
(in the form of acetyl CoA) and water, reduces NAD+ to NADH and produces CO2 as waste product. The NADH produced in the cycle is fed into
oxidative phosphorylation (electron transport) pathway. The net result of
these two closely linked pathways is the oxidation of nutrients to produce
energy in the form of ATP.
In eukaryotic cells the TCA cycle occurs in the matrix of mitochondria.
In prokaryotic cells, such as bacteria, the TCA cycle is performed in the
cytosol with the proton gradient for ATP production being across the cell
surface. The over all yield from the TCA cycle is three NADH, one FADH2
and one GTP.
11.10
Reactions of the TCA Cycle
The initial step of the cycle is catalysed by the citrate synthase in the matrix
of mitochondria. This highly exothermic reaction commits acetyl groups to
citrate formation and complete oxidation in the cycle. The reactions of the
cycle are carried out by eight enzymes that completely oxidize acetate in
the form of acetyl CoA into two molecules each of CO2 and H2 O.
254
Biophysical Chemistry
NAD
Acetyl CoA
NADH
+H+
CoASH
Malate
dehydrogenase
Oxaloacetate
Citrate
synthase
Citrate
L-malate
H2 O
Aconitase
Fumarase
H 2O
Summary of the citric acid cyle
Fumarate
Inputs
FAD
H2
Succinate
dehyrogenase
FAD
Outputs
2(2C) acetyl groups
4CO2
6NAD+
6NADH
2FAD
2FADH2
2ADP + 2Pi
2ATP
cis-aconitate
H 2C
Succinate
isocitrate
CoASH
GTP
NAD+
Pi
CO2
GDP
CoA S α -keto
glutarate
Succinyl CoA
NADH
+ H+
NAD+
(Mass = 380 kDa)
ucc
Isocitrate
alos
dehydrogenase Ox inate
-
NADH + H+
CO2
Gibbs Free Energy Changes for Glycolysis Reactions and TCA
ΔG ◦ of the reaction = −38 kJ mol−1
ΔG ◦ of isocitrate → α-ketoglutarate = −21.0 kJ mol−1
ΔG ◦ of α-ketoglutarate → succinyl CoA = −33 kJ mol−1
ΔG ◦ of succinyl CoA to succinate is −3.0 kJ mol−1
ΔG ◦ of L-malate → oxalo acetate is +29 kJ mol−1
Bioenergetics 255
This endergonic reaction is pulled in the forward direction by the action
of citrate synthase and other reactions which remove oxaloacetate.
One of the primary sources of acetyl CoA is from the break down of
sugars by glycolysis which yield pyruvate that in turn is decarboxylated by
the pyruvate dehydrogenase complex generating acetyl CoA according to
CH3 C(=O)C(=O)Ō + HSCoA + NAD+
−→ CH3 C(=O)SCoA + NADH + CO2
(11.12)
The TCA cycle is the final path way for breakdown of foods. It may be
stated that the four, five, six carbon intermediates generated in the reactions of TCA cycle are important intermediates in the biosynthetic processes. Succinyl CoA, malate, oxalo acetate, α-ketoglutarates and citrate
are all precursors in the biosynthesis of important cellular compounds.
The TCA cycle acts as a source of precursors for amino acid, fatty acid
and glucose synthesis.
R5P− = R5P2−
−
−
ISCIT3− + NADOX
+ H2 O = AKG2− + NAD2red
+ CO23− + 2H+
−
PGN3− + NADP3− + H2 O = RU5P2− + NADP4red
+ CO23− + 2H+
Reference reactions
Glucose + ATP4− = glucose-6-phosphate−2 + ADP3− + H+
Glucose-6-phosphate (glu-6-p2− ) = Fructose-6-phosphate2− (F6P2− )
F6P2− + ATP4− = F16P4− + ADP3− + H+
F16P4− = DHAP2− + GAP2−
GAP2− = DHAP2−
F16P4− = 2 DHAP2−
PG23− = PG33−
PG23− = PEP3− + H2 O
PYR− + ATP4− = PEP3− + ADP3− + H+
ISCIT3− = CIT3−
−
−
LSCIT3− + NADP3OX
+ H2 O = AKG2− + NADP4red
+ CO23− + 2H+
2−
4
−
2
−
+
3
−
GTP + Suc + CoAS- + H = GDP + HPO4 + SUCCoA−
FuM2− + H2 O = MAL2−
MAL2− + NAD2− = OAA2− + NAD2red
+ H+
−
4−
2−
−
NADOX + NADPred = NADred + NADP3OX
2−
4
−
3
−
+
ATP + H2 O = ADP + HPo4 + H
G6P2− + H2 O = G65 + Pi2−
12.9
−26.3
37.5
Δr H ◦ /
kJmol−1
−23.8
11.5
−9.5
49.0
2.7
51.70
28.0
15.1
5.4
−20.0
−22.2
−30.9
−13.2
51.3
−4.1
−20.5
0.91
Name of reactions
Glucokinase (GLK)
Phosphoglucose isomerase (PGI)
Phosphofructokinase (PFK)
Fructose-1-6-bi phosphate aldolase (FBE)
Triose phosphate isomerase (TPI)
Fructose-1, 6-phosphate aldolase (FPA2)
Phosphoglycerate mutage (PGM)
Enolase (ENO)
Pyruvate kinase (PYK)
Aconitrate hydratase (ACON)
Isocitrate dehydrogenase (IDH)
Succinate CoA ligase (SCA)
Fumarate hydratase (FUM)
Malate dehydrogenase (MDH)
NADP transhydrogenase (NPTH)
ATPase (ATPS)
Alkaline phosphatase hydrolysis G6P
(G6PH)
6-Phosphogluconate dehydrogenase
(PGD)
Ribose-5-Phosphate isomerase (R5PI)
Isocitrate dehydrogenase (IDH2)
Table 11.4 Enthalpy changes of reactions involving ATP and related compounds.
256
Biophysical Chemistry
Species
Δ f Gi◦ /kJmol−1
Species
Δ f Gi◦ /kJmol−1
GLC◦
−916.4
NAD−
0
Abbreviation
ADP
ATP
CIT
CoAS
ACoA
Zi (Charge)
−3
−4
−3
−1
0
NH (No. of H’s)
12
12
5
0
3
ATP4−
−2769.7
NADPH2−
−809.2
P6P2−
−1760.8
HPO24−
−1096.1
F16P2−
−2597.6
PG33−
−1508.8
DHAP2−
−1292.9
PG23−
−1502.1
GAP2−
−1285.9
PZP3−
−1269.4
BPG4−
−2354.5
ADP3−
−1906.1
Table 11.6 Gibbs free energy changes of reference compounds.
Reactant
Adenosine diphosphate (ADP3− )
Adenosine triphosphate (ATP4− )
1, 3-bisphosphoglycerate-citrate (CIT3− )
Coenzyme A-SH (CoAS− )
Acetyl coenzyme A (ACoA◦ )
ACoA◦
−188.5
CO23−
−527.8
Δr + 1
−2
−2
−1.9
—
—
NADH2−
23.9
CoA5
0
pKH1
6.5
6.7
5.7
8.2
—
Table 11.5 Dissociation constants and enthalpy changes for reactions involving ATP.
Bioenergetics 257
258
11.10.1
Biophysical Chemistry
Activity of TCA Cycle
TCA cycle is regulated by different factors. First, the supply of acetyl units,
whether derived from pyruvate (by glycolysis) or fatty acids (by
β-oxidation) is crucial in determining the rate of the cycle.
Regulation of the pyruvate dehydrogenase complex, the transport of
fatty acids into mitochondria and β-oxidation of fatty acids are effective
determinants of cycle activity. Second, because of the dehydrogenases of
the cycle are dependent on a continuous supply of NAD+ and FAD, their
activities are very strongly controlled by the respiratory chain that oxidizes
NADH and FADH2 . The activity of respiratory chain is dependent on the
rate of ATP synthesis which is strongly affected by availability of ADP,
phosphate and O2 .
Symbols Pertaining to CTA Cycle
PG2− = 2-phospho-D-glycerate
PG3− = 3-phospho-D-glycerate
PEP3− = phosphoenol pyruvate
PYR− = pyruvate
ATP4− = adenosine triphosphate
ADP3− = adenosine diphosphate
ISCIT3− = isocitrate
CIT3− = citrate
AKG2− = α, ketoglutarate
GTP4− = guanosine triphosphate
Suc2− = succinate
CoAS− = co-enzyme A-SH
GDP3− = guanosine diphosphate
SucCoA− = succinyl-CoA
FuM2− = fumarate
Bioenergetics 259
MAL2− = malate
OAA = oxaloacetate
G6P2− = D-glucose-6-phosphate
GLC◦ = glucose
PGN3− = 6-phosphogluconate
Ru5P2− = ribulose-5-phosphate
R5P− = ribose-5-phosphate
Questions
(1) The standard Gibbs free energy change for the hydrolysis of ATP is
given by
ATP + H2 O ADP + Pi
where Pi = phosphate group is −31.0 kJ mol−1 . Calculate equilibrium
constant of the reaction at 298K. State whether the reaction is spontaneous under these conditions.
Solution:
ΔG ◦ = − RT ln K = −8.314 × 10−3 × 298 × 2.313 log K
−31.0 = −8.314 × 10−3 × 298 × 2.313 log K
= −5.17 log K
31.0
log K =
= 5.43
5.71
K = 2.70 × 105
The reaction is spontaneous under the standard conditions.
(2) The equilibrium constant of the reaction
Creatine phosphate + ADP + H+ ATP + Creatine
is 162 at 293K. Given that the concentrations of creatine = 13 × 10−3 ,
ATP = 4 × 10−3 , creatine phosphate = 25 × 10−3 and ADP = 1.3 × 10−5
in the muscle of the humans under rest conditions, what is the pH of
the biological fluids therein.
260
Biophysical Chemistry
Solution:
[ATP][Creatine]
[Creatine phosphate][ADP][H+ ]
[4 × 10−3 ][13 × 10−3 ]
162 =
[25 × 10−3 ][1.3 × 10−5 ][ H + ]
52 × 10−6
[ H+ ] =
32.5 × 10−8 [162]
K=
= 0.00988 × 102
= 0.988
pH = − log[ H + ]
= − log 0.988
= −(−0.005) = 0.005
(3) Formulate the fuel cell in which glucose undergoes oxidation at the
anode in alkaline medium as one electrode. The other electrode is an
oxygen electrode with Pt for electrical contact. What are the anodic and
cathodic half cell reactions (Express both as reduction reactions). Given
their half cell potentials as −0.93V and +0.52V respectively. Calculate
the Gibbs energy change of the reaction.
Solution:
Pt|C6 H12 O6 in alkaline medium containg CO23− ion
|alkaline medium|O2 (g, 1 atm)|Pt
Half cell reactions
12H2 O + 6O2 (g) + 24e− 24 OH−
( E = +0.52V )
24H2 O + 6CO23− + 24e C6 H12 O6 + 36 OH−
( E = −0.92V )
Ecell = ER − EL = +0.52 − (−0.93) = 1.45V
ΔG ◦ = −nFE◦ = −24 × 96500 × 1.45
= −33582 × 102 J
−33582 × 102
1000
= −3358.2 kJ
=
Bioenergetics 261
(4) If a kg of glucose produces 3200 kwh of energy, how much energy is
generated by 11.1 moles of glucose. Express your answer in kJ.
Solution: 1 kg= 5.55 moles of glucose or 2 kg=11.10 moles of glucose.
Hence 2 kg of glucose produces 2 × 3200 = 6400 kWh. So
1 kWh = 3.6 × 106 J
2 kg glucose produces
= 6400 × 3.6 × 106 J
= 64 × 3.6 × 108 J = 2304 × 108 J
= 230.4 × 105 kJ
(5) What is metabolism? Explain how glucose metabolises in the body.
(6) Define “catabolic” and “anabolic” reactions. Give examples for each.
(7) What is a coupled reaction?
importance.
Give an example and outline its
(8) Explain the term phosphoryl capacity of ATP. How does it impact
energy changes in a cell?
(9) Write down all the steps in citric acid cycle and outline its importance.
12
Biosensors
12.1
Introduction
Biosensors are defined as analytical tools or devices, which include a system of biological detecting elements e.g., a sensor and a transducer. In
medicinal chemistry, the need for affordable and selective sensing of analytes such as glucose, urea, dopamine, progesterone, anesthetics etc., is of
paramount importance It is well known that L-cysteine is a prostate cancer
biomarker, and its timely measurement is essential. Analogously, the qualitative and quantitative detection of trace elements such as arsenic, lead
becomes essential in water analysis. Thus, it is no wonder that the field of
biosensors today occupies a pivotal place in materials science and biophysical chemistry. The essential touchstones in this context are as follows: (i)
selectivity; (ii) limit of detection; (iii) range of detection and (iv) real time
monitoring.
12.2
Components of a Biosensor
The biosensor has mainly three components: (i) sensory element, (ii) transducer, (iii) necessary arrangement for transport of electrons. A sketch of
biosensor is shown in the diagram below:
Figure 12.1 Sketch of a typical biosensor.
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_12
263
264
Biophysical Chemistry
The sensor in contact with the analyte of biological nature responds
and the detector changes the resulting signal via transducer. It is amplified
and displayed via a signal processor.
12.2.1
Working of a Biosensor
The electrical signal of the transducer is often quite low (due to low concentration of the detectable species) and a fairly long base line is observed.
The signal processing includes the necessary correction for deducting the
base line signal.
12.2.2
Applications
The applications of biosensors include: (i) food industries, (ii) agriculture
and (iii) control of pollution in environment and ecology.
Figure 12.2 Applications of biosensors.
12.3
Different Types of Biosensors
Broadly, there are eight types of biosensors (a) to (h):
(a) Piezoelectric: The platform of a piezoelectric sensor is a sensory element
that works on the law of oscillations leading to a collection pump on
the surface of a piezoelectric crystal. The biosensors have their surface
modified with an antigen or an antibody and the detection parts are
united by using nanoparticles.
Biosensors 265
(b) Wearable: It is a digital device used to wear on a human body in different wearable systems like smart watches, tattoos which allow levels
of blood glucose, rate of heartbeat etc. In humans, these sensors may
allow premature recognition of health condition and prevent hospitalization.
(c) Thermometric: There are various types of biological reactions that are
connected with evolution or absorption of heat and this is the basis
of this class of sensors. This sensor is used to measure or estimate
the serum cholesterol. The heat of generated through the oxidation
of cholesterol by the enzyme cholesterol oxidase is measured. This
sensor is also used to estimate glucose, urea, uric acid, penicillin-G.
A schematic diagram of this sensor is shown in Figure 12.3.
Figure 12.3 Schematic diagram of thermometric biosensor.
(d) Optical: These biosensors use fiber optics as optoelectronic transducers.
The sensors mainly involve antibodies and enzymes as the transducing
elements. A sketch of this set up is also shown in next page.
The optical biosensors permit a secure non-electrical inaccessible sensing of equipment. They also do not need reference sensors because the
comparative signal can be produced by using similar light source like
sampling sensor.
Although a variety of sensors can be envisaged as shown above, the
electrochemical biosensors viz.
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Biophysical Chemistry
Figure 12.4 Sketch of an optical biosensor.
(e) amperometric; (f) potentiometric; (g) voltammetric and (h) impedimetric are more prominent in this context on account of the portability,
ease of measurement and sensitivity. Hence the sensors based on electrochemical measurements are described in more detail below.
12.4
Electrochemical Biosensors
Generally, an electrochemical biosensor depends on a reaction that consumes or generates electrons (i.e., a redox reaction). The reaction could be
an enzymatic or a non-enzymatic reaction. The basic set up in the biosensor involves a three-electrode system, i.e., a working electrode, a counter
electrode and a reference electrode.
The analyte gets reduced (or oxidized) and the product is sensed by
the working electrode and the signal is detected by a device such as an
oscilloscope.
12.4.1
Amperometric Biosensors
These are based on the measurement of current flowing between working
and counter electrodes at a chosen potential. In the Clark’s oxygen electrode (used to measure O2 concentration in a metabolite such as blood),
Biosensors 267
a silver electrode (as anode) and a platinum (as cathode) are immersed in
an electrolyte, containing dissolved oxygen. An optimum voltage of about
0.7 V is applied between the electrodes. Oxygen is reduced at the cathode
and silver is oxidized at the anode. The experimentally measured current reaches a plateau due to the rate being controlled by diffusion of O2
towards the electrode surface. The magnitude of the diffusion current is
directly proportional to the bulk concentration of the analyte.
The analytes (e.g., glucose, dopamine, urea, progesterone, ascorbic acid
etc.) are dissolved in suitable solvents. Since the current is linearly related
to the analyte concentration, an effortless method of estimating the amount
with the help of a calibration curve/chart is envisaged. The practical feasibility of the sensors is studied using the calibration plot for deducing
the linear detection range, by the constant addition method-well known in
analytical chemistry. It is desirable to obtain the range of detection varying
from nanomolar to micromolar. The other sensing parameters viz., sensitivity (S), limit of detection (LOD) and response time (τ) are also required
for point of care analysis.
The method of estimating the sensitivity (S) depends upon the experimental technique employed. For example, in amperometric sensors, S is
calculated from the slope of the calibration curve viz.
S=
ΔI
ΔC
(12.1)
In the above equation S represents the change in amperometric current (or
current density) for unit change in the analyte concentration (C ).
The limit of detection (LOD) representing the lowest measurable concentration within a specified confidence interval is computed from the
equation
LOD = k
σ
S
(12.2)
where σ denotes the standard deviation of the measurements and k = 3
indicates the confidence level of 99.6%, S being the slope of the calibration curve. The values lower than 3 in the above equation reflects a lower
confidence interval.
12.5
Enzymatic Sensing of Glucose
The development of biosensors gained impetus from the need to monitor
glucose levels in the blood of diabetics. In 1962, Leland Clark Jr. developed
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Biophysical Chemistry
Table 12.1 Typical range of detection accomplished for analytes using
chemically modified electrodes.
Analyte
Glucose
Catechol
Urea
Cholesterol
Progesterone
Dopamine
Procaine
Approximate range of detection (mM)
0.001 to 60
0.00015 to 0.075
50 to 250
0.005 to 0.1
1 × 10−6 to 1 × 10−3
0.001 to 0.4
0.0001 to 10
an enzyme electrode to estimate glucose levels in humans which is based
on the reaction
Glucose + O2 −→ Gluconic acid + H2 O2
(12.3)
In order to accelerate the rate of the above reaction as well as provide selectivity, glucose-oxidase (GOx ) enzymes are almost always employed. The
method of immobilization of GOx on electrodes is a crucial factor in determining the efficiency of the sensor, in electrochemical methods.
Since bare metal electrodes do not possess satisfactory sensitivity even
in the presence of enzymes, it is customary to employ conducting polymerscoated electrodes for the sensing of analytes. There are several types of
electrodeposition techniques to prepare such chemically modified
electrodes. The simplest method is to polymerize the desired monomers
(e.g., aniline, pyrrole, indole, thiophene. . ..) on any metal electrodes (e.g.,
stainless steel, Ni, Ag, Au etc.) by applying current for optimum duration
of time. Any enzyme-based biosensor requires efficient immobilization on
the chosen electrode surface. The modified surface should possess desirable characteristics for facile electron transfer.
12.5.1
Experimental Details
In a typical experiment, the desired monomer (e.g., pyrrole) is polymerized on the electrode surface (e.g., Au) by a suitable electrodeposition technique (potentiostatic/potentiodynamic/galvanostatic) in electrolytes such
as para-toluene sulphonic acid. This procedure yields the modified electrode designated as (Au/PPy), where PPy refers to polypyrrole. A small
amount (∼ 5% glucose oxidase enzyme in phosphate buffer solutions) is
drop cast on Au/PPy, yielding the enzyme coated surface. This electrode
then serves as the sensor for glucose.
Biosensors 269
Figure 12.5 Schematic variation of amperometric current with time for
equal additions of glucose when an enzyme-coated electrode is dipped in
a suitable medium.
A careful systematic addition of equal volume (often microliters) of
glucose is then added to the solution and the chronoamperometric current is measured (Fig. 12.5). The stepwise decrease in current shown in
Figure 12.5, for various concentrations are the typical features for the feasibility of amperometric sensors. The total number of steps then indicates
the linear concentration range in which the sensor is functional.
12.5.2
Mechanism of Detection
The enzyme-catalyzed oxidation of glucose may be represented in the following manner:
β-D-Glucose + GOx -FAD −→ GOx -FADH2 + δ-D-gluconolactone (12.4)
GOx -FADH2 + O2 −→ GOx -FAD + H2 O2
H2 O2 −→ 2H+ + O2 + 2e−
(12.5)
(12.6)
FAD refers to Flavin adenine dinucleotide (quinine form) and is a coenzyme essential for the functioning of glucose oxidase. FADH2 indicates the
hydroquinone form of Flavin adenine dinucleotide. The gluconolactone
hydrolyses to form gluconic acid. Upon adding the above three equations,
eqn. (12.3) is again obtained.
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Biophysical Chemistry
The sensing of glucose can also be accomplished by the reduction of
H2 O2 viz.
H2 O2 + 2H+ + 2e− −→ 2H2 O
(12.7)
When eqn. (12.1) is applied for sensing of glucose using chemically
modified electrodes, the sensitivity varies from 0.2 to 60 (μAcm−2 mM−1 )
depending upon the nature of the electrode and operating conditions. This
number refers to the variation of the current density for one millimolar
addition of glucose.
12.6
Estimation of Michaelis-Menten Constants
It is possible to estimate the Michaelis-Menten constants for ascertaining
the efficacy of the chosen enzyme from amperometric measurements using
a graphical procedure as shown below:
K
I (max )
= M +1
I
C
(12.8)
where I and I (max ) denote respectively, the current and the maximum
current respectively, C being the concentration of the glucose.
The currents I and I (max ) are analogous to the initial and maximum
velocities respectively in the Lineweaver-Burke plot. By plotting the ratio,
Table 12.2 Illustrative examples of analytes and corresponding enzymes.
Analyte
Enzyme
Reaction
1. Glucose
Glucose oxidase
β-D-glucose + O2 −→
gluconic acid + H2 O2
2. Ethanol
Ethanol oxidase
Ethanol + O2 −→
acetaldehyde + H2 O2
3. Lactic acid Lactase oxidase
L-lactate + O2 −→
Pyruvate + H2 O2
4. Lactose
Galactose oxidase Lactose + O2 −→ galactose
dialdehyde derivative + H2 O2
5. Uric acid
Uricase
Uric acid + 2H2 O+ O2 −→
Allantoin + CO2 + H2 O2
6. Catechol
Catechol oxidase
Catechol + O2 = 1,2-benzoquinone
+ H2 O
7. Dopamine Tyrosinase
Dopamine + O2 −→
indole 5,6 quinone
Biosensors 271
I (max )/I versus 1/C, the Michaelis-Menten constant K M is deduced. Since
the interaction of the chemically modified electrodes with the glucose oxidase enzyme is not identical, K M varies from 4 to 55 mM. These values
pertain to different chemically modified electrodes.
12.7
Non-enzymatic Sensing of Glucose
Although enzyme-based biosensors exhibit remarkable selectivity and sensitivity, they suffer from high cost as well as inadequate storage stability. Hence recent investigations have focused on the fabrication of nonenzymatic sensing of analytes. In the case of glucose, a variety of electrochemical biosensors have been developed, without the use of glucose oxidase. Among them, mention may be made of the following: noble metals,
oxides and hydroxides of transition metals.
12.7.1
Interference Studies
Although sensing of glucose may be the main objective, other analytes
such as ascorbic acid, dopamine, urea, uric acid etc., may also be present in
the sample. Hence, selective detection of glucose is of paramount importance. For ascertaining the selectivity of any biosensors, it is customary
to add very large concentrations (about 50 times that of the analyte) of
the so-called interfering agents. To evaluate the selectivity for sensing of
glucose, various concentrations of other compounds ascorbic acid (AA),
dopamine (DA), uric acid (UA) will be added and if there is no change in
the chronoamperometric current response, it indicates the selectivity.
12.8
Enzymatic Sensing of Urea
Urea (also known as carbamide) is one of the final products in protein
metabolism, having immense biological significance in clinical analysis
and dairy industry. The normal level of urea in serum varies from 2.5–7.5
mM.
Here too, enzymatic and non-enzymatic methods have been reported.
Enzymatic sensors for urea make use of the enzyme urease on account of
its specific binding characteristics as well as impressive catalytic performance. The sensing principle is based upon the detection of ammonium
ions resulting from the hydrolysis of urea in the presence of the enzyme
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Biophysical Chemistry
urease viz.
NH2 CONH2 + 3H2 O −→ 2NH4+ + HCO3− + OH−
(12.9)
As mentioned earlier, the cost factor and limited storage characteristics
are the main limitations of enzymatic sensing. The shelf life of enzymebased sensors is not satisfactory. Non-enzymatic biosensors of urea either
employ conducting polymers based metal electrodes or different nanostructures of Nickel.
12.8.1
Potentiometric Sensors
The potentiometric analysis is a passive technique wherein the potential
between an indicator electrode and a reference electrode is measured, at
zero current. The analysis is based on the Nernst equation for the reduction
viz.
Ox + ne− −→ Red
(12.10)
The cell potential is given by
E = E0 − ( RT/nF ) ln
[Red]
[Ox]
(12.11)
where E is the overall cell potential, E◦ is the standard potential, R denotes
the universal gas constant, and T is the absolute temperature. Ion-selective
electrodes are well-known examples of potentiometric sensors, and the
choice of ion-selective membranes is the most essential component. A variety of liquid membranes are available to achieve selectivity and sensitivity.
Among various ion-selective electrodes, the following deserve mention:
(i) Glass membrane electrode for estimating pH and (ii) LaF3 electrodes
for sensing of F− .
A few the major limitations of potentiometric sensors are: (i) the linear
range obtainable is very limited since the potential depends upon log of the
activity or concentration (a change of ten times the analyte concentration
changes the potential by about 59 millivolts); (ii) limited selectivity and (iii)
cumbersome protocols for synthesis of ion-selective membranes.
12.8.2
Voltammetric Biosensors
In recent times, the voltammetric biosensors have gained prominence.
Among the voltammetric techniques, the two most effective ones are: (i)
cyclic voltammetry and (ii) differential pulse voltammetry. In cyclic
Biosensors 273
voltammetry, current-potential response of an electrochemical system is
recorded at an optimum potential and scan rates (typically 100 millivolts/
sec). The peak current (i p in amperes) is linearly related to the bulk concentration of the analyte (in mol cm −3 ) and is given by the so-called RandlesSevcik equation viz.
i p = 0.4463
F3
R
1/2
n3/2 AD01/2 C0∗ v1/2
(12.12)
where A denotes the area of the electrode in cm2 , D denotes the diffusion coefficient of the analyte (in cm2 /sec) and v denotes the scan rate (in
V/sec), with R being the universal gas constant (in J K−1 -mol−1 ). The subtle difference between chronoamperometry and cyclic voltammetry should
be noticed. In cyclic voltammetry, the potential is scanned at selected scan
rates and the ratio (i p /v1/2 ) is often constant. In amperometric sensing,
equal volume of the analytes were added in a controlled manner. In cyclic
voltammetry, the construction of cyclic voltammograms (current vs. potential response) is feasible at all concentrations of the analyte with the proviso
that the peak current should show appreciable change as the concentration
is varied.
Although this is a very simple and powerful technique, it is not selective since other analytes also interfere in sensing and hence distort the
cyclic voltammograms. Nevertheless, this technique has been employed
for sensing of analytes such as dopamine, urea, catechol, glucose, progesterone etc. Among voltammetric biosensors, the differential pulse voltammetry (DPV) occupies a pivotal role in view of its discriminating the voltammetric responses among analytes. This being a pulse technique wherein
small amplitude voltage pulses are superimposed, by optimizing pulse
width and pulse duration, selective sensing of analytes can be accomplished
in a systematic manner.
12.8.3
Impedimetric Biosensors
The impedimetric biosensors constitute a specific application of the electrochemical impedance spectroscopy, often abbreviated as EIS. By applying a
sinusoidal perturbation of a range of frequencies (ω ) to the potential ( E)
viz.
E = E0 sin(ωt)
(12.13)
different types of impedance spectra can be experimentally obtained. In
view of the sinusoidal pulse, the resulting impedance behavior has a real
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Biophysical Chemistry
and imaginary component. The most popular method of analyzing EIS
data consists in drawing the Nyquist plots (imaginary part of the impedance
vs. real part of the impedance). The frequencies are varied from 10−3
Hertz to 106 Hertz, thus providing a complete ‘spectrum’ of all the events
happening in any electrochemical system. Hence, this technique is often
called as ‘impedance spectroscopy’. The Nyquist plots provide semi-circles
from which the charge transfer resistance and solution resistances can be
deduced. Although both these resistances are directly proportional to the
bulk concentration of the analyte, the solution resistance is more sensitive.
The EIS analysis has been employed for the sensing of glucose, catechol,
urea, dopamine etc.
Questions
(1) The sensing of Dopamine was carried out using chronoamperometry
at a carbon paste electrode. The steady state current was 0.71μA for a
5 mL solution containing unknown concentration of the analyte. Upon
adding 0.005 mL of a solution containing 1500 ppb of Dopamine, the
steady state current reached 1.54μA. Estimate the approximate concentration of Dopamine in the original solution.
Solution: It is well known that the steady state current is linearly proportional to the bulk concentration of the analyte viz.
Iss = constant × C (Dopamine)
From the given two data, it is easy to construct the following equations:
Iss (original) = kC (Dopamine)
Iss (upon spiking)
=k
C (added)V (added)
C (Dopamine)V (initial)
+
V (initial) + V(added)
V (initial) + V (added)
Upon combining the above two equations and substituting the given
data,
1.54
0.71
= C(Dopamine)5
C (Dopamine)
+ 1500 × 0.005
5.005
5.005
Thus, the concentration of dopamine in the given solution = 1.25 ppb.
Biosensors 275
(2) The quantitative analysis of progesterone using a pencil graphite electrode yielded the following linear regression equation for the peak current in cyclic voltammetric studies:
I p (mA) = 73.1C + 56.2
where C ranges from 0.105 to 1.825μM. For an unknown solution of
progesterone, the peak current was 100.5 mA. Estimate the concentration of progesterone.
Solution: Substituting the given data,
100.5 = 73.1C + 56.2
Concentration of progesterone = 0.606 μM.
Part II
Biochemical Techniques
13
Surface Plasmon Resonance
Spectroscopy
13.1
Introduction
While well-known physico-chemical techniques can in principle be
employed to all biochemical reactions and processes, there are certain
experimental procedures which are especially applicable in the context of
biophysical chemistry and molecular biology. This field is quite exhaustive, and hence a brief outline is provided here. The nature of the technique
varies with the information sought. For example, the Surface Plasmon Resonance spectroscopy (SPR) provides valuable insights into non-covalent
interactions of proteins. It also yields mechanistic details of the drug delivery process as well as immune response.
Among the several techniques available for the study of biochemical
reactions, the surface plasmon resonance spectroscopy (SPR) may be cited
as a versatile and unique technique. It is a powerful, label free technique
to monitor noncovalent molecular interactions in real time and in a noninvasive manner too. Being a label free technique, it does not require tags,
dyes or special reagents (like enzyme-substrate complexes) to obtain a visible or fluorescent signal. It has important applications in the areas of electrochemistry, biochemistry, and medicinal chemistry.
During the last 20 years, SPR has been applied to study non-covalent
interactions of protein-DNA, DNA-DNA, DNA-RNA and cell-protein interactions. In addition, other studies such as protein-protein, proteincarbohydrate, protein-peptide and self-assembled monolayers and their
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_13
279
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Biophysical Chemistry
interactions have been investigated. SPR has also been employed to study
drug delivery and monitor an immune response against a therapeutic agent.
It has been used to study the kinetics, affinity and changes in concentrations of selected molecules of a reaction.
13.2
Details of SPR Set Up
The following figure shows the essential details of this technique.
Figure 13.1 Air-Solution interface.
A metal film (Ag or Au) is placed at the interface of two dielectric
media. Medium 1 with a higher refractive index (n1 ) is a prism and medium
2 with a lower refractive index (n2 ) may be air or a solution of interest. A
parallel beam of polarized light impinges on the medium of higher refractive index (n1 ) and travels to the medium of lower refractive index (n2 ).
In doing so, the total internal reflection (TIR) can take place within the
medium 1 as long as the incident angle θ is greater than the critical angle
θc , where sin θ = n2 /n1 .
Quickly fading (or evanescent) waves are formed in the medium of
lower refractive index under conditions of TIR. The amplitude of these
standing waves decays exponentially with distance upto the interface
(interface of media 1 and 2). The evanescent wave is enhanced in the presence of a non-magnetic gold film (as in this case) of sufficient thickness,
and it penetrates the gold film and enters the medium 2. The magnitude
of the parallel wave vector, Levan,II is given by
Levan,II = 2πn1 sin θ/λ
(13.1)
where λ = wavelength of incident wave, n1 = refractive index of medium
1 and θ = incident angle.
Surface Plasmon Resonance Spectroscopy 13.2.1
281
Surface Plasmon
They are quanta of plasma, a surface electromagnetic wave whose propagation is confined to metal-dielectric interface. The magnitude of the wave
vector of the surface plasmon ( LSP ) is related to the dielectric constants
of both medium 2 and gold film. For non-absorbing media, the dielectric
constant, ε = n22 . Thus LSP is determined by n2 and n g (n g = refractive
index of gold film) and
LSP
13.3
2 2
n n
2π 2 g
=
2
λ
n2 + n2g
(13.2)
Surface Plasmon Resonance and
Refractive Indices
The surface plasmon can be excited by the evanescent wave and this phenomenon is referred as surface plasmon resonance. When resonance
occurs, the intensity of the reflected wave decreases sharply. The decay of
the excited surface plasmon includes energy conversion to photons
(or phonons). For SPR to occur, LSP = Levan,II . Thus from equations (13.1)
and (13.2),
2 2
n n
2π 2π
2 g
n1 sin θ =
2
λ
λ
n2 + n2g
2 2
n n
1
2 g
sin θ =
2
n1 n2 + n2g
⎛ ⎞
2
2
n
×
n
1
g
2
⎠
θSPR = sin−1 ⎝ 2
n1 n2 + n2g
(13.3)
(13.4)
(13.5)
The angle required for the resonance, θSPR , is related to n2 when n1 and
n g are fixed. Adsorption, desorption processes on gold surface changes
the refractive index of medium 2 near the metal-dielectric interface and
thus the resonance angle changes. Hence, by monitoring change in SPR,
adsorption–desorption, association–dissociation reactions occurring on
gold surface can be studied.
The intensity of reflected wave as a function of incident angle for gold
film in air is shown in Figure 13.2.
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Biophysical Chemistry
Intensity of reflected
light (mV)
300
250
200
SPR angle (35.2°)
150
25
50
75
Incident angle (deg)
Figure 13.2 Reflection of wave for gold film in air.
The variation of SPR angle with refractive index is shown in Figure 13.3.
Figure 13.3 Variation of SPR angle with refractive index.
Figure 13.4 Binding of rabbit lgG to protein A and anti-rabbit to lgG to
FAB
Surface Plasmon Resonance Spectroscopy 283
Some applications of SPR spectroscopy
(1) Bovine rhodopsin (the light activated receptor) was incorporated into
an egg phosphatidylcholine bilayer and this was deposited on a thin
silver film. The tight binding and activation of its associated G-protein
was established by SPR.
(2) In another experiment, Streptavidin was bound to biotinylated-thiols
in a mixed self-assembled monolayer (SAM) with an excess of ωhydroxy-undecanethiol (HTA) on the metal surface, which then get
bound the biotinylated receptor to the surface through its extracellular N-terminus in a defined orientation. Following immobilization of
the receptor and thorough washing with detergent, a supported lipid
bilayer was formed around the receptors. The activity of the immobilized receptor was observed in SPR data following its illumination with
light which was achieved by SPR. Illumination-induced activity of the
G-protein was followed by its desorption from the membrane. Ligand
binding was monitored and quantified from SPR data by adding IIcis-retinol with increasing concentrations to the immobilized and completely photolysed receptor.
The above experiment is pictorially demonstrated below:
(1) SPR has been used to characterize ligand binding to the human
chemokine receptors CCR-5 and CXCR-4. These receptors have also
been used to demonstrate important developments in SPR methods
for purification, solubilization, reconstruction and functional analysis
of GPCR’s.
Figure 13.5 Illumination-induced activity of G-protein and its desorption
form the membrane.
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Biophysical Chemistry
(2) A novel receptor analyte configuration was used to characterize neurotensin receptor-I binding to the neurotransmitter peptide neurotin
using 5PR.
(3) Using the adenosine A2A receptor, a new approach called Biophysical
Mapping (BPM) has been developed that combines a thermostabilized
GPCR with SPR analysis of ligand binding to binding site mutants.
13.4
Kinetic Applications of SPR Spectroscopy
For a bimolecular reaction between two species S and L according to
k1
BG SL
S + L FGGGGGG
GGGGG
k −1
(13.6)
d[SL]
= k1 [S][ L] − k −1 [SL]
dt
(13.7)
where k1 and k −1 are forward and reverse rate constants. The concentration of SL is monitored by measuring the change in the refractive index at
the surface of the sensor.
By varying the concentration of L the SPR response for the system is
fit to the integrated rate equation
Rt =
Rmax k1 [ L] 1 − e−(k−1 [ L]+k1 )t
k 1 [ L ] + k −1
(13.8)
where Rt is the response measured at time t, Rmax is the maximum response
obtained upon saturation of S with L. By varying the concentration of L,
and by fitting the data to equation (13.7), k1 and k −1 can be determined.
The binding of a target ligand (mass = 50, 000 a.m.u.) to immobilized chymotrypsin was studied.
A number of antibodies-antigen interactions have been studied using
SPR. The interaction rates of DNA-based aptamers with human immune
globulin E have been studied. SPR has also been used to examine the interactions of human carbonic anhydrase-I with various sulfonamide inhibitors.
SPR has been employed to investigate biological interactions that include
reactions with second order rate constants in the range of 102 to 108 M−1
sec−1 and first order rate constants from 10−6 to 1 sec.
Surface Plasmon Resonance Spectroscopy 285
Questions
(1) The magnitude of the wave vector of surface plasmon Lsp is given by
2 2
2 2
n
n
n n
λ 2 g
λ
2 g
(c)
L
=
(a) Lsp =
sp
2
2
2π n2 + n2g
π n2 + n2g
2
2 2
2
n
+
n
n n
2π 2
2π g
2 g
(b) Lsp =
(d)
L
=
sp
2
2
λ
λ
n2 n2g
n2 + n2g
(n2 = refractive of medium; n g = refractive index of gold film; λ =
wavelength of the surface wave)
(2) If the refractive indices of glass and gold film are 1.5 and 0.181 respectively, the SPR angle for resonance is
(a) 10.5◦
(b) 7.4◦
Solution:
(c) 22.9
(d) 20.9
2
n × n2g
1
2
sin θ =
n1 n22 + n2g
where n1 is refractive index of glass, n2 is refractive index of gold film,
therefore
1
0.33 × 2.25
sin θ =
1.5 0.33 + 2.25
1
0.7425
=
1.5
2.58
= 0.667 × 0.536 = 0.357
θ = 20.9
(3) Explain the principle of surface plasmon resonance spectroscopy and
discuss some applications of this technique.
(4) Outline the applications of SPR spectroscopy in ligand binding to
human chemokine receptors.
14
Affinity Chromatography
14.1
Introduction
Affinity chromatography is one of the most diverse and important methods for the purification of a given molecule or a group of molecules from
complex mixtures. It is based on highly specific biological interactions
between two molecules such as interactions between an enzyme and substrate, a receptor and ligand or antibody and antigen. These interactions,
which are typically reversible, are used for purification by placing one of
the interacting molecules, referred as affinity ligand, onto a solid matrix
to create a stationary phase while the target molecule is in the mobile
phase. A successful affinity purification requires some understanding of
the nature of interactions between the target molecule to help determine
the selection of an appropriate affinity ligand and purification procedure.
Because this technique relies on this interaction between molecules, it can
purify a molecule on the basis of its biological function or individual chemical structure.
This technique can also be used for separation of biomolecules based
on highly specific biological interactions between two molecules such as
an enzyme and substrate. The biological interactions between the ligand
and a target molecule can be a result of electrostatic or hydrophobic interactions, van der Waals forces or H-bonding.
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_14
287
288
14.2
Biophysical Chemistry
Methodology
The separation procedure in affinity chromatography is illustrated in the
following diagram which consists of four steps.
The sample is applied under optimum conditions that form specific
binding of the target molecules to the binding molecules (ligand). The
desired molecules bind specifically but reversibly to the ligand and
unbound molecules wash through the column.
Target molecule is recovered by changing conditions that favour elution of the bound molecules. Elution is carried out specifically using a
competitive ligand or non-specifically by changing pH, ionic strength or
polarity. Target protein is collected in a purified, concentrated form.
Affinity medium is re-equilibrated with binding buffer. In the following figure, a typical scheme is shown which shows the application of affinity chromatography. Affinity chromatography with biological ligands is
referred as “bioaffinity chromatography”.
A number of names have been proposed depending upon the wide
application potential of affinity chromatography. Some of them are:
(i) Immuno affinity chromatography,
(ii) High performance affinity chromatography,
(iii) Lectin affinity chromatography,
(iv) Dye-ligand affinity chromatography,
(v) Affinity electrophoresis,
(vi) Avidin-Biotin immobilized system based affinity chromatography,
Figure 14.1 Separation procedure in affinity chromatography.
Affinity Chromatography 289
Figure 14.2 Variation of absorbance with time.
(vii) Membrane based affinity chromatography,
(viii) Covalent affinity chromatography, and
(ix) Hydrophobic chromatography.
Some factors that influence the success of an affinity chromatographic
experiment are:
(i) Selectivity of ligand,
(ii) Recovery process,
(iii) Reproducibility,
(iv) Stability, and
(v) Economic criteria.
Before the start of an experiment, the following factors need, therefore, to
be considered: (i) Support material, (ii) Activation method, (iii) Ligand,
(iv) Immobilization method, and (v) Conditions for adsorption and desorption. A brief description of each of the above factors is given below.
14.2.1
Support Material
It is necessary that the support in the column contains an affinity ligand
capable of forming a strong complex with the solute of interest. Also, the
support material must be chemically and biologically inert to avoid unspecific bindings. The material must also be hydrophilic as most reactions are
carried in aqueous solution. Uniformity of particle size and ease of activation of support material are also important. The support materials can be
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Biophysical Chemistry
divided into three categories: (a) natural, such as agarose, dextrose, cellulose, (b) synthetics such as acrylamide, polystyrene, polymethyl methacrylate, (c) inorganic, such as silica, glass etc. Among the above materials,
agarose is widely used. The structure of agarose may be represented as
shown below.
CH2OH
H
O
HO
O
H
H
O
OH
H
O H
H
H
H
CH2OH
O
O
H
O
OH
Figure 14.3 Structure of agarose.
Cellulose is another example of polysaccharides which is used as support in affinity chromatography. Other supports used are polystyrene,
porous glass, silica. Silica based materials are used in high performance
affinity chromatography because they are basically hydrophilic as can be
seen by the structure of silica.
OH
Si
OH
O
Si
OH
O
Si
O
O
O
Si
Si
Si
Figure 14.4 Structure of silica.
Membranes have been used in various forms such as stacked sheets, in
rolled geometrics or as hollow fibers. Materials commonly used for membranes are cellulose, polysulfone and polyamide.
14.2.2
Ligand
Ligands are molecules that bind reversibly to a specific molecule enabling
purification by affinity chromatography. These molecules play a major role
in the specificity and stability of the system. The selected ligand must be
capable of selectively and reversibly binding to the substance to be isolated and have also some groups which are available for modifications to
Affinity Chromatography 291
be attached to the support material. There are a variety of ligands like
dyes, amino acids, protein A, lectin, metal chelates as well as specific ligands such as enzymes, substrates, antibodies and antigen. These ligands
can be synthetic or biological too. Biological ligands include RNA, DNA
fragments, nucleotides, coenzymes, vitamins, binding or receptor proteins.
Synthetic affinity ligands are generated by synthetic methods or by modification of existing structures like purine, pyrimidine structures, unnatural
peptides, triazine based ligands, oligosaccharide ligands etc. Many parameters such as cost, selectivity, stability, toxicity have to be considered.
Despite the advantages of the techniques its use is limited due to the
high cost of affinity ligands and their biological and chemical instability.
Biological ligands have high selectivity but their binding capacity is low.
Synthetic ligands are in that sense preferable because they provide selectivity and are also inexpensive. Ligands which possess affinity to the immobilized protein are suggested as ligands. The selection of the ligand may
be designed according to the structure of the target protein as well.
14.2.3
Immobilization of Ligand
Immobilized ligand is an essential factor that determines the success of the
method. Several methods are available to couple a ligand to a preactivated
matrix. Before the ligands are coupled, the matrix is activated. Commercially preactivated products are available in the market and a few are listed
in Table 14.1.
Table 14.1 Examples of pre-activated products for immobilizing ligands.
Product name
Functional group specificity
Carbolink coupling resins
CHO, C=0
Epoxy activated agrose 6B
−NH2 , −OH, −SH
Tresyl chloride activated agarose
−NH2 , −SH
Ultra link iodoacetyl resin
−SH
Profinity epoxide resin
−NH2, −OH, −SH
EAH sepharose 4B
−COOH, −CHO
Thiopropyl sepharose 6B
−SH
After the activation of the support material, it is ready for immobilization process of ligand. If the ligand is a small molecule, steric hindrance will occur between the immobilized support and the compound
of interest. Use of supports having spacer arms before immobilization is
recommended.
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Biophysical Chemistry
Ligand
Amt
eluted
Spacer
arm
0
5
10
15
20
25
Elution volume (ml)
Figure 14.5 Dependence of eluted amount with elution volume.
Spacer arms are used to improve binding between ligand and a target
molecule by overcoming effects of steric hindrance. Compounds which
have diamine groups such as hexanediamine, propane diamine, ethylenediamine are preferred spacer arms. The next step is the immobilization
of ligands on the activated matrix. For this, esters like N-hydroxy succinimide esters are used. Immobilisation methods can be classified as follows.
General types of immobilisation methods
Covalent
methods
Nonspecific
adsorption
Noncovalent
methods
Biospecific
adsorption
Co-ordination
methods
Entrapment
Figure 14.6 Different types of immobilization methods.
The simple adsorption of ligand to surface, binding to a secondary ligand, or ligand immobilization through a co-ordination complex belong to
non-covalent immobilization method. In covalent immobilization method,
it is necessary to activate the ligand or support first. Activating of the ligand can be carried when it is desired to couple this ligand through a specific region. An example is the immobilization of proteins through their
amino group to supports activated with N-hydroxy succinimide or carbonyl diimidazole. Amine groups are often used to immobilize proteins
and peptides. Reductive amination (also known as Schiff’s base method)
couples’ ligands to activated periodate is used to oxidize diol groups on
the surface of the support to give aldehydes.
Affinity Chromatography 14.2.4
293
N-hydroxy Succinimide Method (NHS)
The NHS method is often employed when immobilizing biomolecules
through amine groups. This gives rise to the formation of a stable amide
bond. The immobilization via NHS is depicted below:
O
O
O
NH2 +
O
N
O
C
(CH2)n
C
O
O
N
O
DMF
O
O
NH2
C
O
(CH2)n
C
O
N
O
NHS activated support
pH 7–8 Ligand
O
NH2
C
NH2
O
(CH2)n
C
O
NH
Ligand
Figure 14.7 Immobilization by N-hydroxy succinimide method.
14.2.5
Carbonyl Diimidazole Method (CDI)
This reagent can be used to activate supports for the immobilization of
amine containing ligands. This method is simple and easy to carryout.
The immobilization using CDI is shown in Figure 14.8.
There are several other methods for preparing affinity supports such
as sulfhydryl reactive methods, haloacetyl method, maleimide method,
pyridyl disulfide method etc.
14.2.6
Elution
Elution is a critical step for success of the experiment. After all non-retained
components are washed off the column, the retained solute with the ligand as ligand-solute complex can be eluted by treating with solvent. Step
gradient elution is the most common method employed in affinity chromatography.
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Biophysical Chemistry
O
COOH
+
N
Carboxyl
containing matrix
O
C
N
C
N
N
Carbonyl diimidazole
O
O
C
N
N
Carbamate support
pH 9–10 Ligand
NH2
O
C
NH
Ligand
Figure 14.8 Immobilization using carbonyl diimidazole method.
Step elution method is employed if the ligand has high affinity for target molecule. There are also other factors to be considered such as strength
of solute-ligand interaction, amount of immobilised ligand present and the
kinetics of solute-ligand association which have influence on retention and
elution of the compound. Obtaining stable biomolecules in high yield and
purity is of utmost importance in elution process.
14.3
Types of Affinity Chromatography (A.C.)
Depending on the type of ligand used, several names have been given to
affinity chromatography, such as Immuno affinity chromatography,
Protein-A or Protein-G A.A, Lectin A.A, Dye-ligand A.A, metal-chelate
A.A, etc. A brief account of these techniques is given below:
• Lectin A.C.: Certain types of carbohydrate residues may be separated by this method because all lectins have the ability to recognize
and bind this type of compounds. The commonly used columns are
concanavalin A, soybean lectin, and wheat germ agglutinin. Concanavalin A is specific for α-D-mannose and α-D-glucose while wheat
germ agglutinin binds to D-N-acetyl glucosamine. Some commonly
used lectins for isolation of compounds containing carbohydrates and
polysaccharides are listed in Table 14.2.
Affinity Chromatography Table 14.2 Examples of commonly used lectins
drates and polysaccharides.
Lectin Source
Sugar specificity
Con A Jack bean
α-D-mannose,
seeds
α-D-glucose
WG A Wheat germ N-acetyl-β-Dglucosamine
PSA
Peas
α-D-mannose
LEL
Tomato
N-acetyl-β-Dglucosamine
STL
Potato
N-acetyl-β-Dtubers
glucosamine
PHA
Red kidney
N-acetyl-β-Dbean
glucosamine
295
for isolation of carbohyEluting sugar
α-D-methyl mannose
N-acetyl-β-Dglucosamine
α-D-methyl mannose
N-acetyl-β-Dglucosamine
N-acetyl-β-Dglucosamine
N-acetyl-β-Dglucosamine
Enzymes, cofactors, inhibitors, nucleic acids can be used as ligands
in bio-affinity chromatography.
• Immuno A.C: In this technique, the stationary phase comprises of
an antibody or antibody related agent. This technique is useful for
analyzing natural food contaminants such as aflatoxins. First, the
antibodies are immobilized on a support. To bind the ligand on the
support properly, protein-A or protein-G is often used as a bridge
which provides enough space for ligand protein binding. Initially, the
antibodies should be purified prior to preparing the immunoaffinity
column. Both large and small analytes can be determined using direct
detection in A.C. Immuno A.C. is a highly specific form of bioaffinity
chromatography.
• Metal-Chelate A.C.: This technique exploits selective interactions
and affinity between transition metal ions immobilised on a solid
support (resin) via a metal chelator and amino acid residues in the
protein of interest. Amino acids such as histidine, tyrosine, phenylalanine, tryptophane form complexes with transition metal ions.
Adsorbents may be prepared by binding chelators onto the surface
and metals to the chelators. Zn2+ , Na2+ , Cu2+ and Hard Lewis acids
like Ca2+ , Mg2+ , Fe3+ , soft Lewis acids like Ag+, Cu+ are some of
the common used metal ions. Ligands containing −COOH groups,
aliphatic nitrogen groups in compounds like glutamine, phosphorylated amino acids, cysteine are the ligands of choice. For metal ions
like Cu(II), Ni(II), Co(II), Zn(II) the target amino acids on the protein
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Biophysical Chemistry
surface are imidazoyl, thiol and indolyl groups. A list of chelating
agents and the metal ions are given in Table 14.3.
Table 14.3 List of chelating agents and corresponding metal ions.
Chelating
Salicyl aldehyde
8-hydroxy quinolone
compound
Metal ion
Ca(II)
Al(III), Fe(III), Yb(III)
Co-ordination
Bidentate
Bidentate
Imino diacetic acid
Ortho phosphoserine Nitrilotriacetic acid
Cu(II), Zn(II), Ni(II),
Fe(III), Al(III), Yb(III), Ni(II)
Co(II)
Ca(II)
Tridentate
Tri-dentate
Tetra-dentate
N-N-Nt’-(tricarboxy methyl) ethylene diamine
Cu(II), Zn(II)
Penta-dentate
• Protein-A or Protein-G A.C.: These ligands are capable of binding to
many types of immunoglobulins near about neutral pH.
These two ligands differ in their ability to bind to antibodies from different species. They are good ligands for the separation of immuneglobulins.
• Dye ligand A.C.: Some proteins bind triazine dye and this allows it
to be used as an affinity adsorbent by immobilization. This method is
especially popular for enzyme and protein purification. Procion Red
HZ3b, cibacron Blue F3GA are some examples of dyeligands use for
purification.
14.3.1
Applications
Affinity chromatography finds applications in pharmaceutical and biomedical analysis, drug discovery. This technique is useful in isolating and identifying target molecules for a specific ligand utilizing affinity between biomolecules such as antigen-antibody reactions. An example of a target protein isolated and identified by using this technique is given in Figure 14.9.
14.3.2
Purification of Specific Groups of Molecules
The diversity of antibody-antigen interactions available makes them useful
for therapeutic and diagnostic applications as well as for immunochemical
techniques. Affinity chromatography provides an important method for
purification of antibodies and antibodies.
Affinity Chromatography NH
H2N
O
H
N
N
H
297
OH
O
N
H
O
O
Figure 14.9 Target protein identification using affinity chromatography.
Protein-A and Protein-G are bacterial proteins, which upon coupling
to Sepharose create very useful media for many applications. Examples
include purification of monoclonal IgG type antibodies, purification of
polyclonal IgG subclasses and adsorption and purification of immune complexes involving IgG.
14.4
Kinetic Applications of Affinity
Chromatography
An important advantage of using AC or high-performance AC for kinetic
investigations of biological reactions is its ability to use the same immobilized binding agent for many experiments. Two important advantages
Figure 14.10 Application of high performance affinity chromatography in
estimating rate constants.
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Biophysical Chemistry
that this method offers are: (i) its reproducibility and (ii) variety of methods available for kinetic experiments. Second order rate constants ranging
from 103 to 107 M−1 sec−1 and dissociation constants in the range 10−2 to
10−1 sec−1 have been measured.
Various supports and surfaces can be used for immobilizing binding
agent since detection is done after the target or other sample components
have eluted from the column. For detection, absorbance, fluorescence,
mass spectrometry have been used. The immobilisation methods involve
the use of amines, thiols for immobilization of proteins.
Two methods have been employed in H.P.A.C. and AC for determining the rate constants. They are depicted in the Figure 14.11.
Analysis of band broadening in affinity chromatography provides
information on the kinetics of interaction of an analyte with a given binding agent. The plate height method for kinetic studies makes use of a
small quantity of target that is injected onto an affinity column as well as
onto control column at several flow rates.
This method was used to analyze the binding of D-tryptophan with an
HPAC column containing immobilized HAS as shown in Figure 14.11.
Figure 14.11 Binding of D-tryptophan with a HPAC column containing
immobilized HSA.
Affinity Chromatography 299
Questions
(1) (a) Explain the application of affinity chromatography in the separation of biomolecules.
(b) What are the different types of affinity chromatography employed
in the separation procedure. Give an account of support materials.
(2) Describe the application of affinity chromatography in kinetic studies.
(3) The technique of affinity chromatography is most useful for
(a) synthesis of a compound from the required reactants.
(b) purification of a compound from complex mixtures.
(c) separation of a compound from mixtures containing high concentration of contaminants.
(d) none of the above.
(4) SPR spectroscopy is generally useful to study the rates of reactions of
(a) first order reactions with rate constants of the order 10−8 sec−1 .
(b) zero order reactions with rate constants of the order of 105 moles
l−1 sec−1 .
(c) second order reactions with the rate constants of the order of 102 to
108 l mol−1 sec−1 .
(d) none of the above.
15
Capillary Electrophoresis
15.1
Introduction
This technique employs narrow bore capillaries whose internal diameters
range from 20 to 200 μm to perform separations of a variety of molecules
with high efficiency. The separations are carrier out using high voltage
which generates electro osmotic and electrophoretic flow of buffer constituents and ions within the capillary.
The format of Capillary Electrophoresis (CE) requires the following: (i)
capillary tubing of the dimensions mentioned above, (ii) high electric field
strengths of the order of 500 V/cm and (iii) modern detector technology.
Its advantages are
(i) requirement of very small samples,
(ii) can be automated for precise quantitative analysis,
(iii) user-friendly,
(iv) use of small amounts of reagents, and
(v) applicability to a wide selection of analytes.
15.2
Basic Instrumentation
(i) A fused silica capillary with an optical viewing window, (ii) A controllable high voltage power supply, (iii) Two electrode assemblies, (iv) Two
buffer reservoirs, (v) An ultraviolet detector are the required components.
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_15
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Biophysical Chemistry
A schematic diagram of a CE, unit is given in Figure 15.1.
Figure 15.1 Diagram of a capillary electrophoresis unit.
After filling the capillary with buffer, the sample is introduced by dipping the capillary into sample solution and raising the capillary above the
detector side buffer reservoir.
Useful Terminology in CE
(i) Migration time (tm ), it is the time taken by a solute to move from the
beginning of the capillary to the detector window. (ii) electrophoretic mobility (μeq which has units cm2 V −1 sec−1 ), (iii) electrophoretic velocity
Vep (cm sec−1 ) and (iv) electrical field strength E (V/cm). The relation
among them is given by
μeq =
Vep
L /tm
= d
E
V/Lt
(15.1)
where Ld = length of capillary upto the detector, V = potential, Lt = total
length of capillary.
Electro Osmosis
This phenomenon is a consequence of the surface charge on the wall of the
capillary. The fused silica capillaries have ionizable groups in contact with
the buffer in the capillary. The electro osmotic flow is given by
Vε0 =
εZJ
E
4πη
(15.2)
ε = dielectric constant of the medium, η = viscosity of the buffer, J = zeta
potential close to liquid-solid interface.
Capillary Electrophoresis 303
The negatively charged wall attracts the +vely charged ions from the
buffer which results in the electrical double layer. Under the influence of
an applied potential across the capillary, cations in the diffuse portion of
the double layer move towards the cathode carrying water with them. The
net result is flow of buffer solution in the direction of negative electrode.
This flow enables the simultaneous analysis of cations, anions and neutral
species in a single analysis.
The effect of pH on electro osmotic flow is shown below.
Electroosmotic flow
High pH
+ O+ O+ O+ O+
–
Low pH
+ OH O
+
OH O OH –
+
Figure 15.2 Effect of pH on electro osmotic flow.
15.3
Capillary Diameter and Joule Heating
The production of heat in CE is the result of application of high field
strengths. The quantity of heat generated is proportional to the square
of the field strength. Temperature gradients across the capillary are a consequence of heat dissipation.
Both the electro osmotic flow and electrophoretic velocities are directly
proportional to the field strength. Hence, use of highest possible voltages
will result in shorter times of separation.
15.4
Various Types of Electrophoresis
The various types of electrophoresis have different operative and separative characteristics. The techniques are: (i) Capillary zone electrophoresis (CZE), (ii) Isoelectric focusing (IEF), (iii) Capillary gel electrophoresis
(CGE), (iv) Isotachophoresis (ITP), and (v) Micellar electrokinetic capillary
chromatography (MECC). A brief account of these types is given below.
(i) Capillary Zone Electrophoresis (CZE): The separation mechanism in
this technique is based on differences in charge to mass ratio. In CZE,
it is important to maintain homogeneity of the buffer solution and
constant field strength throughout the length of the capillary.
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Biophysical Chemistry
After injection and application of voltage, the components of a simple
mixture separate into discrete zones as shown below:
Figure 15.3 Separation of components using electrophoretic.
The fundamental parameter, the electrophoretic mobility μeq is given
by
q
μeq =
(15.3)
6πηRhyd
where q = net charge, Rhyd = hydrodynamic or Stokes radius, η =
viscosity of the medium.
The movement of a species in a CZE experiment depends upon the
charge which in turn depends on pH of the medium. Zwitterions
like those of amino acids, proteins, peptides exhibit charge reversal
at their isoelectric points and hence shifts in the direction of electrophoretic mobility occur. For capillary material, fused silica is preferred because of its UV transparency and durability. The capillary
must be conditioned before its use.
Buffers: A wide variety of buffers are employed in CZE. Zwitterionic
buffers such as bicine, tricine, MES and Tris are commonly employed
in protein and peptide separations. These buffers have the advantage
of low conductivity and reduced Joule heating. Sometimes, buffer
additives can be employed to change the selectivity of separation.
They also change the electrophoretic mobilities.
Applications: Among the analytes hitherto-studied using CZE, mention may be made of the following: (i) proteins; (ii) β-lactoglobulin;
(iii) myoglobin; (iv) ribonucleases; (v) Adenosine-5 [α − 32P] Triphosphate; (vi) α-chymotrypsin and (vii) collagens.
(ii) Isoelectric Focusing (IEF): It is assumed in IEF that a charged
molecule will migrate under the influence of an applied field. A gradient of electric field with low pH at anode and high pH at cathode
is maintained in IEF. The pH is generated with a number of zwitterionic compounds known as carrier ampholytes. The ampholyte
mixture gets separated under the field and they migrate to cathode
Capillary Electrophoresis 305
or anode depending on the charge they carry. The pH will decrease
at cathodic section and increase at anodic section. Ampholyte migration will cease once the isoelectric point of the ampholyte is reached.
The migration of charges under the field is shown in Figure 15.4.
−
pI (Isoelectric point)
Low pH
⊕
High pH
Figure 15.4 Migration of charges using isoelectric focusing method.
The electro-osmotic flow and other convective forces must be suppressed for effective IEF. For this purpose, the capillary walls are
coated with methyl cellulose or polyacrylamide IEF is used for high
resolution separation of proteins and polypeptides.
There are three basic steps in IEF: (i) loading, (ii) Focusing, and (iii)
mobilization. After loading and focusing are completed, the gel is
stained using traditional methods. In IEF, the bands must migrate
past the detector. As mentioned earlier, a pH gradient is formed
along the capillary.
Applications: IEF is useful for separating immunoglobulins, hemoglobin variants, recombinant proteins. The isoelectric point of a protein can be determined. A diagram showing the separation of a protein mixture is given below:
Figure 15.5 Separation of proteins using isoelectric focusing method.
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Biophysical Chemistry
(iii) Capillary Gel Electrophoresis (CGE): CGE is conducted in an anticonvective medium such as polyacrylamide or agarose. The gel suppresses the EOF. The composition of the media can also serve as a
molecular sieve to perform size separations as shown below:
Figure 15.6 Size separation using capillary gel electrophoresis.
Two classes of gels are employed in CGE: (i) physical gels and (ii)
chemical gels. The physical gel attains its porous structure by entanglement of polymers and is quite rugged to changes in environment.
An example of such gels is hydroxy propyl methyl cellulose. Chemical gels use covalent attachment to form porous structure.
Physical
gel
Chemical
gel
Figure 15.7 Physical and chemical gels.
Cross linked polyacrylamide is commonly used as the gel forming
agent along with urea and other buffer agents like Tris-borate-EDTA.
CGE is usually carried in 50–100 μm capillaries of length 10 cms to
1 m. The applied voltage is restricted to less than 500 V/cm.
Applications:
(i) Protein separations: Proteins are initially denatured using 2mercapto ethanol and treated with SDS. Under these conditions,
all the proteins have the same charge to mass ratio because the
native charge is obscured by SDS binding.
In the presence of SDS, all proteins become negatively charged
and migrate towards anode. They unfold and have rod like
structure allowing uniform molecular sieving for size separation. A calibration graph of mobility vs. molar mass permits size
assignments of the fragments. Usually, 10–20 cm size capillaries
Capillary Electrophoresis 307
are employed with field strengths in the range of 400 V/cm.
Under these conditions the correlation of mobility with molecular weight is linear.
The denaturing of proteins is usually done in 1% SDS and 2%
mercapto ethanol for 30 minutes at 363 K.
(ii) DNA: Separation of oligonucleotides and DNA sequence products are carried out in poly acrylamide gels. Separation of deoxy
oligonucleotides such as poly (dA) 40–60 is accomplished in 8%
T gel using a buffer of Tris-borate at pH = 8.3 in presence of
2 mMEDTA and 7 M urea. Determination of the purity of synthetic oligonucleotides is an important application of CGE. The
CGE of thymidine synthetic homopolymer using buffer of Tris
(25 × 10−3 M), boric acid (25 × 10−3 M), 7 M Urea and polyacrylamide gel, 7.5% T, 3.3% C is shown below:
Figure 15.8 Capillary gel electrophoresis of thymidine synthetic polymer.
Double stranded DNA can be separated with physical gels. The
separation of Hae-III restriction digest of OX174 DNA has also
been carried out using CGE.
(iv) Isotachophoresis (ITP): Like IEF, ITP is based on zero EOF and the
buffer system is heterogeneous. The sample is injected after filling
it with a leading electrolyte that has a higher mobility than any of
the sample components to be determined. A terminating electrolyte,
whose ionic mobility is lower than any of the sample components,
occupies the opposite reservoir. The separation occurs in the gap
between the leading and terminating electrolytes based on individual mobilities of analytes. Highly efficient separations result due to
stable boundaries formed between individual components.
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Biophysical Chemistry
Differential conductivity and an UV detection are commonly
employed as detection techniques. The selection and optimization
of the buffer are important factors to be considered in these studies.
The ITP of a mixture of anions with conductivity detection is shown
below:
Figure 15.9 Isotachophoresis of a mixture of anions.
Capillary size: 105 μm id;
fluorinated ethylene-propylene co-polymer leader;
10 × 10−3 HCl titrated to pH = 6.0 with histidine,
2 × 10−3 hydroxyethyl cellulose terminator;
5 × 10−3 MES,
current = 10μA.
Anions measured: L; Cl− ; 1: SO24 ; 2 : ClO3− ; 3: CrO24− ;
4: malonate; 5: adipate; 6: benzoates; 7: impurity; 8: acetate;
9: β-bromopropionate; 10: naphthalene, 2-sulfonate;
11: glutamate; 12: enantate, T, MES.
In isotachophoresis, all bands move with the same velocity and bands
are clear cut. EOF can be suppressed with 0–25% hydroxypropyl
methyl cellulose. Leading electrolyte 5 × 10−3 H3 PO4 and 10−2 M
Valine as terminating electrolyte are commonly employed.
(v) Micellar Electrokinetic Capillary Chromatography (MECC): The
use of micelle forming surfactant solutions can give rise to separations resembling reverse phase L.C.
Capillary Electrophoresis 309
Among the surfactants, the anionic surfactant, SDS and cationic surfactant CTAB are most useful in MECC. Naturally occurring bile salts
are also useful. Micelles have the ability to organize analytes at molecular level based on hydrophobic and electrostatic interactions. Even
neutral molecules can bind to micelles since the hydrophobic core
has strong solubilizing power. Surfactant solutions can serve as chromatographic mobile phase carriers with respect to MECC. The analyte can partition between micellar phase and bulk phase or between
micellar and stationary phase as well.
Separation mechanism: In neutral or alkaline pH, a strong EOF
moves in the direction of cathode. In the presence of an anionic surfactant like SDS, the electrophoretic migration of the anionic micelle
is towards anode. When an analyte is associated with a micelle, its
overall migration velocity is slowed. Analytes that have greater affinity for micelle have slower migration velocities compared to analytes
that spend more time in bulk phase. When using a cationic surfactant the EOF is reversed. Therefore, the electrode polarity must also
be reversed to detect the analyte.
Order of migration: In presence of SDS, the general migration order
is anions, neutral molecules, cations. Anions spend more time in the
bulk phase than in micellar phase because of electrostatic repulsions
between the −ve surfactant and anions. Neutral molecules get separated due to hydrophobicity. Cations elute last due to strong electrostatic attraction due to the formation of ion pairs with micelles.
Applications: The separation of some corticosteroids using NaClO3
as a surfactant has been demonstrated by MECC.
Use of organic modifiers reduces EOF and the overall peak capacity
of separation also increases. Modifier also makes the bulk solution
more receptive to hydrophobic analytes. The commonly used organic
modifiers are methanol and acetonitrile in the range 5 to 25%. Other
solvents like DMSO, DMF are also used as modifiers.
15.5
Chiral Recognition
Additives such as optically active bile salts and cyclodextrins permit chiral
resolution by stereo selective interaction with the solute. The interaction
occurs with in the molecular cavity in the case of cyclodextrins by forming an inclusion complex. When an analyte is complexed with micellar or
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Biophysical Chemistry
cyclodextrin additive, its migration velocity is slowed down relative to the
bulk phase.
Another approach to chiral selectivity is precapillary derivatisation.
The analyte is derivatised with an optically active reagent to form covalently bound diasteromers. They are easily separable by MECC.
The separation of chiral amino acids derivatised with Marfey’s reagent
(1-fluoro-2, 4 dinitrophenyl-5-L-alanine) is shown in the Figure 15.10.
Figure 15.10 Separation of chiral amino acids using MECC.
MECC has been applied to a variety of separation studies involving
modified nucleic acids, penicillin, urinary porphyrins, water-soluble vitamins, β-lactum antibiotics, sulphonamides etc.
Questions
(1) (a) Give a sketch of the capillary electrophoretic unit and indicate the
various parts.
(b) What is capillary zone electrophoresis and describe some of its
applications.
(2) Electroosmosis is due to
(a) flow of ions of the buffer to the respective electrodes.
(b) flow of ions of the analyte to the respective electrodes.
Capillary Electrophoresis 311
(c) the presence of surface charges on the walls of the capillary.
(d) the absence of electrical double layer in the analyte.
(3) Explain the principle of capillary gel electrophoresis and outline its
uses in separation of proteins and DNA.
(4) What is Micellar electrokinetic capillary electrophoresis and discuss its
applications.
(5) Calculate the electrophoretic mobility of blood given its viscosity is
4.5 × 10−2 pascals and its hydrodynamic radius as 3 nm.
(6) Calculate the electrophoretic mobility of a solute moving under an electrical field of strength 250 V cm−1 in which the length of the capillary
is 10 cm (upto detector window) with the total length of the capillary
being 15 cm. Under these conditions, the solute takes 30 min to migrate
from the beginning of the capillary to the detector window. What is its
mobility if the electric field strength is doubled?
16
NMR Technique in the
Elucidation of Biochemical
Problems
16.1
Introduction
NMR is a spectroscopic technique concerned with the magnetic properties
of certain nuclei (NMR active) such as the nucleus of H atom or the C-13
isotope of carbon. Only those nuclei possessing non-zero nuclear spin
have a nuclear magnetic moment which produces magnetic interactions
with an external magnetic field. The NMR active nucleus will have the
energy level splitting when placed in an external magnetic field. A radio
frequency pulse of the right frequency would induce transitions of nuclei
from the lower to higher energy levels.
After the radio frequency pulse is switched off, the nucleus at the
higher energy states relaxes to the lower energy states giving out signals.
This is referred as Nuclear Zeeman effect. Since the energy level splitting is different for different nuclei, the frequency of the signal becomes a
characteristic for a specific atom (Larmor frequency). Since the resonance
frequency W0 of the radiation is proportional to ΔE, the energy of splitting
which in turn is proportional to magnetic fields strength H, one may write
W0 = γH
(16.1)
where γ is a proportionality constant known as gyromagnetic ratio and is
a constant for a given atomic nucleus.
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_16
313
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Biophysical Chemistry
Atoms such as 1 H, 2 H, 13 C, 15 N, 31 P are common nuclei studied using
NMR in biomedical research.
16.2
Basics of NMR
The following diagram illustrates the basic features of NMR.
B0
(Magnetic field
strength)
off
1≠0
(nuclear spin)
on
on
ΔE = r B0
ΔE = hν
Energy level
splitting ΔE
on
Relaxation
NMR
signal
+ hν
Radio frequency
pulse (υ )
Figure 16.1 Basic features of NMR.
The nuclear energy level splits in an external magnetic field. The splitting ΔE is determined by the field strength B0 and the gyromagnetic ratio
γ. The radio frequency pulse induces a transition of nuclei from low to
high energy level. The nuclei at high energy level (excited state) would
relax back to lower energy level and give NMR signal.
It may be pointed out that the nuclei that exhibit NMR are those for
which the spin quantum number I is greater than zero and this, in turn, is
associated with the mass number and atomic number as shown below:
Table 16.1 Mass numbers, atomic numbers and spin quantum numbers.
Mass number
Even
Even
Odd
Atomic number
Even
Odd
Even or odd
Spin quantum number
0
1, 2, 3, . . .
1 3 5
2, 2, 2, . . .
Hence, nuclei such as 12 C, 16 O have I = 0 and hence are non-magnetic.
Nuclei like 13 C, 31 P are magnetic nuclei.
16.3
Applications of NMR
NMR can be used to identify molecules and to study molecular interactions. It has been applied to the structure and function determination of
NMR Technique in the Elucidation of Biochemical Problems
315
important biological molecules such as drugs and their interaction with
targets. NMR has also been employed for identification of abnormal
metabolites as biomarkers for specific diseases, also diagnoses of disorders
by imaging particular tissues or organs.
The interactions within pFMRP—Caprin-I biological condensates were
proved by solution state NMR.
It may be mentioned that there are four major interactions besides, the
Zeeman interaction (discussed earlier), which include chemical shift, dipolar, paramagnetic and quadrupole interactions. These interactions are susceptible to molecular motion and chemical environment (such as chemical
bonding, spatial arrangement of atoms in a molecule etc.) which in turn
affect the position, intensity and splitting of NMR peaks. The size and
motion of molecules also affect NMR relaxation properties. For example,
the relaxation behavior in tissues has been utilized in magnetic resonance
imaging (MRI) to take images of different parts of the body.
16.4
Applications of NMR in Biomedical Research
It is known that the main building blocks of our body are composed of
proteins, nucleic acids, lipids and carbohydrates. Each class of these body
blocks metabolise in different ways to provide energy to the body.
16.4.1
Determination of Protein Structure
Proteins account for more than 75% of the drug targets. They also are used
as drugs to maintain our body. Knowledge of the structure of a particular
protein helps in our understanding how it achieves its specific function.
For example, the binding of APP9mer to the GABAB RI a single domain has
been investigated with NMR. Proteins are biopolymers made of different
amino acids in different orders and lengths and thus form different threedimensional structures. An example of a protein sequence is shown below:
H
H
R1
N
C2
C′
N
C2
C′
N
C2
H
R1
O
H
H
O
H
RN O
C′
Figure 16.2 Protein sequence formed by connecting peptide bonds.
It is not easy to assign each chemical shift to a specific atom in the
amino acid sequence of amino acids due to the crowded nature of the one
dimensional NMR (see Fig. 16.3). By using high field NMR one can obtain
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Biophysical Chemistry
a spectrum of higher resolution and sensitivity at higher fields. Multidimensional NMR techniques enable correlation between neighboring atoms
or residues. They facilitate the sequential assignment of proteins which
is a first step in solving protein structure. These correlations are usually
obtained through J coupling interactions between neighbouring atoms or
through some relaxation mechanisms. Although chemical shifts have been
used to predict protein secondary structures, more structural constraints
are needed to build the structure of a protein.
300
200
100
13C
0
100
200
frequency (ppm)
While Karplus coupling predicts three bond scalar coupling values to
molecule dihedral angles, RDC (residual dipolar coupling) enables dipolar coupling values 15N− H or 13C− H of proteins which helps us to figure out the orientation of these bonds with respect to molecular alignment
axis. H-NOE (nuclear overhauser effect) is used to measure the distance
between two nuclei upto 5A◦ .
The final protein structures are generally obtained by running a simulated annealing process such as Xplor with all chemical shift assignments
and structural constraints.
Questions
(1) An NMR instrument operates at 500 MHz frequency. Calculate the
magnetic field strength required to observe resonance lines in the case
of 13 C nucleus (g for 13 C nucleus = 1.404) β N = 5.02 × 10−27 J T−1 .
Solution:
r=
g · βN
× BZ
h
NMR Technique in the Elucidation of Biochemical Problems
BZ =
317
rh
gβ N
500 × 106 × 6.63 × 10−34
1.404 × 5.02 × 10−27
33.15 × 1026
=
7.05 × 10−27
= 4.7 × 10 = 47 gauss
=
(2) Discuss briefly the application of NMR technique in the elucidation of
structure of proteins.
17
Applications of ESR
Spectroscopy for the Solution
of Biological Problems
17.1
Introduction
EPR spectroscopy is a very powerful tool that can provide valuable structural and dynamic information on a wide ranging biological systems. EPR
spectroscopy requires the presence of an unpaired electron spin and a simple EPR active system consists of a single unpaired electron spin in a molecular orbital. The electron can exist in one of two spin quantum states, + 12
and − 12 . In the absence of a magnetic field, the two states are degenerate
and have the same energy. When a magnetic field is applied, the − 12 state
decreases in energy and the + 12 state increases in energy as shown in the
Figure 17.1.
EPR transitions occur when the energy in the microwave photons
matches the splitting between two electron spin states. In the simplest case,
this splitting is a function of the magnetic field.
In a typical continuous wave (CW) EPR, a constant microwave frequency is applied and Bo , the magnetic field swept with spin-flop transition occurring when the energy separation between the two election spin
states matches the constant microwave energy. Further to sweeping Bo , the
field is modulated to enhance the signal to noise ratio. This gives rise to
the derivative line shape (see Fig. 17.1) observed in EPR spectra. The block
diagram of a simple ESR spectrometer is shown below.
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_17
319
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Biophysical Chemistry
Figure 17.1 Energy-changes in electron spins in the absence and presence
of a magnetic field.
The magnetic field at which this signal appears depends on the g value
which governs the slope at which the energy levels for the two spin states
changes are function of the magnetic field. In most biological systems, the
effective g value depends on the orientation of the molecule with respect
to the magnetic field. The EPR spectrum also depends on interactions
between electron spin and any NMR active nuclei in the vicinity. This
interaction is referred as for MRS, a one-dimensional spectrum of a particular location of the body is obtain using the magnetic field gradients
in x, y, z direction. Many nuclear like 1 H, 31 F, 19 F, 13 C have been studied.
The chemical stiff information can be used to distinguish between different species. In the MRI of brain, the commonly observed metabolites are
N-acetyl aspartates creatinine, choline etc. MRI and MRS have been used in
Alzheimer’s disease to diagnose and monitor the tumour in the brain. The
relative changes in concentration of some MRS detectable metabolites like
NAA, creatinine, choline have been studied and found useful in diagnosis
of AD.
17.2
Advances in Magic Angle
Spinning (MAS) NMR
This method allows the identification and characterisation of protein complexes such as transmembrane proteins and metalloproteins. MASNMR
can be used to identify structural details at atomic level for T-355, DSbB,
and FimA proteins. In addition to protein complexes, MASNMR has been
used for analysis of nucleic acids and nucleic acid-protein interactions.
MASNMR is also useful for analysis of molecular interactions in cellulose membranes, cytoskeleton components and activity of viral proteins
Applications of ESR 321
and viral protein complexes. It was also used to study functions and conformational modifications of cyclophilin-A, a human protein which forms
a composition with HIV capsid and affects HIV-I infecting capacity. MASNMR has also been employed for structural and functional analysis studies
of bacteriophage proteins.
Phospholamban can be studied by solid state NMR because it is a
membrane protein and most of its interacting partners are membrane proteins too. Solid state NMR has also been used to study α-synuclein
oligomers.
Solid state NMR has successfully been used to study β-amyloid peptide structure which is of great use in understanding Alzheimer’s disease.
NMR has been used to develop new drugs and also in synthesizing new
molecules. In pharmaceutical research too, NMR contributed extensively
NMR drug screening has been carried out by observing ligand resonance
changes.
NMR Study of Metabolites
Metabolites are intermediates or final products of different biochemical
reactions occurring in a biochemical system.
Metabolomics is a subset discipline of systems biology where the
metabolites from a specific biological system are assessed, quantified to
gain information on the system. NMR is a good tool to investigate the
metabolites. Biofluids like urine, blood have been analysed by using NMR
with strong magnetic fields and better probe designs. NMR has been successfully used to identify potential biomarkers in Alzheimer’s disease. HNMR spectroscopy has been used to compare the metabolomic profiles of
different brain regions in wild type mice.
MRI or Magnetic Resonance Spectroscopy (MRS)
of Body and Tissues
MRI has now been developed to such an extent that it is now widely used
in clinical diagnosis and spectra could be obtained on any part of a body
non-invasively.
In such studies, the sample has to be a kept in a homogeneous magnetic field to make NMR peak line width as narrow as possible. In MRI,
the field gradients are purposely introduced across the sample so that the
NMR frequency of the nucleus at different positions in the sample is slightly
different and this information is used to map the location of the nucleus in
the sample.
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Biophysical Chemistry
z
MRI
imaging
principle
–
+
Field gradient Gx
Bz = B0 + Gxx
x
–
+
x
NMR frequency
ν (x) = ν0 + yGx x/h
Figure 17.2 Magnetic Resonance Imaging principle of body and tissues.
Optimizations and structural refinements, and averaging them the
final 3D structure of the protein is arrived at. An example relates to the
solution NMR study of the structure of Phospholamban (a protein in the
sarcoplasmic reticulum (SR) membrane of cardiomyocytes) and its variants. NMR distance and dihedral angle constraints were used as inputs in
X-plor and the final structure was generated.
The structures obtained were used to predict the binding mode
between phospholamban and calcium ATPase and explain why critical
mutations are critical in phospholamban’s function.
17.3
Protein Structure Determination
A lot of biochemical reactions occur in solution phase. However, there
are situations where studies have to be made in solid phase, for example,
bones, teeth whose structure could be elucidated through NMR.
Most protein drug targets are membrane proteins. They act as receptors or transporters to pass information in and out of membranes. There
are interesting solid state protein structures in humans, bacteria, and
viruses as well. For example, the virus capsid formed by capsid proteins
has a unique structure and it has been studied by solid state NMR. Such
studies may ultimately provide a cure for AIDS.
In solid state NMR, the chemical shift is referred as chemical shift
anisotropy. The chemical shift depends on orientation of each molecule
causing broadening of peaks. This can be circumvented by aligning the
molecules in the sample to have the same orientation. This method has
been applied to study membrane proteins and phospholipid bilayer membranes. The protein molecules in the aligned sample have similar chemical
shifts and narrower line widths.
Hyperfine interaction (A) and it depends on three factors: (i) amount
of election spin density on the nucleus, (ii) distance between electron spin
and nucleus, (iii) angle between the two with respect to the magnetic field.
Applications of ESR 323
Figure 17.3 Protein structure determination using solid state NMR.
Figure 17.4 Block diagram of a EPR spectrometer.
It is possible to extract information on electron-electron couplings
between two sets of spins and this provides valuable distance information
between biological systems and zero field splitting in higher spin systems.
17.4
Electron Nuclear Double Resonance
Spectroscopy (ENDOR), Electron Spin Echo
Envelope Modulation (ESEEM) and
Hyperfine Sub-Level Correlation (HYScore)
Spectroscopic Techniques
In these spectroscopies, the electron spin is used as a detector to probe
nuclei that are coupled to the unpaired electron spin. In ENDOR, nuclear
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Biophysical Chemistry
transitions are directly driven using either continuous or pulsed radio frequency radiation. The effect on the EPR signal as a function of RF is monitored. Similar to ENDOR, ESEEM and HYScore are pulsed methods that
are used to obtain information about nuclei in the vicinity of unpaired electron spin.
Double Electron-Electron Resonance (DEER) or
Pulse Electron Double Resonance (PELDOR) Spectroscopy
When there are two or more EPR active species in a system, there will be
electron-electron dipolar interaction present. This interaction depends on
the distance and the angle between the two paramagnetic species relative
to the externally applied magnetic field. In DEER spectroscopy, this dipolar coupling is measured and thereby the distance between the two species
can be measured in the range 20–80 A◦ . This technique has emerged as a
powerful tool in structural biology.
Spin Label EPR
With the advent of spin labelling, the potential use of EPR has been extended to any biological system because biological techniques are available to
place stable radicals at specific locations in biological macromolecules.
EPR spectroscopy has emerged as a powerful technique to obtaining
structure and dynamic information on peptides, proteins, nucleic acids
and macromolecules. EPR spectroscopy offers high sensitivity (at μM concentrations) and is not limited by the size of the protein.
EPR measurements can be made on a variety of samples such as solutions of proteins, densely packed membrane suspensions, tissue samples.
Analysis of EPR data for a series of spin labelled protein sequences allows
modelling of protein structure.
Site-Directed Spin Labeling (SDSL) Methods
Native biological systems cannot be studied by EPR because they are not
EPR active. A reporter group or a label such as spin probe needs to be
incorporated into the desired system to be detected by EPR. The site specific incorporation of unpaired electrons into biomolecules is known as
SDSL.
In SDSL experiments, all native non-disulfide bonded cysteines are
removed by replacing them with another amino acid like alanine. A unique
cysteine residue is then introduced into a recombinant protein via site
Applications of ESR O
S
N
O
S
O
r4
CH3 + HS
CH2
r5
Protein
325
r2
S
r3
C
r1
Protein
N
O
Figure 17.5 Structure of MTSL and the resulting side chain produced by
reaction with cysteine residue of the protein. r1 , r2 , r3 , r4 , r5 represent locations of five rotations about the chemical bonds.
directed mutagenesis and subsequently reacted with a sulfhydryl specific
nitroxide reagent to generate a stable paramagnetic EPR a dive side chain.
17.5
Structural and Dynamical Information of
Biological Systems
The overall mobility of the spin label attached to a protein, or a peptide is
a superposition of the contributions from: (1) the motion of the label relative to peptide back bone, (2) fluctuations of α-carbon back bone, and (3)
rotational motion of the entire protein or peptide. Under the experimental conditions, these motions can be isolated from the EPR spectrum. The
spin label side chain motion is used to study tertiary contacts and protein
structure.
For β-sheet proteins, the spin label motion is influenced by steric interactions with the nearest neighbours. The inverse line width of the central line provides a measure of relative stability. Scanning the inverse line
width of the EPR spectrum against the amino acid sequence yields a periodic data profile that reflects the local secondary structure of the protein.
This method also provides a strategy for identifying functional domains in
high molecular weight proteins, membrane proteins and supra molecular
complexes.
The work done on Escherichia coli ferric citrate transporter FecA constitutes an excellent example of application of SDSLEPR. This FecA protein is involved in ferric iron—iron translocation across the outer membrane. Another example of SDSLEPR to probe the structural and dynamic
properties of proteins is Vimentin. Vimentin is a type III intermediate filament protein found in many cells of mesenchymal origin. Mutations in
IFgenes have been linked to different human diseases. Vimentin’s head
domain structure is dynamic and changes with filament assembly.
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Biophysical Chemistry
Figure 17.6 Molecular structure of vimentin.
Figure 17.6 shows vimentin molecular structure with a representation
of protein domains of Vimentin. At the amino terminus is the head domain
leading into rod domain-I. Black boxes represent linked 1-2 Rod 2B and
Rod 1B are parallel helical structures of Rod 2A/linker 2 Panel.
17.6 Topology of Proteins
Membrane proteins control bioenergetics, movement of ions across a cell
and initiate signaling of pathways. Nitroxide base site directed spin labeling EPR can be used to obtain structural and dynamic information on
membrane—protein assembly. EPR spin labels are very sensitive to the
presence of other paramagnetic species which alter the relaxation properties. There are several biologically important membrane protein systems
(e.g., Bacteriorhodopsin, ABC cassette transporter MsbA, cytochrome C
oxidase subunit IV (Cox IV) and Ferric enterobactin receptor FepA) which
have been studied using spin label EPR to investigate structural topology.
A typical example of using CW-EPR power saturation data is to study
the topology of KCNE-I membrane protein in proteoliposomes (KCNE-I
is a single transmembrane protein that modulates the activity of KCNQI voltage gated K channel). These data showed that KCNE-I spans the
full width of lipid bilayer with Leu-59 residue located near the center of
membrane. Analysis of SDSLEPR line showed that the dynamic motion
of the nitroxide spin label is slower in the membrane environment than
in micelles and that residues within membrane are less mobile than that
outside.
An excellent example of using accessibility and mobility data to identify α-helical secondary structure is the study of lactose permease protein.
Site directed spin labeling EPR studies to obtain periodicity of side chain
mobility and accessibility to O2 showed that the transmembrane domain
XII in the lactose permease protein adopts and α-helical conformation.
Applications of ESR 327
CW-EPR at X-band has been used to study membrane topology of
integrated membrane proteins inserted into aligned phospholipid bilayers. The membrane alignment technique coupled with dipolar broadening
CW-EPR was used to determine the distance and relative orientation of
two nitroxide spin labels on M-28 peptide of acetylcholine receptor (Ach
R) in DMPC vesicles.
17.7
SDSLEPR Methods Under High Fields/
High Frequencies
EPR spectral line shapes can provide information on the secondary structure of a protein and on the orientation of its sub-units relative to each
other. If the experiment is carried at more than one frequency, additional
information about the tumbling of the protein and internal motion consisting of back bone fluctuations and side chain isomerization can be obtained.
EPR spectra obtained at multiple frequencies provide unique perception
on the molecular motions and give accurate descriptions of the dynamics
of spin probe environment.
EPR, as a biophysical tool, can be used to tackle the following three
areas:
(1) Structure and dynamics of large molecular weight proteins in solution,
(2) Membrane and membrane associated proteins, structure, location,
interaction with other membrane components or DNA or RNA,
(3) Fast conformational transitions of proteins and RNA’s in solution
protein folding and unfolding. Extending conventional EPR to higher
frequencies, it is possible to obtain the following additional features:
(1)
(2)
(3)
(4)
(5)
enhanced spectral resolution,
enhanced orientational selectivity in disordered samples,
enhanced low temperature electron spin polarization,
enhanced sensitivity for probing fast motional dynamics, and
enhanced detection sensitivity for restricted volume samples.
Multi frequency EPR has provided pertinent structural and dynamic information on myosin. It is known that myosin are involved in muscle contraction and a wide range of eukaryotic mobility processes. Multi frequency
of EPR has also been used to study the dynamic properties of T4 lysozyme
in solution. It is a globular protein composed of 164 amino acid residues
with a molar mass of 18.7 kDa (i.e., 18700 atomic mass units).
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Biophysical Chemistry
SDSL-ESEEM Methods
ESEEM is a powerful pulsed EPR technique to study many different biological systems. It has been used to probe the site specific secondary structure of membrane peptides using microgram amounts of sample.
The three pulse ESEEM data obtained for AChRM28α-helical peptide
in a membrane and an ubiquitin β-sheet peptide in solution is shown in
Figure 17.7.
Figure 17.7 Three pulse ESEEM spectra with T = 200 ns of the I + 2
and I + 3 labelled Leu for AchR M-282 helical peptide in lipid bilayer and
Ubiquitin β-sheet peptide in solution.
Applications of ESR 17.8
329
Double Site-Directed Spin Labeling Methods
It is possible to use EPR for measuring the distances between two spin
labels in terms of either intramolecular distance between sites on different
proteins. Using double labeling EPR techniques, distances can be measured to probe secondary, tertiary and quaternary structures.
The CWEPR line broadening approach has been used to study the bacterial K + -translocating protein KtrB. Such proteins are found in bacteria
fungi, plants and trypanosomes.
In DEER, one set of spins are monitored, and another set of spins are
excited with a second microwave frequency leading to the measurement of
the coupling between the two spins and hence the distance between them.
DEER can be used to probe the structure of biomacromolecules, globular proteins, membrane proteins, oligomer states and RNA. At high fields,
DEER can be used to measure relative orientation of spins. The protein
fluctuation dynamics and spin label rotameric motions have a significant
contribution to DEER distribution width. The transmembrane region of
α-helical nature C-99 Amyloid precursor protein in proteo-liposomes was
identified by DEER.
C-99 is a transmembrane carboxyl terminal domain of the amyloid
precursor protein that is cleaved by r-secretase to release amyloid βpolypeptides which are involved in Alzheimer’s diseases. The curved
nature of the transmembrane is important for C-99 interactions with rsecretase. CW-EPR power saturation data were used to confirm the spanning of transmembrane domain of C-99 and revealed that N-helices and Chelices are associated with membrane surface. DEER measurements on a
dual labeled construct of CBD-12 indicated that the β-sandwich regions of
CBD-1 and CBD-2 domains are widely separated at their N- and C-termini
and are largely insensitive to Ca2+ binding.
Figure 17.8 Identification of transmembrane region of α-helical nature of
C99 amyloid precursor protein in proteoliposomes.
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Biophysical Chemistry
An illustrative example for measuring distance constraints with DEER
is the Na+ /Proline transporter Put P Escherichia coli system. Proteins of
this family utilize a sodium motive force to drive uphill transport of substrates such as sugars, amino acids, vitamins, myo-inositol and urea.
Protein-Protein Interactions
EPR spectroscopy is a powerful technique to study protein-protein interactions and oligomeric states. It is pertinent to point out that protein-protein
interactions are involved in all biological processes such as immune
responses, cell signaling, translocation and regulation. DEER has emerged
as a powerful tool to measure distance between spin labeled binding sites
in a protein-protein complex. An example of application of CW-EPR and
DEER spectroscopy is associated with the study of complicated proteinprotein complexes involving Cdb3|AnKD34 proteins. Both techniques
were used to map the binding interfaces of these two proteins in the complex and to obtain the inter-protein distance constraints. Saturation transfer EPR was used to probe the homo- and hetero oligomeric interactions of
the sarcoplastic reticulum Ca-ATPase (SERCA) and phospholamban (PLB).
Unpaired Spins in Biological Systems
Structural information in biological processes can be obtained by EPR since
electrons are almost intimate participant of these processes.
Naturally Occurring Radicals
Many common biological radicals are those stemming from amino acid
residues such as tyrosine, tryptophane, cofactors such as flavine and pigment molecules such as chlorophyll. They typically contain one unpaired
electron spin residing in an aromatic molecular orbital. Higher field frequency instruments can be used to determine small anisotropies in “g”
values. The deviation of “g” values is a characteristic for a given system
and hence can be used to obtain information on identity and environment
of electron spin.
A few illustrative examples of how EPR and related methods have
been used to answer important biological questions in systems containing
organic based biological radicals are given below.
(1) Ribonucleotide reductase: The Ribonucleotide Reductase (RNR) family of enzymes catalyses the conversion ribonucleotides to deoxyribonucleotides that are used in the synthesis of DNA in every living
Applications of ESR 331
organism. The ribonucleotide reaction is believed to proceed via the
creation of a tyrosine radical in the R2 submit by the di-iron center in
the R1 sub-unit, followed by an electron transfer reaction. This creates a thyil radical near the substrate binding site and the subsequent
reduction of ribonucleotide.
(2) Phycocyanobilin—Ferredoxin oxidoreductase (PCYA)
Phycocyanobilin: PCYA catalyses the reduction of biliverdin to
3Z/3E phycocyanobulin via a 4e− process. This system functions via
a two step 4e− reduction with 181, 182-dihydro-biliverdin (DHBV) as
an intermediate in the reaction. The small amount of “g” anisotropy
present in many biological radicals precludes determination of the g
values by conventional low field/frequency EPR. This can be seen in
the case of radical intermediate in PCYA reaction as shown in Figure 17.9.
Figure 17.9 EPR spectra and corresponding simulations at three fields
(or frequencies) of the radical intermediate in the Phycocyanobilin—
Ferredoxin oxidoreductase system.
The principal factor that gives rise to deviations in g values from that
the free electron value is spin orbit coupling within the molecule. Spin
orbit coupling is the interaction between the unpaired electron spin
and low lying unoccupied excited states.
17.9
Other Important Biological Systems
Quinone based electron transport systems have been widely studied using
EPR and ESEEM methods. These studies have helped in understanding
Q-cycle in the complex III family of membrane bound electron transport
systems. EPR methods were also used in furthering our understanding of
two redox active tyrosine residues with in photosystem II. Many biological
cofactors such as chlorophylls, flavins have also been studied by EPR.
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Biophysical Chemistry
Oxygen Evolving Complex (OEC) in Photosystem-II (PS-II)
Photosystem-II is a multi-submit membrane associated enzyme system
which catalyses the light driven oxidation of water to molecular O2 releasing 4 protons. It has helps fuel the pH gradient that drives ATP synthesis
and form e− in the form of reducing equivalents which eventually participate in the reduction of CO2 to sugar. The water oxidation occurs at the
oxygen evolving complex (OEC) which consists of 4 Mn atoms and one
atom each of Ca and Cl− .
While EPR spectra can detect interactions between an unpaired electron spin and other electrons and nuclei in the vicinity, those interactions
are often lost in the large inhomogeneously broadened EPR spectra of transition metal systems.
The multi-line signal associated with S2 state of photosystem-II was
investigated with 55 Mn ESE-ENDOR. The pattern of multiline signal arises
from the coupling between S = 12 electrons spin and all of the nuclei in
the vicinity with the isotopically prevalent 55 Mn I = 52 nucleus being the
dominant interaction.
ESEEM and ENDOR techniques have been very powerful in determining ligand identity to the OEC. Multifrequency ESEEM studies have shed
light on the hyperfine couplings of the directly bound and second nitrogen
of the imidazole ring and the nature of the histidine ligation to the OEC.
The FeMo Cofactor of Nitrogenase
Nitrogen fixation is catalysed by the enzyme Nitrogenase. This process
consists of two parts: (i) an electron delivery system, the Fe protein, which
delivers an electron upon hydrolysis of two molecules of ATP and (ii) the
FeMo protein which contains an iron-molybdenum cluster where the catalysis takes place. As with PS-II, many intermediate states have EPR spectra which are informative and also allow techniques such as ENDOR to
be used to characterize them. The FeMo cluster where nitrogen fixation
occurs is of interest. This cluster exists as a rhombic S = 32 system with of
values as 4.32, 3.64 and 2.00. These EPR signals associated with nitrogenase system yield unpaired electron spins that can be used by techniques
such as ESEEM, ENDOR and HYSCORE.
Metal Replacement
Although there are large numbers of EPR active metal-based systems in
nature, there are still many that are EPR silent. Of them, Mg and Zn are
important. Many systems require Zn for function. It is a d10 system and
Applications of ESR 333
has no unpaired electrons to study by EPR. However, they can be functionalized by substituting with Co (II) to give an EPR active probe within the
system.
One important biological system that has been studied in this manner
is metallo-β-lactamase family of enzymes. These enzymes are a primary
target for antibiotic drug design and have been well characterized by
replacement of Zn (II) with Co (II) for EPR studies. This metal replacement
has also enabled EPR studies of interaction of nucleic acids with Mg2+ .
Thus, replacement of Mg2+ (which is EPR silent) by Mn2+ , gives a spectroscopic probe which has been widely utilized to understand the role of
counter ions in the structure and function of nucleic acids.
Questions
(1) (a) The g value of a species is independent of the magnetic field.
(b) The g value depends on the orientation of the molecule with respect
to the magnetic field.
(c) The g value varies between + 12 and − 12 .
(d) None of the above.
(2) For a certain radical, the magnetic field strength is 3350 gauss and the
frequency of the microwave is 9500 MHz. Calculate its g factor.
Solution:
g=
71.4484 in GHz
B (in nT)
71.4484 × 9500 × 10−3
3350 × 10−1
= 2.026
=
The number of lines from the hyperfine interaction can be determined
by the formula
2N I + 1
where N is the number of equivalent nuclei and I is the spin.
(3) Calculate the number of lines to be expected for methyl radical CH3 .
Solution:
2N I + 1 = 2 × 3 ×
1
+1 = 4
2
334
Biophysical Chemistry
(4) Discuss briefly the usefulness of EPR spectroscopy in understanding
the structure of biologically important radicals like ribonucleoside
reductase.
(5) Explain how the multi line signal associated with S2 state of photo system II can be understood using ESE-Endor?
18
Flow Methods for the Kinetic
Study of Fast Biochemical
Reactions
18.1
Introduction
The kinetic study of any reaction involves the start of the reactions by mixing the reactants and following the course of the reaction by monitoring the
concentration change of a species by titrimetry or by following the change
in physical property of the system as a function of time.
Flow techniques, developed by the pioneering work of Hartridge and
Roughton, are used to study reactions occurring on time scales of the order
of seconds to milliseconds.
18.2
Experimental Arrangement
A simple experimental flow-set up or apparatus is given below:
Reactant 1
Reactant 1
Fixed
detector
Reactant 2
Reactant 2
Movable detector
Movable
injector
Figure 18.1 A simple experimental set up for flow methods.
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_18
335
336
Biophysical Chemistry
In the experimental set up shown above, reactants are mixed at one
end of a flow tube and the composition of the reaction mixture is monitored at one or more positions along the tube. If the flow velocity is known,
then measurements at different positions provide information on the concentrations at different times after initiation of the reaction.
A different version of the method has the detector in a different position but a movable injector may be used to inject one of the reactants
into the flow tube at different positions relative to the detector in order
to study the time dependence of the composition of the reaction mixture.
In a stopped flow method, a fixed volume of reactants flow rapidly in a
reaction chamber and are mixed by the action of a syringe fitted with an
end stop. The composition of the reaction mixture is then monitored by a
physical technique (Spectrophotometrically or otherwise) as a function of
time after mixing at a fixed position in the reaction vessel.
Figure 18.2 Technique of stopped flow method.
It may be mentioned that the continuous flow methods have the disadvantage of requiring large quantities of the reactants as also high flow
velocities of reactants to study fast reactions.
Importance of Study of Biological Reactions
and their Rates
Many biological reactions occur in the living systems. For example,
enzymes catalyse reactions by binding and modifying substrates, transport proteins bind to and carry lipids, hormones within circulatory systems. Further, antibodies are utilized by the immune system to bind and
remove foreign substances from the body.
18.3
Reactions Studied Using this Technique
Examples of biological interactions that have been studied using this technique include: (i) kinetics of protein folding, (ii) enzyme inhibition and
(iii) binding of proteins or DNA to hormones or drugs. In these studies, a
variety of molecules have been used like drugs, hormones, proteins, metal,
ions etc.
Flow Methods for the Kinetic Study of Fast Biochemical Reactions
337
Commonly used detection techniques are absorbance and fluorescence.
Reactants or products with a specific chromophore or fluorophore as in
NADH, pyridoxal phosphate on tryptophan residues have been studied.
Other techniques like NMR, small angle X-ray scattering, circular dichroism have been coupled to the stopped flow apparatus.
18.4
Applications to Unimolecular Reactions
Consider a reversible reaction involving a molecule which changes its conformation from one form to another. The reaction may be represented by
k1
BG y
x FGGGGGG
GGGGG
k −1
(18.1)
If the observed signal (b) is obtained as a function of time (t), it can be
fitted to the equation
b(t) = beq − (beq − b0 )e−kobs t
(18.2)
where b(t) = signal measured at time t, b0 = signal observed at the beginning of the experiment (i.e., at t = 0), beq = signal obtained after a sufficiently long time i.e., at equilibrium and beq − b0 = total change in signal during the reaction. k obs is equal to the sum of k1 and k −1 and if the
equilibrium constant K of the reaction is known, both k1 and k −1 can be
calculated. Reactions involving conformational changes in proteins often
follow this type of kinetics.
The dis oxygenation of haemoglobin according to
Oxyhaemoglobin −→ Haemoglobin + O2
(18.3)
follows a first order kinetics with a t1/2 = 64.8 × 10−3 sec and k = 10.7
sec−1 .
The reaction between the apoprotein and excess of Zn2+ has been followed by following the change in fluorescence intensity with time after
mixing them. The fluorescence intensity changes with time are shown
below. The rapid interaction given in curve (a) may be described by the
equation
k1
BG SL
S + L FGGGGGG
GGGGG
k −1
(18.4)
338
Biophysical Chemistry
Figure 18.3 Rapid interaction between apotransferrin and Zn2+ .
while second and third processes can be expressed by the equation
k1
BG S∗
S FGGGGGG
GGGGG
k −1
(18.5)
k obs = k1 [ xtotal ] + k −1
M−1
sec−1
(18.6)
sec−1 .
and k −1 = 6
A
Analysis of data gives k1 = 21 ×
stopped flow kinetic study of the binding of high mobility group domain
proteins with cisplatin modified DNA was made using fluorimetric
detection.
The second order rate constant of the reaction from the slope is 1.1 ×
8
10 M−1 sec−1 . The variation of fluorescent intensity with time is shown
in Figure 18.4.
104
Figure 18.4 (a) Variation of intensity of fluorescence with time in the
kinetic study of high mobility protein with cisplatin modified DNA and
(b) Variation of k obs with concentration of HMGI domain.
Flow Methods for the Kinetic Study of Fast Biochemical Reactions
339
Other bimolecular reactions under pseudo first order conditions were:
(1) binding of warfarin with human serum albumin,
(2) reaction of isonicotinic hydrazide and its analogues
mycobacterium tuberculosis catalase peroxidase (kat G),
with
(3) interaction of enzymes or coenzymes with peptides,
(4) protein-protein interactions or protein binding with ATP.
18.5
Applications Involving Competitive
Reactions
Consider the reaction
k1
BG SL −→ (4);
S + L FGGGGGG
GGGGG
k −1
k2
S + C FGGGGGGGB
GGGGGGG SC
k −2
(18.7)
In this scheme, “C” competes with “L” for the same site on “S”. Competition experiments in stopped flow analysis have been performed to study
drug-protein, DNA-protein, protein-protein and enzyme peptide binding.
As a specific example, phenyl butazone was used as a competing agent
in kinetic studies of interactions of warfarin with HSA. In another case,
podophyllotoxin (POD) was used as a competing agent to study binding
by tubulin to two analogues of colchicine, TCB and TKB (2, 3, 4, trimethoxy4’-acetyl-1, 1’-biphenyl).
18.6
Applications Involving Multi-step Reactions
Consider the reaction scheme
k1
k2
∗
BG SL FGGGGGGGB
S + L FGGGGGG
GGGGG
GGGGGGG SL
k −1
k −2
(18.8)
when the concentration of L >> S,
k obs2 =
k2 [ Ltot ]
+ k −2
K−1 + [ Ltot ]
(18.9)
where k obs2 = observed rate constant for the slower unimolecular reaction
and K−1 is the dissolution equilibrium constant for the fast bimolecular
reaction, i.e., K−1 = k −1 /k1 .
340
Biophysical Chemistry
Several reactions such as:
(1) J-binding protein 1 interaction with J-DNA- binding domain with DNA
oligomers that contained glycosylated 5-hydroxyl methyl cytosine,
(2) binding of phosphatidyl serine containing vesicles to lactadherin,
(3) binding of Fe2+ and Zn2+ to human serum transferrin. Stopped flow
method can be used to study first order reactions with rate constants
ranging from 10−6 to 106 sec−1 and from 1 to 109 M−1 sec−1 for second
order reactions.
The rate of association of E (binding site of Lysozyme) with NADH viz.
E + NADH ←→ E − NADH
was studied by stopped flow method. The rate constant for the forward
reaction was found to be 5 × 106 M−1 sec−1 at 276 K.
The equilibrium and kinetic constants for the dissociation of
ko f f
LADH − NADH FGGGGGGGGB
GGGGGGGG LADH + NADH
k on
(18.10)
were also estimated, with the following values:
Buffer
Phosphate buffer pH = 7.0
Phosphate, pH + 50 mM NaCl
k on /M−1 sec−1
1.7 × 107
2.5 × 107
k o f f /sec−1
3.2
9.0
Questions
(1) (a) Continuous flow methods required only small volumes of reactants.
(b) Stopped flow methods can be conveniently used to study reactions
in the time scale of a few seconds to milliseconds.
(c) The reaction between H+ and OH− ions can be studied by stopped
flow technique.
(d) Continuous flow methods can be employed to study reactions with
low flow rates.
(2) (a) Pressure jump technique is used to study relatively faster reactions
as compared to temperature jump method.
Flow Methods for the Kinetic Study of Fast Biochemical Reactions
341
(b) There are more methods for following the rates in pressure jump
method than in temperature method.
(c) The extent of displacement of equilibrium in a temperature jump
experiment is dependent on the enthalpy change of the reaction.
(d) The rate of neutralisation of H+ by OH− ions in aqueous solution
can be studied by P-Jump method.
(3) The reaction between myoglobin (MG) and O2 is given by
R1
MG + O2 FGGGGGGB
GGGGGG MGO2
R −1
for this reaction,
1
= R−1 (MG + Ō2 ) + R1 .
τ
1
= R1 (MG + Ō2 ) + R−1 .
(b)
τ
(a)
1
= R1 [MG] + R−1 [Ō2 ].
τ
1
= R1 [Ō2 ] + R−1 [MG].
(d)
τ
(c)
(4) The deoxygenation of oxyhemoglobin, according to the equation,
oxyhemoglobin −→ hemoglobin + O2
is a first order process with a t1/2 = 65 millisec. Calculate the time for
90% deoxygenation to take place.
Solution:
0.693
0.693
=
= 10.66 sec−1
t1/2
65 × 10−3
a
2.303
2.303
2.303
log
log 10 =
k=
=
t
a − 0.9a
t
t
2.303
t=
= 0.216 sec = 216 m sec
10.66
k=
(5) Carbon monoxide reacts with haemoglobin (Hg) according to
CO(g) + Hg(l) COHg(l)
The rate constant for forward reaction is 0.51 × 10−6 M−1 sec−1 .
Assuming that the concentration of CO in blood is 1 × 10−3 M and
the concentration of Hg is 2 × 10−2 M, calculate the time required for
the concentration of CO to be reduced to 1 × 10−4 M.
342
Biophysical Chemistry
Solution: For a second order reaction
k=
2.303
b( a − x )
log
t( a − b)
a(b − x )
In this case, let [HG]= a = 0.02 M; [CO]= b = 0.001M,
x = 0.001 − 0.0001 = 0.0009
substituting the data
0.51×−6 =
or
0.51×−6 =
or
2.303
0.001(0.02 − 0.0009)
log
t(0.02 − 0.001)
0.02(0.001 − 0.0009)
0.0000191
2.303
2.303
log
=
× 0.98
t × 0.0019
0.000002
0.019 × t
2.303
× 0.98 = 2.32 × 106 sec
0.019 × 0.51 × 10−6
232.9 × 106
=
= 6.47 × 104 hrs
3600
t=
(6) The association of E binding site of Lysozyme ( E) with NADH according to the reaction
E + NADH E − NADH
was studied by a stopped flow method and the rate constant was found
to be 5 × 106 liters mol−1 sec−1 . Assuming that both the species are at
an initial concentration of 1 × 10−3 M, calculate the time required for
the binding to be 75 percent complete.
Solution:
1
t
1
5 × 106 =
t
1
=
t
k=
·
·
x
a( a − x )
0.75
1 × 10−3 (0.01 − (75/100) × 0.001)
0.075
0.001 × 0.0002
0.075
t=
6
5 × 10 × 0.001 × 0.0025
0.075
=
5000 × 0.00025
0.075
=
= 0.06 sec
1.25
×
Flow Methods for the Kinetic Study of Fast Biochemical Reactions
343
(7) The activation energy of the reaction between CO2 and ammonia
according to the reaction
CO2 + NH3 −→ NH2 COOH
is 46.5 kJ mol−1 . Given that its rate constant at 313 K is 1000 L mol−1
sec−1 , what will be its value at 293 K?
Solution:
1
E
1
k2
=
−
log
k1
2.303 × R T1
T2
46.5
1
1
1000
=
−
log
k1
2.303 × 8.314 × 10−3 293 313
46.5
20
=
×
= 0.5295
0.01915 293 × 313
1000
= 3.385
k1
1000
= 295.4 L mol−1 sec−1
k1 =
3.385
(8) The reaction between Zn2+ ions and apoprotein (AP) given by
Zn2+ + AP Zn2+ AP
was studied fluorimetrically. The variation of fluorescence intensity
in presence of excess of Zn2+ and at a concentration of apoprotein of
1 × 10−3 M is as follows:
Time (sec)
0.01
0.015
0.020
0.030
Fluorescence intensity (arb units)
5
62.5
61.2
60.8
Calculate the rate constant of the reaction.
19
Temperature Jump
Relaxation Technique for the
Kinetic Study of Fast
Biochemical Reactions
19.1
Introduction
A unique technique for rapidly disturbing the position of a chemical equilibrium is through a sudden change of temperature of the system and the
temperature jump method (T-jump) is based on this principle.
The displacement of the equilibrium of a reaction is based on the thermodynamic relation
∂ ln K
ΔH ◦
=
(19.1)
∂T
RT 2
P
where K denotes the equilibrium constant of the reaction and ΔH ◦ is the
standard enthalpy change of the reaction.
This method is most versatile among all the relaxation methods
because of several advantages, associated with this technique, as enumerated below.
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_19
345
346
Biophysical Chemistry
Advantages of this Technique
(1) It covers a wide range of relaxation times from about 1 μsec to 1 sec.
(2) A variety of experimental techniques such as:
(i)
(ii)
(iii)
(iv)
spectrophotometry,
fluorimetry,
conductivity, and
polarimetry are available for following the course of the reactions.
(3) Various methods are available for bringing about a rapid heating of the
solution. Some of these are:
(i)
(ii)
(iii)
(iv)
19.2
joule heating,
optical heating,
dielectric heating, and
heating by flash lamp.
Schematic Diagram of the Apparatus
A schematic diagram of T-Jump apparatus developed originally by Eigen
and co-workers in Germany is given below:
Figure 19.1 Schematic diagram of a temperature jump apparatus developed by Eigen and his group.
In any given experiment, the condenser (C) is charged to a voltage of
20 to 100 kV. The spark gap (S.G.) is next fired so that the capacitor discharges through the cell containing the experimental solution. A temperature jump of 5 to 10◦ may be obtained in a time of 0.05 μsec to 0.5 μsec.
Temperature Jump Relaxation Technique 347
Simultaneously, the oscilloscope is triggered and the relaxation curve is
traced.
19.3
Follow Up of the Change in Concentration of
Reactants by Spectrophotometry
The change in the concentration of a species ’i’ is related to the change in
the intensity of the transmitted light ΔIi as
ΔIi = Ii (t) − Ii (ref) = Ii (ref)[e−ε j ΔCi l − l ]
(19.2)
where ε j denotes the extinction coefficient of the species ’i’; ΔCi is the
change in the concentration at time t and l indicates the path length of
the cell. Upon linearizing the exponential term,
However,
ΔIi
= −ε i ΔCi l
Ii (ref)
(19.3)
ΔCi = ΔCi (t = 0)e−t/τ
(19.4)
where τ denotes the relaxation time. Substituting eqn. (19.4) in eqn. (19.3).
ΔIi
= −ε i l
Iref
∂T
∂ ln K
ΔH
ΔCi (0)e−t/τ
RT 2
(19.5)
The variation of the concentration of a species I with time following a TJump may be shown below.
Ii (ref)
Ii (t)
Ci (ref)
Ci (t)
Ci
Ci (0)
Time
Figure 19.2 Variation of concentration of species with time following a
temperature jump.
348
19.4
Biophysical Chemistry
Applications
Many enzyme catalysed reactions and biochemical reactions involving
phospholipids, dispersions have been studied. Other reactions include
metal complex formation reactions, proton transfer reactions etc.
Specific Examples
(1) The mechanism of an enzyme-substrate reaction may be given as
k1
k2
BG ES FGGGGGGGB
E + S FGGGGGG
GGGGG
GGGGGGG ES
k −1
k −2
(19.6)
where ES and ES are the enzyme-substrate complex and another rearranged species of ES. The rate constants k1 and k −1 are of the order of
106 − 108 lmol−1 sec−1 and 10−4 to 106 sec−1 respectively. k2 and k −2
are found to be of the order of 103 − 104 sec−1 . For example, the conformational changes in β-lactoglobulin have been studied using spectrophotometric detection.
(2) T-Jump kinetic study of the binding of imidazole by sperm whale Metmyoglobin. The relaxation of metmyoglobin ( Mb) containing imidazole ( Im) at constant pH has been expressed in terms of the mechanism
k1( app)
Mb + Im FGGGGGGGGGGB
GGGGGGGGGG MbIm
k −1( app)
(19.7)
for which the relaxation time τ is given by
1
= k −1(app) + k1(app) ( M̄b + Īm)
τ
(19.8)
The bars on Mb and Im represent equilibrium concentrations. τ was
found to vary from 0.2 to 0.04 sec depending on pH and concentration
of Im. In all experiments [ Īm] ≥ [ M̄b]. At pH = 7.0 and T = 298 K,
the k1( app) = 170 M−1 sec−1 and k −1( app) = 5.4 sec−1 using imidazole
as one reactant. When benzimidazole was used instead of imidazole,
k1( app) = 509 M−1 sec−1 and k −1( app) = 8 sec−1 .
(3) The biochemical reaction involving glyceraldehyde-3-phosphate dehydrogenase + β-NAD was investigated by T-Jump method. Three relaxation times τ1 = 1.43 × 10−4 sec. τ2 = 1.45 × 10−3 sec, τ3 = 5 sec have
been observed. A general mechanism was put forward to explain the
observed relaxation times.
Temperature Jump Relaxation Technique 349
(4) (a) The kinetic study of the reaction of sperm whale myoglobin with
O2 at pH 7.0 ( T = 293 K) was made by T-Jump method.
The relaxation times were found to range from 0.0125 sec to 3 ×
10−3 sec depending upon the equilibrium concentrations of the
myoglobin and O2 . The following scheme
k1
BG MGO2
MG + O2 FGGGGGG
GGGGG
k −1
(19.9)
was proposed where MG = myoglobin and MGO2 is its complex
with O2 . The relaxation time is related to the rate constants k1 and
k −1 according to
1
= k −1 + k1 (MḠ + Ō2 )
τ
(19.10)
From the variation of 1/τ with [(MG + Ō2 )], the rate constants k1
and k −1 were found to be
k1 = 1.9 × 107 M−1 sec−1 and k −1 = 11 sec−1
(19.11)
(b) A temperature jump kinetic study of the reaction of O2 with β−SH
and β PMB sub-units of human haemoglobin was made at pH = 7.0
(temperature = 293 K). The relaxation times ranged from 4.3 × 10−3
sec to 1.5 × 10−3 sec in the former case and from 1.13 × 10−3 to
4.8 × 10−4 sec in the latter case.
The rate constants for the reaction of β−SH with O2 were calculated
to be k1 = 6.5 × 107 M−1 sec−1 and k −1 = 16.0 sec−1 . The rate
constants for the reaction of β PMB with O2 were found to be k1 =
8.3 × 107 M−1 sec−1 and k −1 = 156 sec−1 . The temperature was
maintained at 293 K in all cases.
Questions
(1) Derive an expression for the reciprocal relaxation time (1/τ ) for a general chemical reaction given by
k1
BG C + D
A + B FGGGGGG
GGGGG
k −1
350
Biophysical Chemistry
(2) The relaxation time of a polypeptide chain represented by
YXXXX YYYYY
containing the amino acid alanine was measured by laser T-Jump technique and found to be 160 nanosec. If the relaxation is due to helix-coil
transformation of the chain and its equilibrium constant is 1.10, calculate the forward and reverse rate constants for this change.
(3) A certain proton transfer reaction involving an indicator, HIn, was
studied by T-Jump method at 298 K. The reaction may be represented
by
k1
BG In− + H2 O
HIn + OH− FGGGGGG
GGGGG
k −1
The relaxation times observed at two concentrations of the OH− ion
and the calculated equilibrium concentration C̄HIn + C̄OH are given
below:
−
COH
1.29 × 10−4
2.45 × 10−4
C̄HIn + C̄OH
3.20 × 10−4
4.35 × 10−4
1/τ × 10−4 (sec−1 )
3.90
4.30
Derive the equation for 1/τ and calculate the rate constants k1 and k −1 .
(4) Derive the equation
∂ ln K
∂P
=−
T
ΔV ◦
RT
which relates the displacement of equilibrium by application of pressure on a system. ΔV ◦ represents the difference between standard
molar volumes between the products and reactants.
(5) The hydration of propionaldehyde, given by the reaction
k1
BG CH3 CH2 CH(OH2 ) + H3 O+
CH3 CH2 CHO + H3 O+ + H2 O FGGGGGG
GGGGG
k −1
was studied by P-Jump technique and a relaxation time (τ ) of 2.5 sec
was observed at pH = 3.30. τ is given by the expression
1
1
CH +
= k1 1 +
τ
K
where K is C̄hydrate /C̄aldehyde = 480. Calculate k1 and k −1 .
20
Flash Photolysis Technique
for the Kinetic Study of Fast
Biochemical Reactions
20.1
Introduction
The initiation of a reaction by photo irradiation is a powerful method for
the study of rates and for detecting transient intermediates formed in the
reaction. In this context, the flash photolysis technique, developed by Norrish and Porter in UK has proved to be very convenient not only to initiate
the reaction but also for bringing about a considerable extent of reaction in
a very short interval of time.
20.2
Principle of the Method
The reactant system is irradiated with an intense flash of light in UV or
visible region to produce a measurable change of concentration in the system. The flash is usually of a very short duration, of the order of a few
microseconds (i.e., short compared to the reactions that follow).
The subsequent chemical changes are followed by what is known as
kinetic spectrometry. In this method, the absorption is measured at a given
wavelength as a function of time.
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_20
351
352
Biophysical Chemistry
Experimental Arrangement
The following diagram illustrates the basic experimental setup.
Photolytic flash
Photoelectric
detector
Collimalting
lens
Source for
for following
reaction
Mono
chromator
Reaction tube
Display
device
G
Figure 20.1 A flash photolytic unit.
A high intensity flash with energies ranging upto 2000 J is generated
in an interval of 1 to 100 μsec or microsec. The emerging light is collimated
onto reaction vessel through a series of mirrors. The change in absorbance
of the solution is monitored by means of a monochromator and photocell
and displayed on an oscilloscope. In recent years, lasers have been used as
sources of photolysis flash.
20.3
Applications
(1) Reactions such as the triplet state of naphthalene in hexane, decay of
duroquinone radical in viscous paraffin liquid have been studied.
(2) The reaction of myoglobin with carbon monoxide has been investigated by this method at a pH = 7.1 and temperature of 296.5 K. The
reaction is
k1
BG MgbCo
Mgb + Co FGGGGGG
(20.1)
GGGGG
k −1
and was monitored at 540 μm. The reaction was found to be second
order and the rate constant k1 is 5.5 × 105 l mol−1 sec−1 .
Flash Photolysis Technique
353
(3) The reaction between myoglobin and oxygen was also studied by this
method and the rate constant for the combination reaction
Mgb + O2 ←→ MgbO2
(20.2)
has been reported as 1.28 × 107 lmol−1 sec−1 at 296.5 K.
(4) Flash photolysis technique was employed to generate a reactive aryl
nitrene from N-(4-azido-2-nitrophenyl) 2-aminoethyl sulfonate (NAPtaurine) in the presence of the protein ribonuclease A. The reactive
nitrene is inserted in about 2 ms into those C-H-bonds of the protein
that are exposed to the solvent. On the basis of amino acid analysis,
it appears that the residues of the native protein that are buried in the
interior of the molecule do not react with the nitrene. But when these
residues (even the non-reactive ones such as valine and proline) are
exposed by the denaturation of the proteins, they react with nitrene. It
is observed that the native ribonuclease A retains 90% of its enzymic
activity when flashed in the absence of NAP-Taurine. This small loss
in activity arises from the disruption of a limited portion of the native
enzyme structure. The site of this limited disruption may be a portion
of the enzyme surface near the cys-26-cys-84 disulfide bond.
Questions
(1) The decay of the triplet state of naphthalene was followed by a flash
photolysis technique at 410 nm. The first order rate constant for this
process was found to be 6.3 × 105 min−1 . Calculate the time required
for its concentration to be one tenth of its original value.
(2) The reaction between myoglobin (Mgb) and carbon monoxide was studied by flash photolytic technique at 540 nm and the rate constant of the
forward reaction
k1
BG MgbCO
Mgb + CO FGGGGGG
GGGGG
k −1
was found to be 5.5 × 105 l mol−1 sec−1 . Assuming that their initial
concentrations are 1 × 10−4 M, calculate the time required for the “CO”
concentration decrease to 1 × 10−5 M.
21
Pressure Jump Relaxation
Method for the Kinetic Study
of Fast Biochemical Reactions
21.1
Introduction
The use of pressure as a perturbing parameter to initiate and understand
biochemical reactions has a long history. In the pressure jump technique,
the pressure on a reaction system in solution is changed rapidly and the
position of equilibrium thereby shifts adiabatically according to
ΔV ◦
Vα ΔH ◦
∂ ln K
(21.1)
=−
+
·
∂P
RT
C p RT
S
where ΔV ◦ = change in molar volume between products and reactants
under standard conditions, ΔH ◦ refers to difference in enthalpy between
products and reactants under standard conditions, α is the coefficient of
thermal expansion
1 ∂V
V ∂T P
C p is the heat capacity, R is universal gas constant.
In aqueous solutions at 298 K, for moderate pressure changes of the
order of 50 atm (≈ 50 bar), the contribution due to second term of equation
(21.1) is quite small and hence the equation is adopted.
∂ ln K P
ΔV ◦
(21.2)
=−
∂P
RT
S
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_21
355
356
21.2
Biophysical Chemistry
Experimental Arrangement and Methodology
There are two essential parts of a P-Jump unit: (i) a device for the production of a fast P-Jump, (ii) a suitable Wheatstone bridge net work with a fast
recording device such as an oscilloscope to follow the change in conductivity (there are other physical parameters such as colour change which can
be followed in a spectrophotometer) of the solution with time.
The following diagram shows the set up commonly used in Pressure
Jump experiments. The Wheatstone bridge employed in these studies is
such that it measures only relative changes of cell resistance and hence its
design is different from the bridge normally used in resistance measurements. It is also to be noted that the bridge frequency must be greater than
the reciprocal of the smallest relaxation time to be measured.
Conductance cells
Bursting
memberance
Auto
clave
Inlet for
pumping
kerosene
50 kHz
Oscillator
Potentiometer
Oscilloscope
Figure 21.1 Experimental arrangement of a pressure jump unit.
Pressure Jump Relaxation Method 357
Two small conductivity cells (1 ml capacity) are placed inside the autoclave one filled with the experimental solution and the other with a reference non-relaxing solution like KCl or MgSO4 such that they nearly have
the same resistance. The rest of the autoclave is filled with a non-conducting
liquid like kerosene and the pressure is built up by pumping kerosine into
the autoclave which is covered with a thin metallic disc. The disc ruptures when the pressure reaches around 60–70 atms and simultaneously
the oscilloscope is triggered. The release of pressure to the atmospheric
pressure takes about 50 μsec.
21.3
Applications
(1) Conformational changes of serum albumin: In this study, changes in
intrinsic protein fluorescence and absorbance changes due to spectral
shift in the benzyl orange dye spectrum are used to follow the isomerisation. A single relaxation time was observed and the rate constant
was ∼ 6.0 sec−1 .
(2) Equilibrium in myosin assembly: The equilibrium in myosin according to
100 myosin monomers ( M ) ←→ myosin filament assembly ( F )
(21.3)
whose equilibrium constant is given by
K er =
[ F]
[ M]100
(21.4)
The experimental data was fitted to the second order equation
2M ←→ M2
(21.5)
and the second order rate constant was found to be ≈ 4 × 104 l gm−1
sec−1 at 298 K and a pH = 8.3.
(3) The equilibrium concentrations of the reactants in the system
Lactate + NAD+
Lactate
+
FGGGGGGGGGGGB
GGGGGGGGGGG pyruvate + NADH + H
dehydrogenase
(21.6)
were altered by change of pressure on the system by 150 atm. The total
change in [NADH + NAD+ ] concentration was followed by UltravioletVisible spectroscopy at 340 nm. The relaxation time is found to be in
the range of 50 msec in presence of phosphate buffer.
358
Biophysical Chemistry
In another experiment the P-Jump of 200 atm was applied and the overall
lactate dehydrogenase equilibrium was monitored by protein fluorescence
quenching and a relaxation time of 0.56 sec was observed for a slow step.
(1) Several metal complex formation reactions in 2 : 2 and 3 : 2 electrolytes like BeSO4 , aluminium sulphate and gallium sulphate solution were studied by P-Jump technique. The hydration of pyruvic acid
(CH3 COCOOH) was investigated by employing this technique and a
relaxation time of 1 to 2 sec was observed.
(2) The pressure-jump induced relaxation kinetics was used for studying
the protein folding/unfolding of Y115W, a fluorescent variant of
ribonuclease A. Pressure jumps of the order of 350–400 atoms were
achieved and relaxation times of the order of a few minutes were
observed. For the reaction given by
ku
Folded state FGGGGGGB
GGGGGG Unfolded state
kf
(21.7)
The data obtained are shown below:
Temp (K)
303
323
pH
5.0
5.0
ΔGu◦
(kJmol−1 )
34.5
10.2
P1/2
(MPa)
468
225
k obs at P1/2
(sec−1 )
4.2 × 10−3
9.5 × 10−2
ΔVu=
(ml · mol−1 )
−58.2
−17.5
where P1/2 = pressure at half transition. The observed rate constants
at a pressure of 300 MPa are k f = 1.82 × 10−2 sec−1 at 300 K and k u =
8.1 × 10−2 sec−1 .
Questions
(1) For the reaction
k1
k3
BG Be(H2 O)SO4 FGGGGGG
BG Be2+ SO24−
Be2+ + SO24− FGGGGGG
GGGGG
GGGGG
k −1
k4
the first step is very fast (to be studied by P-Jump) and the reciprocal
relaxation time is given by
1
K0 (C̄1 + C̄2 )
= k4 + k3
τ
1 + K0 (C̄1 + C̄2 )
Pressure Jump Relaxation Method 359
where C1 = C̄Be2+ , C̄2 = C̄SO2− . Given that 1/τ at concentrations
4
(C̄1 + C̄2 )1 = 0.002; (C̄1 + C̄2 )2 = 0.004 are 350 sec−1 and 400 sec−1
respectively and K0 = 100 M−1 . Calculate k3 and k4 .
22
Circular Dichroism as a Tool
for the Analysis of
Biochemical Reactions
22.1
Introduction
Circular dichroism (CD) spectroscopy is a spectroscopic technique where
the CD of molecules is measured over a range of wavelengths. CD is
the difference in the absorption of left-handed circular polarized light (LCPL) and right-handed circularly polarized light (R-CPL) and occurs when
a molecule contains one or more light absorbing groups (or chiral chromophores).
(22.1)
CD = ΔA(γ) = A(γ)LCPL − A(γ)RCPL
where γ = wavelength and A = absorbance.
CD spectroscopy is widely used to study chiral molecules of all types
and sizes but it finds most applications in the study of large biological
molecules. Its primary use is in analyzing the secondary structure or conformation of macromolecules particularly proteins as secondary structure
is sensitive to environment, pH and temperature. Structural, kinetic and
thermodynamic information about macromolecules can be derived from
CD spectroscopy.
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_22
361
362
22.2
Biophysical Chemistry
Principle
The differential absorption occurs when a chromophore is chiral (or optically active) either (a) intrinsically by reason of its structure or (b) by being
linked to a chiral centre or (c) by being placed in an asymmetric environment. In practice, the plane polarized light is split into two circularly polarized components by passage through a modulator subjected to an alternating electric field (50 kHz). The modulator consists of a piezoelectric quartz
crystal and a thin plate of isotropic material (e.g., quartz) tightly coupled
to the crystal. The alternating electric field induces structural changes in
the quartz crystal which make the plate transmit circularly polarized light
at the extremes of the field.
If after passage through the sample, the LCPL and RCPL components
are not absorbed, combination of the components would regenerate radiation polarized in original plane. If one of the components is absorbed
by the sample to a greater extent than the other, the resultant radiation
would now be elliptically polarized i.e., the resultant would trace out an
ellipse. The CD spectropolarimeter does not recombine the components
but detects the two components separately. It will then display the dichroism at a given wavelength of radiation as either the difference in absorbance
of
(γA = A L − A R ) or as ellipticity in degrees (θ )
the two components
θ = tan−1 ba , where b and a are minor and major axes of the resultant
ellipse. The relationship between θ and ΔA is
θ = 32.98ΔA
(22.2)
Absorbance
(A)
ACD spectrum obtained when the dichroism is measured as a function of
wavelength is shown below:
1
2
3
Figure 22.1 Different types of CD spectra. Band 1 is not chiral; Band 2 has
a positive CD spectrum with L absorbed more than R; and Band 3 has a
negative CD spectrum.
Circular Dichroism as a Tool for the Analysis of Biochemical Reactions
363
If two or more identical chromophores are in close proximity, the interaction between them can give rise to a sigmoidal CD spectrum. The exciton
coupling model can be used to account for this behavior. Such coupling
can occur from close pairs of Trp side chains or from more extended sets
of chromophores in proteins. The latter situation is seen in the case of the
ring 18B850 of bacterio-chlorophyll molecules in the LH2 light harvesting
complex from photosynthetic bacteria. In much of biological work, the
observed ellipticities are of the order of 10 milli degrees (i.e., the difference
in absorbance between two circularly polarized components of the incident
radiation is of the order of 3 × 10−4 absorbance units).
22.3
Experimental Set Up
The light source for conventional CD measurements is a xenon arc. It gives
output over a range of wave lengths (178 to 1000 nm), a good range for
studies on proteins. It is important to flush the instrument with N2 gas to
remove oxygen from the lamp housing to prevent ozone formation and to
allow measurements below 200 nm.
The protein samples must be homogeneous and should be freed of
scattering particles by centrifugation or passage through suitable filter. It is
also important to minimize absorption due to other components of buffers
(solvents, supporting electrolytes etc.). Sometimes, it may be necessary to
run “blank” CD spectra to ensure that the buffer components do not lead
to excessive noise or other artefacts in the spectra.
It is also essential to know the protein concentration to within ±5% in
order to enable the estimate of the secondary structure content of a protein
from CD.
22.3.1
Units for CD
When CD data are expressed in terms of absorbance, ΔA = difference in
molar absorbance = A L − A R , (i.e., cm−1 · M−1 ). When the data are
expressed in terms of ellipticity, the mean residue ellipticity [θ ]mrw,λ at a
given wavelength is given in units of deg · cm2 · d · mol−1 and is given by
[θ ]mrw,λ =
MRWθ
10d · C
(22.3)
where θ = observed ellipticity (deg), d is the path length, C is the concentration (in gm mol−1 ).
364
Biophysical Chemistry
At any given wavelength, θ and ΔA can be related according to
θmrw = 3298ΔA
22.3.2
(22.4)
Amount of Sample Required
Considering that the cells used for far UVCD are about 0.01 to 0.05 cm,
protein concentrations are in the range of 0.1 to 0.2 mg ml−1 . The CD
signals in the near UV and visible regions are much weaker than in far
UV indicating the much lower concentrations of chromophores compared
to those of peptide bonds. Protein concentrations of the order of 0.5 to
2 mg ml−1 with a cell of length 0.5 to 2 cm are used. Recording of CD spectra at liquid nitrogen temperatures i.e., 77K give very useful information.
22.4
Methodology
Stopped flow technique was adopted in the initial stages and has been used
to study early events in protein unfolding. Concentrations of the order of
0.55 M bring out sufficient changes in ellipticity in the far UV (225 nm) and
near UV (290 nm) to get valuable information in the secondary and tertiary
structure of proteins.
22.4.1
CD Studied by Using Synchrotron Radiation
Synchrotron radiation is generated when charged particles like electrons
moving at velocities close to that of light are accelerated through magnetic
fields. Intense radiation is produced covering a wide range of wavelengths
from X-rays to IR. The key adavantage for CD in the generation of synchrotron radiation is the very large increase (over 1000 fold) in intensity in
the far UV region (below 200 nm) compared with xenon arc sources.
22.5
Disadvantages as Limitations
Studies using CD have certain advantages over X-ray and NMR in that
the measurements can be carried out fast and good quality spectra can be
obtained in the near UV and far UV region in a short interval of 30 mins.
Some examples illustrate this point. The changes in the far UVCD spectrum of a 58-residue DNA binding peptide derived from transcription factor GCN4 over 1000 fold range of concentrations has been interpreted in
terms of a dimeric species with a high helical content (about 70%) dissociating upon dilution into two unfolded chains. Far UVCD studies required
Circular Dichroism as a Tool for the Analysis of Biochemical Reactions
365
only small amounts of material. The technique is non-destructive and
hence one can recover most of the solution and conduct multiple experiments on the same sample. Since cells of differing path lengths can be
used, a wide range of protein concentrations can be employed. CD studies
can be performed over a wide range of experimental conditions including
pH and temperature.
The main limitation of CD is that it only provides low resolution structural information. For example, although few UVCD can give reliable estimates of the secondary structure content of a protein (in terms of proportion of a-new and b-sheets and β-turns), the overall figures do not indicate
which regions of the protein are of what type. CD gives little information
on quaternary structure of protein.
22.6
Applications
CD has been employed widely in studies of protein unfolding and folding.
The secondary and tertiary structural characteristics of a range of mutant
proteins has been assessed rapidly by CD Mutations have not resulted in
significant distortion of the structure when in two mutant forms of isocitrate lyase from Escherichia coli in which CYS 195 has been replaced by
alanine or serine. In the case of phosphoglycerate mutase from schizosaccharomyces pombe, mutations of HIS163 to Gln leads to complete loss of
activity. The CD spectra of isocitrate lyase from E.coli has been obtained.
Although the ligand or cofactor has no CD signal, the observed CD signal
in the complex indicate that the binding site of ligand or cofactor confers
chirality. It should be noted that there is generally a much smaller change
in absorbance of the cofactor on dissociation from the protein.
22.6.1
Effect of Cofactors and Ligands on CD
(i) Pyridoxal-5-phosphate: This cofactor is used by many enzymes such
as amino transferases and decarboxylases in amino acid metabolism.
It is also a cofactor in glycogen phosphorylase.
CD was used in studies of two iso enzymes of aspartate aminotransferase which were used to study unfolding of proteins. The specific
CD peaks at 365 nm (for cytoplasmic isoenzyme) and 355 nm (mitochondrial isoenzyme) were used to monitor loss of cofactor from the
enzyme since free pyridoxal-5 -phosphate shows no significant CD
signal in this region.
366
Biophysical Chemistry
(ii) Flavins: The two flavins (Flavin mononucleotide (FMN)) and flavin
adenine nucleotide (FAN) play important redox roles in the electron
transport chain as well as in a number of enzyme catalysed oxidation
reactions of substrates such as amino and hydroxyl acids. Free flavins
exhibit only very small visible CD and hence dissociation of cofactor
leads to complete loss of the signal. The FMN containing E.coli flavodoxin shows negative ellipticity over 310–450 nm with a maximum
visible signal at 365 nm (−160 deg cm2 · dmol−1 ). However, its redox
partner flavodoxin reductase exhibits a +ve ellipticity over 300–420
nm with a maximum around 390 nm (+95 deg · cm2 · dmol−1 ).
Figure 22.2 CD spectra of flavoproteins in the near UV and visible region.
θ (deg. cm2. d mol–1)
15000
10000
5000
0
–5000
–10000
200
220
240
Wavelength (nm)
Figure 22.3 CD spectra of isocitrate lyase from E.coli.
260
Circular Dichroism as a Tool for the Analysis of Biochemical Reactions
367
2.0
1.0
0
–1.0
260
270
280
290
300
310
320
Wavelength (nm)
Figure 22.4 CD spectra of the same species in the near UV region.
(iii) Haem: The Haem cofactor plays a number of roles in proteins such as
binding of O2 in Haemoglobin cytochrome C-oxidase, a cofactor for
hydro-peroxidases such as catalase or as a redox cofactor as incyto
chromes b and c. CD has been used widely to study cytochrome P450 enzymes. The CD spectrum of a P-450 can be used to predict
active site structural properties such as H-bonding and polarity.
22.6.2
Specific Examples
(1) Peptide bond: Peptide bond absorbs in far UV. Aromatic amino acid
side chains (like phenyl alanine, tyrosine and tryptophan) absorb in
the near UV range 250–290 nm studies in far UV can give information
on the secondary structure. The tertiary folding of polypeptide chain
can place these side chains in chiral environment giving rise to CD
spectra which can serve as characteristic finger prints of the native
structure. This bond absorbs primarily in the far UV region (240 nm
to 190 nm). Studies of far UVCD can be used to assess the overall
secondary structure of the protein quantitatively.
(2) Aromatic amino acid side chains: The near UVCD of proteins arises
from the environs of each aromatic amino acid side chain as well as
possible contributions from disulphide bonds, or non-protein cofactors. Small model compounds of the aromatic amino acids exhibit
CD spectra because the chromophore is linked to the nearby α-carbon
atom. It may be noted that in the case of proteins in their native states
the side chains of amino acids are placed in a variety of asymmetric environments characteristic of the tertiary structure of the folded
protein. Some factors which influence CD spectra of aromatic amino
368
Biophysical Chemistry
acids are: (i) rigidity of protein, (ii) nature of environment such as Hbonding polar groups and polarisability, (iii) interactions among the
amino acids, (iv) number of aromatic amino acids in a protein.
(3) Non-protein chromophores: CD in the near UV, visible and near IR
provides a great deal of information on the environments of cofactors
or other.
(4) Flavocytochromes: CD in the visible region provides a valuable spectroscopic probe of flavins in flavocytochromes. It may be mentioned
that flavocytochromes are enzymes containing two redox cofactors
i.e., FMN or FAD and a Haem group. For example, in nitrate reductase from chlorella, the flavin contribution is masked in the visible
absorption spectrum of this molybdoprotein and Haem b containing enzyme. Positive CD signals from FAD are seen at 311 nm and
387 nm with negative CD signals at 460 nm and 487 nm.
(5) Nucleic acid chromophores: The bases DNA and RNA do not show
any intrinsic CD. The chirality of the sugar residues induces a small
CD in the nucleotide units. The stacking of bases which occurs in
helical structures adopted by oligonucleotides and nucleic acids gives
rise to large CD signals due to exciton coupling between the bases.
The CD spectrum in the range 230 to 300 nm is a very sensitive measure of secondary structure with the various forms of DNA and RNA.
(6) Conformational changes in proteins: CD is an ideal technique to
monitor conformational changes in proteins arising from experimental factors such as temperature, pH, binding of ligands. CD has
proved to be very useful in studying the extent of conformational
changes and the stability of the folded state of the protein. Far UVCD
has been found to be a valuable tool for monitoring the transitions
from α-helical to β-sheet structures in proteins and peptides.
CD was used to monitor the binding of Ca2+ to troponin C, the protein which confers Ca2+ sensitivity to muscle. It was found that addition of Ca2+ led to an increase in the negative CD signal in the range
200 to 230 nm showing the increased stability of the helical regions of
the protein.
CD has been used to monitor the binding of the substrate ATP to a
mutant form of the molecular chaperone GroEL. This protein consists of two stacked rings of seven sub-units with a central cavity
within which the protein folding is assisted. Binding of ATP to K3E
mutant was shown from the observation that addition of nucleotide
led to a 50% increase in the size of the CD signals in the range 230 to
Circular Dichroism as a Tool for the Analysis of Biochemical Reactions
369
190 nm. By contrast, addition of ATP does not lead to any increase
in the far UVCD signal of the wild type protein. The near UVCD of
the hexameric enzyme glutamate dehydrogenase was used to monitor the binding of NAD+ in the presence of a competitive inhibitor
glutarate. Addition of NAD+ was found to change the near UVCD
signal sharply at a point corresponding to half saturation of the sites.
CD has also been employed to monitor the R ←→ T allosteric transition in haemoglobin. The CD spectrum of the protein shows the
bands in the 270–300 nm region due to Tyr and Trp side chains and a
large band at 260 nm due to Haem group. Deoxyhaemoglobin shows
Figure 22.5 CD spectra of α-lactalbumin (a) and (b) represent far UV and
near UV spectra at pH = 7.0.
370
Biophysical Chemistry
a negative band at 287 nm. Oxyhaemoglobin shows weak negative
bands at 283 nm and 290 nm. The marked changes in the near UVCD
spectrum were attributed to changes in environments of Trp-37 on
the β-chain and Tyr-42 on the α-chain.
A particularly important use of CD has been to characterize the overall structural states of proteins. For example, when lactalbumin is
incubated at pH = 2.0, it retains its native secondary structure as
shown by far UVCD. However, under these conditions, the near
UVCD signal of the protein is very much reduced indicating that the
native tertiary interactions are absent.
(7) The refolding of proteins and peptides: An important problem of
molecular biology is the mechanism of protein folding. An understanding of protein folding is necessary to learn about the disease
states of the body which arise possibly from protein misfolding. In
order to establish the mechanism of refolding of a protein, it is necessary to study the time scale of recovery of structure from the denatured state and to correlate the same with the recovery of biological
state. The regaining of secondary structure is monitored by far UVCD
and that of tertiary structure by near UVCD and fluorescence.
Stopped flow CD has been widely used to examine the properties
of early intermediates in protein folding. Studies showed that during
the refolding of denatured ferricytochrome C and β-lactoglobulin, the
native ellipticity in the far UVCD was regained in around 18 msec.
However, the ellipticity in visible region for ferricytochrome C or
near UV for β-lactoglobulin was regained over a period of few minutes. In a study of native and mutant forms of staphylococcus nuclease, it was found that a transient kinetic intermediate formed within
10 msec. This intermediate possessed only about 30% of native ellipticity at 225 nm. The relatively low amplitude indicated formation
of the 5-stranded β-sheet structural core of N-terminal domain of the
enzyme with the helices contributing bulk of the CD signal at 225 nm.
In some detailed studies of protein folding and refolding on hen egg
white lysozyme, it was shown that the form disulphide bonds are
kept intact. Stopped flow CD and fluorescence were used to define
the regions of protein where secondary structure and tertiary interactions have formed during refolding process. Further insights into the
folding of lysozyme were obtained from stopped flow CD at far and
near UV region. The near UV results at 289 nm show that the native
Circular Dichroism as a Tool for the Analysis of Biochemical Reactions
371
ellipticity develops in a single kinetic process with a rate similar to the
protection of β-sheets domain. Stopped flow CD in the far UV shows
that about 80% of native ellipticity was regained at 225 nm. In a second phase which was complete after 80 msec the CD signal increases
to about 150% of the native value in a third phase of t1/2 = 300 msec.
(8) Protein and peptide design: CD is a very useful tool in characterizing peptide and protein fragments which have been designed to
adopt specific structures and thus display special biological functions. Peptides based on a repeating unit AEAAKA having length
in the range of 14 to 50 residues form helices as shown by far UVCD.
The urea induced unfolding of these peptides has been monitored
by CD and the energetics of the unfolding is shown to correspond
with those of unfolding of helices in proteins like myoglobin. The far
UVCD spectrum of peptide of the type AC-KAKAKAKAEAEAGA
EA-NH2 show interesting structural changes at 20◦ C as shown by far
UVCD. It was shown to form a stable macroscopic β-sheet in water
which stains dyes like congored. There is an abrupt change in the CD
spectrum on raising the temperature to 70◦ C which indicates that a
helical structure is formed.
(9) Membrane proteins: CD is an important technique in the study of
membrane proteins especially to study the retention of native structure on extraction and purification. For example, in studies of extraction of myelin basic protein (MBP) from equine myelin, CD was used
to assess the effect of several detergents on the protein. The solubilisation of bacteriorhodopsin in the non-ionic detergent octyl glucoside
led to significant changes in secondary structure as indicated by for
UVCD spectrum. The structural changes in the OMPA protein in the
unfolded and folded state were monitored by far UVCD and protein
fluorescence. The changes in CD signal at 206 nm showed that there
were three phases to the folding and insertion process. Far UVCD
studies showed that peptides are largely helical (78%).
(10) Interactions between domains in proteins: Most polypeptide chains
larger than 30 kDA (30,000 amu) tend to exist in multiple domains i.e.,
as independent folding units. Preparation of large quantities of protein corresponding to defined domains has been achieved by recombinant DNA technique. CD has been employed as a structural technique in such studies. Using CD signal at 218 nm to monitor the loss
372
Biophysical Chemistry
of structure caused by Gdmcl, the unfolding of intact enzyme and
isolated N- and C-domains were found in phosphoglycerate kinase.
CD studies helped to unravel the low rate of electron transfer
between the redox centers of separate flavin and P-450 domains of
BM3 flavocytochromes. The significant differences CD spectra in the
near UV and visible regions in intact BM3 were ascribed to characteristic environments of the aromatic amino acid side chains and flavin
and haem cofactors. In experiments with P-450 BM3, CD was used to
compare the structural stability of the protein as measured by CD in
the near and visible regions.
CD studies of the LH2 complex from Rhodopseudomonas acidophila
10050 in the far UV region have shown that α- and β-polypeptides
have high (50–60%) α-helical content. The absorption and CD spectra
of LH2 show three main absorption peaks between 450 and 550 nm
and they were ascribed to carotenoid molecules. The absorption peaks
at 380 nm, 595 nm and in 750 to 900 nm region are due to Bchla (also
known as B-800) and that at 863 nm is due to the inner set of 18 Bchla.
When the carotenoid (of LH2 complex) is extracted and dissolved in
organic solvents, there is no CD signal which reflects the symmetry
of the molecule. CD can be used to assess the structural integrity of
LH2 complexes.
Comparison of CD spectra of native LH2 , B-850 complexes in both
visible and near IR spectral regions showed that the inner core of
carotenoids and Bchla-B850 were very similar. In addition, the CD
of the reconstituted and native complex around 800 nm showed that
the Bchla-B800 molecules had bound in the correct orientation in the
reconstituted complexes.
(11) Ligand binding and drug design: The binding of ligands to proteins
gives rise to a CD signal due to asymmetric binding of the ligand.
Such binding leads to conformational changes in proteins which can
be detected by far UV and near UVCD. Studies of the helical content and thermal stability using CD were used to examine the effects
of mutating amino acids involved in contact with the N-helix in the
hydrophobic groove.
Circular Dichroism as a Tool for the Analysis of Biochemical Reactions
373
Questions
(1) In circular dichroism, the technique used is
(a) light scattering
(c) NMR
(b) UV-Vis
(d) ESR
(2) The relation between ΔA (A-absorbance) and θ (ellipticity in degrees)
is
(a) θ = 35 × ΔA
(c) θ = 3298 × ΔA
Φ
(b) ΔA =
(d) θ = 45.82 × ΔA
40.5
(3) The far UVCD spectrum of α-lactalbumin (around pH = 2.0) shows a θ
maximum (in nm) around
(a) 310
(c) 305
(b) 250
(d) 420
(4) Circular Dichroism is best used for study of
(a)
(b)
(c)
(d)
acid-base dissociation
potential changes in electrolytic solutions
electro reduction of a species at metal or mercury surface
conformational changes in proteins
(5) Far UV circular dichroism gives valuable information on
(a)
(b)
(c)
(d)
secondary structure of proteins
lipid bilayer composition
carbohydrate metabolism
Phosphate groups in ATP
(6) Discuss the advantages and disadvantages of the technique of circular
dichroism in the study of biomolecules.
(7) Enumerate the applications of circular dichroism giving specific
examples.
23
Applications of Isothermal
Calorimetry in the Study of
Biochemical Reactions
23.1
Introduction
Isothermal calorimetry (ITC) is a physical technique that measures directly
the heat released or consumed in a bimolecular reaction. In this analytical
technique the ligand comes in contact with a macromolecule (or any other)
at constant temperature. From basic thermodynamic principles, whenever there is contact between two molecules, there is either heat evolution
or absorption depending on the type of binding. The heat generated or
absorbed during an interaction is measured. It is widely used for measuring the binding energetics of biological macromolecular interactions
including protein—ligand and protein—protein interactions. Kinetic data
of enzyme-catalysed reactions have also been obtained by employing this
method.
In recent years, ITC has been recognized as a reliable tool for obtaining thermodynamic parameters (of intermolar interactions) such as ΔH, ΔS
and binding constants of protein-ligand interactions.
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_23
375
376
23.2
Biophysical Chemistry
Experimental Set Up
The schematic presentation of a simple isothermal heat conduction
calorimeter is given below.
(a) Syringe holder
(b) U-shaped holder
(c) Ampoule made of glass
(d) Aluminium cup (for sample)
(e) Aluminium cup (for reference)
(f) Thermocouples
Figure 23.1 Experimental set up of an isothermal heat flow calorimeter.
In the above instrument there are two identical heat flow sensors, one
for sample measurement and the other for a reference. On top of the sensors, aluminium cups are mounted into which the ampoules with the sample and reference are mounted and inserted for a measurement. The sensors are in intimate thermal contact with a relatively large aluminum heat
sink. A syringe is mounted above the sample cup to enable introduction of
a solution or liquid as reagent. The complete instrument is shielded from
the surrounding by an insulation jacket.
The voltage from the reference heat flow sensor is subtracted from the
voltage from the measurement heat flow sensor. Heat that enters through
the jacket will influence the measurement and the reference sides identically and thus will produce no net signal. Only a signal produced by
the sample will be recorded. It is advantageous for the calorimeter to be
equipped with a thermostatting position where a sample may be held for
Applications of Isothermal Calorimetry 377
some time before being introduced into thermal contact with the thermocouples. Isothermal calorimeters are calibrated electrically with a resistance heater inserted into the measurement ampoule or attached to the
ampoule holder.
23.3
Applications
(1) Thermodynamic data for characterization of ligand—target binding:
According to the equation
ΔG = ΔH − TΔS
(23.1)
changes in enthalpy and entropy account for the Gibbs free energy of
ligand binding. When the conformation of a ligand becomes more
rigid, ΔS becomes smaller indicating that the entropic loss of ligand
target binding is getting smaller. If the ΔH becomes more negative, it
means that more stronger interactions occur between the ligand and
target. To evaluate which ligand is more enthalpy favourable, a quantity enthalpic efficiency (EE) equal to ΔH/Q is proposed where Q is
number of heavy atoms or molar mass of a ligand. By analysing the
thermodynamic profiling of inhibitors of HIV protease and the
hydroxymethyl glutaryl co-enzyme A (HMG-CoA) reductase, it was
possible to differentiate between the best in drug classes. The enthalpy
change data serve as a key indicator to qualify ligands as candidates
for best ligand-target studies.
(2) ITC combined with ligand—protein complex structure: Rapid development of structural biology, especially in X-ray protein crystallography, provides a powerful approach to solving protein-ligand complex
structures and reveal the actual binding site of compounds in the binding pocket of targeting proteins. Introduction of a H-bond or presence
of hydrophobic interactions in protein ligand exchange are responsible
for enthalpy/entropy gain or loss. The importance of thermodynamic
data in the understanding of protein ligand interactions is explained
through the following examples.
(a) Phosphodiesterase type 5, an enzyme catalyzing the hydrolysis of
CGMP to 5-GMP is an important drug target for treatment of diseases associated with low level of CGMP such as pulmonary arterial hypertension. ITC experiments were used to supplement the
experimental results of the bonding of halogen (F, Cl, Br, I) substituted (in the 5th position) monocyclic pyrimidinones with the
378
Biophysical Chemistry
residue of Y612 of PDE5. The crystal structures of PDES complex
with compounds involving halogens (in pyrimidinones) showed a
similar binding mode for the compounds with the enzyme. The
binding free energies of the Cl, Br, I substituted compounds in
pyrimidinones with PDE5 were found to be −34.9, −36.4, and
−38.9 kJmol−1 respectively.
A second example which takes advantage of the thermodynamic
data supported by complex structures is the discovery of FABP4,
which is a fatty acid binding protein responsible for transportation
of saturated fatty acids to mediate cell signal path ways. It was
shown that FABP4 (knocked out from mice) protects the mice from
diseases such as atherosclerosis and type-2 diabetes.
(b) A typical ITC experiment consists of three steps:
(i) a ligand is titrated into a solution containing the bio macromolecule (e.g., a protein),
(ii) measurement of the heat released or absorbed that is associated
with the binding event,
(iii) the resulting data is processed to obtain binding constant, Kb ,
free energy of binding, ΔGbin , enthalpy and entropy of binding
(ΔHbin and ΔSbin ). A representative ITC data for the binding
of cytidine 2’-monophosphate (2’ CMP) to RNase A is shown
in Figure 23.2.
Figure 23.2 Binding curve obtained from unprocessed data.
Heat of reaction per injection as a function of the ratio of total ligand
concentration to the protein concentration.
At pH = 5.5 and T = 301K, it was found that K B = 2.5 × 105 M−1 ,
ΔH = −12.98 kcal mol−1 for the reaction between 2’ CMP and
RNASEA.
Applications of Isothermal Calorimetry 379
(3) Application of ITC data in kinetic studies: ITC measurements can be
used to obtain binding constant Km , k cat and Vmax of enzyme-substrate
reactions and inhibition constant (Kc ) of enzyme-inhibitor binding. The
development of a method, known as kinetic ITC technique, led to the
possibility of measuring binding kinetics (k on and k o f f ) and the
inhibitory constant Ki . The simple competitive enzyme inhibition
model given by the following kinetic scheme was used.
ko f f
k1
k cat
EI FGGGGGGGGB
GGGGGGGG E + I + S FGGGGGGGB
GGGGGGG ES −−→ E + P
k on
k −1
(23.2)
where E = enzyme, I = inhibitor, S = substrate, P = product, EI =
enzyme inhibitor complex), k on = association rate constant, k o f f = dissociation rate constant, k cat = catalytic rate coefficient.
The instantaneous rate of enzyme catalysis is given by MichaelisMenten equation as
d[ P]
d[S]
k cat [S]([ E0 ] − [ EI ])
=−
=
dt
dt
Km + [ S ]
(23.3)
where [ E0 ] = total enzyme concentration, [ EI ] = concentration of
enzyme-inhibitor complex, Km = Michaelis-Menten constant.
Assuming that the inhibition follows 1st order kinetics, given by
d[ EI ]
= k on [ E][ I ] − k o f f [ EI ]
dt
(23.4)
where [ E] = concentration of free enzyme.
The heat flow generated Q(t) by catalysis is given by
Q(t) = ΔHcat × Vcell ×
d[ P]
dt
(23.5)
where ΔHcat = enthalpy of catalysis, V = Volume of the cell. The basis
of this method is that ITC detects heat flow in real time and then gives a
direct measurement of enzyme activity and its variation in response to
inhibitor according to equations (23.3) and (23.5). The kinetic parameters of inhibitor binding to enzyme can be calculated from equation
(23.4). The kinetic ITC technique enables the kinetic constants of inhibition, k on ≈ 103 to 107 M, and Kc in sub-nM values to be determined.
380
Biophysical Chemistry
(4) Enzyme catalysed reactions: The enzyme glucose-6-phosphate dehydrogenase (G6PD) uses NAD+ or NADP+ as a cofactor and the
enthalpy changes of this enzyme catalysed reaction varied with the
type of buffer used in the reaction. When tris was used as buffer,
ΔH ITC = −22.93 kJ mol−1 , but ΔH ITC (when phosphate buffer) was
19.4 kJ mol−1 for NADP+ linked reaction but the values were −11.7 kJ
(tris) or 30.6 kJmol−1 (phosphate) for NAD+ reaction.
(5) ITC application in RNA biochemistry: ITC has been used in the areas
of small molecules binding to RNAs, RNA—protein interactions and
in fundamental studies of protein folding.
ITC was employed to study ligand binding to both natural purine
riboswitch as well as synthetic riboswitch that responds to tetracycline.
Binding has been shown to be enthalpically favourable and entropically opposed for all the species that can be accommodated in these
riboswitches.
Another group of small molecules—RNA interactions that has been
studied by ITC are the aminoglycoside antibiotics. These agents are
known to bind the 16SrRNA, HIV responsive elements and many other
RNA’s. It was shown by ITC studies that the binding of neomycin to
the 16SrRNA depends strongly on pH and buffer sensitivity.
ITC was used to probe individual steps in the assembly pathway of the
small subunits of bacterial ribosomes.
ITC was employed to probe the binding of ELF4E to a 7-methyl-GpppG
cap analogue. The temperature dependent analysis of this binding
showed significant enthalpy-entropy compensation. The following
table shows some data related to this binding.
Table 23.1 Enthalpy–entropy compensation in the binding of F4E to 7
methyl-GpppG.
Temperature (K)
290
300
310
ΔG ◦ (kJmol−1 )
−42.0
−40.0
−40.0
ΔH ◦ (kJmol−1 )
−70.0
−55.0
−40.0
(6) ITC use in biopharmaceutical development: Prox is a drug (prescribed
as tablets) used for treating bacterial infection of skin tissue. ITC has
been used to aid the development of additives for Prox. In this process,
polysorbate-80 and phenol were examined as the additives. It may be
Applications of Isothermal Calorimetry 381
mentioned that polysorbate-80 is a surfactant to prevent non-specific
adsorption and aggregation of proteins.
Fitting of the binding curve to a particular binding model can yield
Kb , ΔH and n (number of ligands) from a single experiment. Kb can
be obtained from ΔGbin . ΔSbin and ΔCP(bin) may similarly be obtained
using the following relationships.
ΔH(T ) = ΔH(T0) + ΔCP(bin) ( T − T0 )
ΔS(T ) = ΔS(T0) + ΔCP(bin) ln
T
T0
(23.6)
(23.7)
ΔG(T ) = ΔH(T0) − TΔST0 + ΔCP(bin)
T − T0 − T ln
T
T0
(23.8)
where T = experimental temperature, T0 = reference temperature. ITC
has been used to optimize HIV-I protease inhibitor binding by considering the thermodynamics of binding interactions. The figure below
shows the thermodynamic profiles for a pair of HIV-protease inhibitors
with the difference in the pair being a functional group. In this case, the
replacement of a thioether on KNI-10033 by a sulfonyl on KNI-10075
results in a more negative enthalpy change (−3.9 kcal mol−1 ) and less
entropy gain (−4.2 kcal mol−1 ).
ΔG = −14.9, ΔH = −8.2; TΔS = −6.7 (all in kcal mol−1 )
HO
H
N
O
O
O
N
H
O
O
N
N
H
CH
S
S
Figure 23.3 Thermodynamic profiles of HIV proteanase inhibitors.
Another example of an ITC experiment is a study of exothermic interaction between a specific phosphotyrosyl peptide (concentration = 210μM)
and Fyn SH2 domain (concentration = 21.0μM) at 293K. For this interaction, it was found that n = 1.1, K B (binding constant) = 2.42 × 106 M,
ΔHB◦ = 30.9 kJmol−1 .
ITC studies were performed on S-protein binding to seven forms of Speptide in which the methionine at position 13 (Met 13) was substituted by
382
Biophysical Chemistry
different amino acids. At pH = 6.0 and 298K, ΔΔGB◦ values for the Met13 →
isoleucine (ile) and Met13 → gly substitutions were 0.8 and 21.0 kJmol−1
respectively. (Note that ΔΔGB◦ = ΔGB◦ (mutant peptide) −ΔGB◦ (wild type
peptide)).
ITC studies were carried out on the formation of complex DNA structures corresponding to the intersection points of oligonucleotides. ITC has
been used to determine the Δ B H ◦ at 298 K for the interaction of the Δ and Λ
isomers of [Ru(phen)2 DPPZ]2+ with calf thymus DNA. Δ B H ◦ values of 0.8
kJmol−1 and 12.1 kJmol−1 were observed for Δ and Λ isomers respectively.
ITC studies proved to be invaluable in understanding the specificity
of proteins involved in intracellular signal transduction. Analysis of ITC
data showed the subtle differences between the thermodynamic consequences of interactions between selected SH2 domain and specific (and
non specific) tyrosyl phosphopeptides. Some thermodynamic data relating to these interactions is given below.
ITC has been used to explore the interaction of molecules in receptor
systems to elucidate the stoichiometry and thermodynamic parameters of
interactions. The mode of binding of acidic fibroblast growth factor (aFGF)
to its receptor was also investigated by ITC. It was shown that aFGF forms
a 1 : 1 complex with its receptor.
ITC data for the high affinity interaction of the immunosuppressive
agent FK506 with the protein FKBP-12 also supported the relation between
ΔCP( B) and surface area burial. The interactive surface between these two
molecules is entirely hydrophobic.
In the ternary complex TK: dT: ATP, the substrate dT and cofactor ATP
are located in separate and well defined binding pockets of the enzyme.
Table 23.2 Binding constants and thermodynamic data of tyrosyl phosphopeptides with different SH2 domains at 298 K.
K B × 10−6
ΔB G◦
ΔB H◦
Δ B S◦
−
1
−
1
−
1
(M )
kJmol
kJmol
Jmol−1
SH2 domain-specific
1.6
−35.4
−35.3
+0.43
peptide
Fyn SH2 domain-specific
1.4
−35.0
−35.6
−0.02
peptide
P85SH2 domain-specific
2.3
−36.2
−39.2
−10.1
peptide
LCK SH2
0.32
−31.3
−31.5
−0.37
domain-non-specific
peptide
Applications of Isothermal Calorimetry 383
The formation of the ternary complex may proceed through two sequential pathways (TK → TK: dT → TK: dT: ATP or TK → TK: ATP—TK: ATP:
dT). In a random binding mechanism, all these reactions take place. To distinguish between ordered and random mechanism, ITC was used which
confirmed the ordered binding mechanism (i.e., TK: dT → TK: dT: ATP) by
titrating the preformed TK: binding complex with ATP. The reaction was
found to be exothermic with K B = 3.9 × 106 M−1 and ΔHbin = −13.8 kcal
mol−1 at 298 K.
23.4
Mutational Studies
The mechanism of substrate diversity observed with HSVTKL was investigated by a thorough mutational study, Kinetic measurement and ITC
study. The residue triad H58/M128/Y172 was found to give a distinctive
binding of a large variety of substrates to HSVTKL. Mutations in this trial
have been prepared and analyzed by ITC.
Table 23.3 Thermodynamic data for binding in ATP in presence of various
mutants.
Mutant (of HSVITK)
ΔH/
ΔG/
TΔS/
KB
kcal mol−1 kcal mol−1 kcal mol−1 (105 )
Wild type
−26.3
−9.9
−16.4
229
M128F
−8.1
−6.8
−1.3
1.04
M128F/Y172F/H58L
−19.8
−7.4
−12.4
2.6
Specificity of Citrate Binding to the Histidine
Autokinase CitA Receptor
ITC was applied to describe the thermodynamic properties of citrate and
citrate analogue binding to CitAPhis . Citrate binding to CitAPhis was found
to be an exothermic process and could befit to a single-site binding model,
yielding K D value, where K D = K1B of 5.5 × 10−6 M and ΔHobs = −18.3 kcal
mol−1 . Depending on the pH, citrate exists in four species i.e., H3 -Citrate,
H2 -Citrate, H-Citrate2− and Citrate3− with pK1 = 3.13, pK2 = 4.76 and
pK3 = 6.40. In order to determine the preferred ligand species, the pH
dependency of citrate binding constant to CitAPhis was studied in the pH
range 4.0–9.0.
384
Biophysical Chemistry
Table 23.4 Thermodynamic parameters of citrate binding to CitAPhis at
298 K in 50 mM phosphate buffer at different pH’s as determined by ITC.
K B (104
KD
ΔHobs
ΔG (kcal
TΔS
pH
N
M−1 )
(μ M) (kcal mol−1 )
mol−1 )
(kcal mol−1 )
4.0 0.98
6.26
16.7
−24.7
−6.5
−18.2
7.0 0.89
18.25
5.5
−18.3
−7.2
−11.1
9.0 0.83
6.47
15.5
−17.9
−6.6
−11.3
Table 23.5 Thermodynamic parameters of citrate binding at different concentrations of MgCl2 .
ΔHobs
ΔG
TΔS
MgCl2
K B (104
(kcal
(kcal
(kcal
(mM) pH
N
M−1 )
K D (μ M) mol−1 ) mol−1 ) mol−1 )
2.0
7.0 0.83
7.78
12.9
−18.9
−6.7
−12.2
10.0
7.0 0.85
2.47
40.5
−21.3
−6.0
−15.3
20.0
7.0 0.89
1.74
57.5
−21.4
−5.8
−15.6
The inhibitory influence of Mg2+ ions on citrate binding as seen from
the above data (decrease of K B ) has been confirmed by ITC. From the tenfold decrease in K B at 20 mM Mg2+ , it was confirmed that that all citrate is
complexed as Mg-Citrate2− .
ITC was also used to localize amino acids involved in citrate binding
by studying the influence of point mutations in CitAPhis .
ITC was applied for investigating membrane bound protein-ligan interaction especially the serine receptor of Escherichia coli chemotaxis. ITC
was employed to shed light on human apo- and holo-transferrin binding
to the neisseria meningitides transferrin receptor.
23.5
Interaction of Transducer Fragments
Phototaxis of nitrosobacterium pharonis is mediated by two sensor
rhodopsin SR-I and SR-II. SR-I enables bacteria to seek light conditions
optimal for the function of light driven ion pumps and to avoid UV light.
SR-II conveys negative phototaxis, which might enable the bacteria to
evade harmful conditions of high oxygen concentration in presence of light.
Both receptors are bound to membrane proteins (halobacterial transducers of rhodopsin, Htr-I and Htr-II). Light excitation of the SR-II: NpHtr-II
complex leads to conformational changes in both proteins. Both SR-II and
NPHtr-II form a tight 2 : 2 complex on membranes where as in micelles
it dissociates to a 1 : 1 homodimer complex. ITC was used to elucidate
the dimerisation as well as to determine the size of the receptor binding
Applications of Isothermal Calorimetry 385
domain of transducer and also to determine K D of a series of the transducers to the receptor.
Binding affinities of four shortened transducer fragments (NpHtr-II82, . . . ,101, NpHtr-II-114, . . . ,tr-II-157) to the receptor SR-II were
determined using ITC. The relevant thermodynamic data are given below.
Table 23.6 Thermodynamic parameters pertaining to binding affinities.
ΔHobs
ΔG
ΔS
Temp K B (106
KD
(kcal
(kcal
(cal K−1
NpHtr-II
(K)
M−1 )
(nM) mol−1 ) mol−1 ) mol−1 )
157
318
6.2
160
−4.3
−9.9
−17.6
114
318
4.32
240
−4.2
−9.6
−17.0
101
295
0.1
104
−1.4
−6.7
−18.2
5
82
295
< 0.01 > 10
—
—
—
These data show that ITC is suitable to study membrane-protein
interactions.
Questions
(1) In an isothermal calorimetric experiment carried out in a cell of capacity 2 ml, the rate of the reaction in presence of an enzyme was found
to be 0.1 moles liter−1 min−1 . Calculate the rate of heat flow of this
enzyme catalyzed reaction.
(2) Draw a neat sketch of an isothermal calorimeter and indicate the parts.
(3) Describe the
biochemistry.
application
of
isothermal
calorimetry
in
(4) Discuss the use of isothermal calorimetric data in kinetic studies.
RNA
24
Principles of Differential
Scanning Calorimetry and its
Applications in the Study of
Biochemical Reactions
24.1
Introduction
It is a thermal analysis technique used to measure enthalpy changes due
to changes in physical and chemical properties of a material as a function
of temperature or time. It allows one to characterize materials and is a fast
sensitive technique.
In this technique the difference in energy inputs into a substance and
a reference material is measured as a function of temperature while the
substance and reference material are subjected to a controlled temperature
program.
24.2
Experimental Set Up
Block diagram of a heat flux DSC is given in the Figure 24.1. The apparatus
consists of a sample and reference holder, heat resistor heat sink and heater.
The heat from the heater is supplied to the sample and reference through
heat sink and heat resistor. The heat flow is proportional to the heat difference of heat sink and heat holders. Heat sink has enough heat capacity
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_24
387
388
Biophysical Chemistry
Reference
Sample
Heat sink
Heater
Heat driven
C.P.U
Thermo couple
Temp. control
Heat
resistor
Amplifier
Temp.
recording
Amplifier
Temp diff. that
flux recording
Thermo
couple
Figure 24.1 Block diagram of a heat flux differential scanning calorimeter.
compared to the sample. In a case where the sample undergoes endothermic or exothermic process such as transition or a reaction, this process is
compensated by heat sink. Thus the temperature difference between the
sample and the reference is kept constant. The difference in the amount of
heat supplied to the sample and reference is proportional to the difference
in temperature of the two holders. By calibrating the standard material,
quantitative measurement of unknown sample is possible.
There are two types of DSC systems which are commonly used, i.e.,
heat flux DSC and power compensation DSC. A simple version of a single
heat flux DSC is given below.
Figure 24.2 Single heat flux source in DSC.
Principles of Differential Scanning Calorimetry
389
In heat flux DSC, the sample and reference are connected by a low
resistance heat flow path (a metal disc). The assembly is enclosed in a
single furnace. Enthalpy or heat capacity changes in the sample cause a
difference in its temperature relative to the reference; the resulting heat
flow is small because the sample and reference are in good thermal contact.
The temperature difference is recorded and related to enthalpy change in
the sample using calibration experiments.
In power compensation DSC the temperatures of the sample and reference are controlled independently using separate but identical furnaces.
The temperatures of sample and reference are made identical by varying
the power input to the two furnaces; the energy required to do this is a
measure of the enthalpy or heat capacity changes in the sample relative to
the reference.
24.3
Methodology
In a scanning calorimeter, one measures the specific heat of a system as a
function of temperature. For a solution, the apparent specific heat of the
solute, C2 is given by the equation.
C2 = C1 +
1
(C − C1 )
w2
(24.1)
where C1 = specific heat of solvent, C = specific heat of solution and w2 =
weight fraction of the solute. In DSC, (C − C1 ) is directly measured. As an
example, the DSC curve obtained in the reversible thermal denaturation
of a globular protein is shown below. The apparent specific heat of the
native form of the protein increases while that of the denatured form is
independent of temperature.
The integral of the Cex (where Cex = excess apparent specific heat is the
amount by which the apparent specific heat during a transition involving
the solute exceeds the base line specific heat) over the temperature gives
the specific calorimetric enthalpy, Δhcal for the transition.
The interpretation of DSC data is based on the thermodynamic relation
∂ ln K
∂T
=
P
ΔH
RT 2
where ΔH denotes the van’t Hoff enthalpy.
(24.2)
390
Biophysical Chemistry
Figure 24.3 Curve trace obtained with the solution of Arg96 → His
mutant of the lysozymes of T4 phage.
24.4
Illustrative Application of DSC
Thermal Denaturation of Proteins
Calorimetric analysis of thermal unfolding of proteins by DSC provides
information concerning the fundamental nature of this process. The forces
involved in the stabilization of native structures of proteins can be elucidated as well.
(a) Two state denaturations with self dissociation or association: DSC
was employed to study the dissociative unfolding of the protein streptomyces subtilisin inhibitor (SSI). At ordinary temperatures, this protein is dimeric. Its denaturation curves are slightly asymmetric and
the values of tm (the temperature at which Cex reaches its maximum
value Cex,max ) increase with increasing protein concentration. Analysis of DSC curves showed that the denaturation process follows the
scheme A2 ↔ 2B. The native protein remains dimer upto 80◦ C.
The core protein obtained by partial proteolysis from lac repressor of
E.coli is tetrameric at ordinary temperatures. DSC data of the thermal
denaturation of this protein, which is irreversible, gives three values of
ΔHV H according to the methods given as follows: (i) curve fitting to
the model A4 ↔ 4B gave ΔHvan t Hoff = 520 kcal mol−1 , (ii) calculation
according to the equation
2
ΔHV H = ART1/2
Cex,1/2
Δhcal
(24.3)
where Δhcal = calorimetric specific enthalpy, T1/2 = t1/2 + 273.15
where t1/2 is the temperature in deg C at which the process is half
Principles of Differential Scanning Calorimetry
391
completed, Cex,1/2 = excess specific heat at half life t1/2 , R denotes the
universal gas constant and A = 4.00 gave ΔHV H = 585 kcal mol−1
and (iii) slope of van’t Hoff plot of ln ( L0 ) (where L0 is the total protein
concentration) vs. 1/T1/2 with n = 4 in the equation
ln K1/2 = constant t + (n − 1) ln( A0 ) + m ln( L0 ) =
gave ΔHV H = 498 kcal mol−1 .
−ΔHV H
+ constant
RT1/2
(24.4)
(b) Two-state denaturations with ligand dissociation: DSC was applied
to protein/ligand association reactions involved in the binding of LArabinose and D-galactose to the Arabinose binding protein (ABP)
of E.coli. The thermal denaturation of ABP is reversible both in the
absence and presence of ligands. van’t Hoff plot of ln( L0 ) vs. 1/Tm ( L0 =
Total ligand concentration) yielded the following values for both ligands: ΔHV H = 137 kcal mol−1 at 58◦ C and ΔHV H = 161 kcal mol−1 at
65◦ C. The value for ΔHcal in presence of glucose at 59◦ C was estimated
as 200.7 kcal mol−1 .
(c) Multistate denaturations: The thermal denaturation of taka-amylase A
gives a DSC curve with a single asymmetric peak with ΔHV H /ΔHcal
≈ 0.17 indicating a multistate transition. This protein contains a single
tightly bound Ca2+ ion in the absence of any added Ca2+ , its denaturation curves can be accurately resolved into the sum of two in dependent two state transitions including the dissociation of Ca2+ in the last
step.
Aspartyl transcarboxylase is a complex protein of six catalytic polypeptide chains and six regulatory chains per molecule of 310 × 104 a.m.u.
The C6 r6 molecule can be separated into two catalytic subunits, C3 and
three regulatory subunits, r2 . A DSC study of these units showed that
the denaturation curve of C3 can be resolved into three two state components and that of r2 into two components. C6 r6 in composed of two
separate peaks and was resolved into five two state components. The
tm values for denaturation of r2 and C3 were found to be independent
of concentration indicating that the poly peptide chains do not separate
on denaturation. The tm for denaturation of C6 r6 increase with increasing concentrations showing that dissociation into C3 and r2 subunits
accompanies denaturation.
(d) Denaturation of mutant proteins: DSC measurements of Arg 96 →
His mutant of lysosome of T4 bacteriophage indicated that the unfolding of this protein is reversible and the following results were obtained.
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Biophysical Chemistry
Type of mutant
Wild type
Arg96-His
ΔH/kCal mol−1
5.97 + 2.35t
−8.58 + 2.66t
T1/2
2.11 + 17.29t◦ (C)
−19.84 + 21.31t◦ (C)
A DSC study of glutamine and serine replacements of Glu49 in the
α-submit of tryptophane synthase showed decreases in the tm at pH
= 7.0 and increases at pH = 9.3. Both proteins showed enthalpy of
denaturation between 81 heal mol−1 at 45◦ C and 124 kCal mol−1 at
60◦ C.
The denaturation curve for the α repressor of E.coli showed two separate peaks. The one at lower temperature is due to denaturation of the
N-terminal portion of the molecule and the other at higher temperature is due to denaturations of the C-terminal portion. Seven mutant
forms of the tail spike protein of phage P22 were studied by DSC. In
this case, a maximum destabilization energy of +17.0 kCal mol−1 was
determined.
The Asp27 → Asn and AsP—Ser mutant forms of dihydrofolate reductase were studied using DSC. In each case, tm raises by 3.8◦ and 5.2◦ C
respectively. The denaturation enthalpy increases by 15 kcal mol−1 in
the former case and by 7 kcal mol−1 in the latter case.
(e) Conformational transitions of nucleotides: The helix-coil transitions
of oligo and polynucleotides have been widely studied by means of
DSC. The enthalpy increase for this transition is about 8–10 kcal (mole
of base pairs)−1 . DSC studies of the unfolding of a tRNA and the melting of several tRNA’s were made. DSC has been applied to the study of
DNA ligand interactions also. The thermodynamics of transition from
the right handed helical B form to the Z form has been determined for
poly (dG-m5 dc) and for poly (dGdC). The B-helix-to-coil and Z-helixto-coil transitions of these polynucleotides occur at 125◦ C depending
on experimental conditions.
(f) Phase transitions of phospholipids and phospholipid mixtures: DSC
has been employed to obtain thermodynamic data relating to phospholipid bilayers because they serve as simple models for complex biological membranes and also because they are interesting quasi two dimensional systems.
Phospholipid phase transitions give two or more closely spaced DSC
peaks. These transitions relate to gel-to-liquid crystal type. The addition of several compounds to a phospholipid bilayer lowers the phase
transition temperature and also broadens the transitions. The cooperativity of a transition of a pure liquid is indicated by its sharpness and
Principles of Differential Scanning Calorimetry
393
may be expressed in terms of the size of lipid molecule of the cooperative unit by the ratio
ΔHV H
ΔHcal
where ΔHcal is the enthalpy of transition per mole of lipid (in calories). DSC has been employed to study phase transitions in twenty
chemically related glycosphings lipids and also their mixtures with
dipalmitoyl-phosphatidyl cholin and myelin basic protein.
(g) Conformational transitions of polysaccharides: A widely studied process by DSC involving polysaccharides is the double helix-coil transition of iota- and kappa-carrageenan. The order-disorder transition of
xanthan poly electrolyte, which is composed of a cellulosic backbone
with trisaccharide side chains carrying carboxyl groups (some of which
are on pyruvate residues condensed at the carboxyl group), has been
investigated by DSC.
It has been observed that the triple helical polysaccharide schizophyllan undergoes a sharp transition at 6◦ C and in studies in water DMSO
mixtures this polysaccharide showed two transitions. The transition at
the higher temperature is attributed to a triple helix-single coil change
in conformation.
(h) Applications to more complex systems: DSC was applied to the study
of transitions involving plasma membrane Acholeplasma laidlawii. Two
endothermic peaks were observed. The one at lower temperature was
reversible and is due to phase transition of gel phase lipids while the
higher temperature peak was attributed to the denaturation of membrane proteins.
DSC was also employed in the study of human erythrocyte membrane.
Four transitions were observed, two of which were attributed to simple
unfolding transitions of proteins.
The heat produced by suspension of murine macrophages was investigated by employing DSC. The total heat evolved in the range 10 − 37◦ C
varied from 300 to 2500 × 10−12 Cal per cell in the scan range 1 K min−1
depending on cell density and glucose concentration. It was shown
that 24% heat liberated is due to the conversion of glucose to lactic
acid and an additional 14–41% is due to hydrolysis of ATP.
The DSC curve observed on heating a suspension of photosystem-II in
the temperature range 30–70◦ C was resolved into 5 two-state systems.
The peak at 48.2◦ C was attributed to functional denaturation of oxygen
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Biophysical Chemistry
evolving complex. The peak is very sharp with
ΔHV H
= 3 × 106
ΔHcal
The thermally induced reversible polymerization of the coat protein of
tobacco mosaic virus has also been studied by using DSC.
Questions
(1) In a study of the denaturation of the protein SSI by differential scanning
calorimetry, the excess specific heat (Cex1 , 12 ) of the calorimeter used is
0.380, the constant A = 4.00, Δhcal = calorimetric specific enthalpy =
0.8 cal and T1/2 = temperature in deg K at which denaturation is half
complete = 383.1. Calculate the enthalpy change for the denaturation
process.
Solution:
Cex , 1/2
Δhcal
4.00 × 0.002 × (383.1)2 × 0.380
=
0.8
= 558 k cal mol−1
2
ΔH = ART1/2
(2) Draw a neat sketch of a differential scanning calorimeter and describe
the components.
(3) Derive the relation
∂ ln K
∂T
=
P
ΔH ◦
RT 2
(4) Discuss the applications of differential scanning calorimetry in understanding the denaturation of proteins.
(5) Enumerate the use of differential scanning calorimeter in understanding (i) conformational transitions in nucleotides and phase transitions
of phospholipids.
25
Applications of Gel Filtration
Technique in the Separation
of Biomolecules
25.1
Introduction
Gel filtration (also known as size exclusion chromatography, SEC) separates molecules based on differences in size as they pass through a gel filtration medium packed in a column. In this method, the molecules do not
bind to the chromatography medium. As a result, one can vary the conditions to suit the type of sample or the requirements for further purification,
analysis or storage without altering separation.
Gel filtration is suitable for biomolecules that may be sensitive to a
variety of conditions such as change in pH, concentration of metal ions or
cofactors. It is used to separate proteins, peptides and oligonucleotides. It
is widely used for molecular size analysis, separation of components of a
mixture.
25.2
Methodology of Gel Filtration
At first the gel filtration medium is packed into a column to form a packed
bed. The medium is a porous matrix of particles which are physically stable and chemically inert. The bed is equilibrated with a buffer which fills
the pores of the matrix and the space between the particles. The liquid
inside the pores is known as the stationary phase and is in equilibrium
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_25
395
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Biophysical Chemistry
with the liquid outside the particles i.e., the mobile phase. The following
figure illustrates the separation process of gel filtration.
(A)
(B)
(C)
Figure 25.1 Schematic picture of (A) a bead with enlargement, (B) sample
molecules diffusing into bead and (C) separation process.
The two steps involved are as follows: (1) Sample is applied on the
column, and (2) The smallest molecule is more delayed than the largest
molecule. The largest molecule is eluted first from the column. Band
broadening causes some dilution of the protein zones during chromatographic separations.
25.3
Principle of Gel Filtration
As shown in Figure 25.2, the separation of molecules is based on the basis
of their molecular sizes. The molecular sieve properties of the porous
resins are made use of. Large molecules elute first because they are completely excluded from pores, interstitial spaces and resin particles. The
smaller molecules get distributed between the mobile phase inside and
outside the beads and pass down the column at a slower rate. Thus they
are eluted last. The distribution of a molecule in a column of cross linked
beads is determined by the total volume of mobile phase inside and outside the beads.
Gel Filtration Media
A polysaccharide, dextran, is cross linked to give small beads of a
hydrophilic and insoluble in nature material. When they are placed in
water, they swell and form an insoluble gel. It is known as Sephadex
Applications of Gel Filtration Technique in the Separation of Biomolecules 397
Figure 25.2 Schematic diagram of elution.
Commercially. Sephadex has the property to exclude solutes of large molecular size but it is accessible for diffusion to molecules of small dimension. There are other types of filtration media such as polyacrylamide (BiogelPTM ), dextran polyacrylamide (SephacrylTM ) and agarose (sepharoseTM
and Bio-gelATM ). They are available in a large range of pore sizes for separation of macromolecules of different sizes. A gel with a smaller range of
pore sizes gives higher resolution while a gel with a wider range gives a
lower resolution than the largest molecule. The largest molecule is eluted
first from the column. Band broadening causes some dilution of the protein zones during chromatographic separation.
Elution Volume Relationships
A fraction collector is attached to the system to collect fractions of elution
and a detector connected to the collector analyses the separated fractions.
The distribution coefficient (KX ) of an analyte may be expressed as
KX =
(Ve − V0 )
(Vt − V0 )
(25.1)
where Ve = volume of buffer that elutes from the column before a particular peak appears in the elution profile; Vt = total column volume (or bed
volume), V0 = volume of the space between gel particles (or void volume).
Thus (Vt − V0 ) = volume occupied by gel including gel matrix (Vgel ).
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Biophysical Chemistry
The distribution coefficient, KX of a molecule between the inner and
outer mobile phase is a function of molecular size. If a molecule is sufficiently large to be completely excluded from the mobile phase between
the beads then K = 0. On the other hand, if a molecule is very small that
it accesses the inner most mobile phase deep in the beads, KX = 1. For
in between situations, KX will vary between 0 and 1. The variation of KX
between 0 and 1, enables separation of the molecules present in a mixture
within a narrow molecular range.
25.4
Applications
Physical parameter
(1) A typical elution pattern of dialysis (on Sephadex G-25) to separate
hemoglobin from the salt NaCl is shown in Figure 25.3:
Haemoglobin
NaCl
Elution volume
Figure 25.3 Typical elution pattern of dialysis (on Sephadex G25).
(2) Another example involves the separation of RNAase from a protease
in pancreatic extract using Sephadex G-75 column. The elution pattern
is depicted in Figure 25.4.
Enzyme activity
Protease
RNAase
Elution volume
Figure 25.4 Separation of RNAase from protease in pancreatic extract
using Sephadex G-75 column.
(3) The distribution coefficient (KX ) varies with molecular weight for different proteins. Gel filtration method can be used for determination of
Applications of Gel Filtration Technique in the Separation of Biomolecules 399
molecular weights. Depending upon the possible molecular weight, a
suitable gel such as Sephadex G-100 (or G-200) is selected and a column is prepared with this gel. A calibration curve is obtained with
pure proteins of known molecular weight by determining their elution
volumes. Next, the protein whose molecular weight is under consideration is placed on the same column and its elution volume determined
under the same conditions used as in calibration experiments. The
molar mass of the unknown is read off from the calibration curve.
Sometimes, instead of elution volume, K D is used. The K D -molecular
weight data is given above.
(4) The gel filtration of hemoglobin: A solution of methemoglobin and
ferricyanide is applied to Sephadex column. Separation of brown
hemoglobin is first observed. The methemoglobin then overtakes a
previously added band of iron (II) salt and is reduced by it to purple
haemoglobin. When the protein emerges from the reducing agent, it
enters buffer saturated by air and becomes oxygenated to form scarlet
oxyhemoglobin. Thus three distinct processes occur and a small tube
of 8 cm is adequate for completing the separation.
(5) Monoclonal antibodies (MAB) separation: An important step in MAB
production and characterization is the analysis of aggregates and determination of purity of monomeric fraction. The separation of a monoclonal mouse lgG from its aggregates has been studied using Superdex
200 Increase 10/300 GL. Buffer used 0.1 M sodium phosphate solution.
Buffer used = 100 × 10−3 Na3 PO4
Questions
(1) Explain the principle of gel filtration technique. Discuss its applications
in the separation of (i) haemoglobin and (ii) monoclonal antibodies.
(2) Explain the principle of gel filtration technique with special reference
to the media employed in this method.
26
Gel Electrophoresis and
its Applications to
Biochemical Analysis
26.1
Introduction
The term ‘Electrophoresis’ denotes the separation of charged molecules
under the influence of an applied electric field. In particular by exploiting the differences in ionic mobilities, separation of species can be accomplished in a facile manner, for various biochemical contexts. Apart from
mobilities, several other factors too play a significant role viz. charge to
mass ratio, size and shape of molecules, viscosity and porosity of the matrix
though which the species migrate.
Principle of the Technique
It is well known that a mixture of charged molecules when placed in an
electrical field of strength E (V/cm), migrates to the oppositely charged
electrode surface. However, their ionic mobilities are not identical, dictated by several factors mentioned above, move at different rates depending upon the physical characteristics of the molecule.
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_26
401
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Biophysical Chemistry
The velocity of v, of a charged molecule in an electric field may be
expressed as
q
v = E×
(26.1)
f
where q denotes the net charge on the species and f is the frictional coefficient which depends on mass, shape and compactness of the migrating
particles, porosity of the matrix and viscosity of the medium. A simple
method of estimating the frictional coefficient is to employ Stokes-Einstein
equation approximately valid for dilute solutions and spherical particles.
From the above equation, it can be inferred easily that the movement
of the molecules will be more rapid if both the net charge and the applied
field can be made large. On the other hand, the frictional coefficient will
retard the velocity.
Hydrated gels provide significant advantages over in gel electrophoresis since: (i) these can be used in horizontal/vertical columns in the form
of slab gels or in tubes/capillaries and (ii) these are chemically inert and
hence do not possess significant chemical interaction with biomolecules.
Consequently, the separation essentially exploits physical rather than
chemical differences between the components of the system.
26.2
Nature of Gels Commonly Employed
The matrix refers to the gel employed in electrophoresis and functions as
a molecular sieve in the separation process based on their size. Among
many gels, the following deserve mention (Table 26.1).
Among several polysaccharide gel matrices, agarose is a typical one.
The polymer agarose consists of repeating disaccharide units (known as
agarobioses) and is extensively used in the separation of DNA constituents.
The molar masses of DNA can be accurately estimated, from the mobility
in agarose gels.
In view of the facile polymerization of acrylamide, a stronger gel matrix
results in this case and is useful in the separation of proteins as well as
nucleic acids. In order to obtain a cross linked gel with tunable porosity,
satisfactory mechanical strength and chemical inertness, it is customary to
Table 26.1 Illustrative examples of various gels.
Gel type
Main uses
Starch
Isolation of proteins
Agarose
Proteins of large dimensions, nucleic acids, nucleoproteins
Acrylamide Nucleic acids and proteins
Gel Electrophoresis and its Applications to Biochemical Analysis
403
add optimal quantity of cross linked acrylamide with the help of N-N ,
methylene bisacrylamide. These gels are particularly suitable in order to
accomplish high resolution separation of DNA and proteins, with varying
ratios of molar masses.
An important protocol in gel electrophoresis is the requiem for ‘staining’ the molecules separated in the gel matrix so as to view their locations.
Several staining agents customarily used are as follows:
(i) Amido black,
(ii) Ponceau red,
(iii) Coomassie blue,
(iv) Nile red, and
(v) Fluorosamine.
26.3
Experimental Arrangement
A sketch of the set up used in gel electrophoresis experiments is shown
below:
−
Sample
wells
⊕
Figure 26.1 Experimental set-up of gel electrophoresis.
The horizontal electrophoresis apparatus consists of two platinum electrodes, one at each terminal. The unit contains removable gel casting trays
with rubber end caps for sealing off the ends of the tray during gel-casting.
After the agarose gel is cast, the filled tray is immersed in an appropriate buffer-filled chamber for loading the sample under investigation. It
has been demonstrated that for best resolution of fragments, a voltage of
5 volts per cm serves as the optimal value.
Buffers used in these studies, especially for electrophoresis of duplex
DNA, are TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA). They
maintain the pH apart from providing satisfactory ionic conductivities.
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Biophysical Chemistry
Types of Gel Electrophoresis
There are two types of gel electrophoresis viz. (i) one-dimensional and
(ii) two-dimensional.
(i) The one dimensional gel electrophoresis may be further classified
into three parts: (a) SDS-PAGE, (b) Native-PAGE, (c) IEF (isoelectric
focusing). Several types of PAGE are known and they yield diverse
information about proteins. SDS (Sodium dodecyl sulfate)—PAGE
(Polyacrylamide gel electrophoresis) is capable of separating proteins
based on their mass.
The so-called Native—PAGE separates proteins using their mass:
charge ratios.
(ii) Two-dimensional PAGE (2D-PAGE) separates proteins from the information on the isoelectric points (IEF) in the first dimension and subsequently by mass in the second dimension.
The samples are often treated with SDS-an anionic detergent. This
denatures the protein by breaking the disulfide bonds and giving
rise to negative charges to each protein in direct proportionality to
its mass.
26.4
Applications of Gel Electrophoresis
Among diverse applications of gel electrophoresis, a few are described
briefly below:
(1) Agarose gel electrophoresis is widely used for the mechanistic analysis
of the DNA cleavage of small molecules and for analysing the binding modes of the molecules with supercoiled DNA. It may be reiterated that an adequate comprehension of DNA cleavage is essential in
drug designing. Natural plasmid DNA consists of (i) supercoiled (SC)
pUC19 DNA; (ii) nicked circular (NC) and (iii) linear conformations
(LC).
It is possible to employ agarose gel electrophoresis for quantifying their
relative efficiencies. By intercalating small molecules to plasmid DNA,
the SC DNA form cleaves, thereby decreasing rate of mobilities. The
DNA cleavage occurs either by hydrolytic or oxidative pathways.
Hydrolytic cleavage agents do not require co-reactants and are thus
more suitable in the designing of drugs. Consequently, they are more
beneficial than oxidative cleavage agents.
Gel Electrophoresis and its Applications to Biochemical Analysis
405
Several metallonucleases have been investigated as regards their DNA
cleavage properties. In particular, copper—amino acid/dipeptides containing complexes have been systematically studied in this context.
Upon electrophoresis of SC DNA, rapid migration was observed for
the intact SCDNA. If scission occurs essentially on only one strand, the
SC form relaxes to yield a slower moving nicked circular (NC) form.
As an illustrative example, the conversion of SC DNA to NC form
has been studied at several concentrations of [Cu(II)(hist)(trp)]+ and
[Cu(II)(hist)(try)] and is schematically represented below:
H2
N
O
O
N
H2
R
oled
erco
Sup NA
D
37°C
7.2,
pH =
Cu
N
N
H
%NC 7 12 21 32 45 56
1 2 3 4 5 6
NC
SC
(a)
[Cu(II)(hist)(tyr)trp]+
%NC 10 21 35 42 49 53
1 2 3 4 5 6
NC
SC
OH
R=
N
H
(b)
Figure 26.2 Agarose gel electrophoresis pattern for cleavage of supercoiled PUC DNA.
In some cases, kinetic data i.e., rate of cleavage of DNA as a function of
time can also be deduced.
(2) DNA analysis: The identification of DNA and DNA fragments constitutes an important application of Agarose gel or acrylamide gel electrophoresis. Upon applying optimal potential gradients, larger and
smaller fragments of DNA begin to separate since they are affected
differently on account of friction from the medium chosen. When the
applied electric field is stopped, the fragments get frozen and can then
be examined at high resolutions.
(3) Molecular cloning: This term refers to the construction of recombinant
DNA molecules which are integrated into diverse organisms so as to
create genetic modifications. The outcome of these modifications is
subtle.
Applications of molecular cloning include adding fluorescent protein
fusions to existing cellular proteins for identifying their locations in
cells and creating new genetic circuits to carry out specific functions
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Biophysical Chemistry
such as breaking-down of toxins. Gel electrophoresis is a crucial step in
quality control and production of DNA fragments in molecular cloning.
It can analyse fragments arising from the polymerase chain reaction
(PCR) to ensure that they are of correct dimensions.
(4) Genetic finger printing: The identification of individuals based on
their genetic codes is often known as genetic finger printing. In combination with with PCR, agarose gel electrophoresis remains as a powerful technique for genetic finger printing. The human genome is composed of several regions of short repeats, whose number varies
uniquely among individuals. By targeting these regions with specific
PCR primers, a profile of band on an electrophoresis gel corresponding to these regions gets created which is unique to an individual. This
technique, designated as DNA finger printing is used in forensic analysis for criminal investigations, genealogy and parentage testing.
(5) Diagnostic applications: Gel electrophoresis is useful for the screening
of genetic disorders and for inferring abnormal proteins. DNA can
be extracted not only from patients but also from embryos. It is then
subjected to PCR and agarose gel electrolysis for qualitative analysis
of genes or genetic abnormalities. In order to ascertain correct medical
treatments, this method is also applied to some proteins to analyse the
composition of blood.
Functioning of SDS-PAGE
In this method, the proteins are dissolved in SDS and then subjected to
electrophoresis. SDS binds to protein in a ratio of one SDS molecule to
two amino acids. This protocol effectively masks the charge on the protein
so that all proteins become uniformly negatively charged. The denatured
proteins are then applied to one end of a layer of poly acrylamide gel which
is submerged in a buffer. When an electric current is applied across the
gel, the −vely charged proteins migrate to the +ve pole of the electrode
surface.
Since proteins of smaller lengths fit more easily in the pores of the chosen gel, they move faster, while the larger ones move slowly. On account of
this difference in rate of migration (caused by size), smaller proteins move
farther down the gel, while the larger ones stay closer to the origin. After
a particular time duration, proteins become separated according to their
sizes.
407
Gel Electrophoresis and its Applications to Biochemical Analysis
26.5
Isoelectric Focusing (IEF)
This technique separates molecules by exploiting their electric charge differences. Here, proteins are separated by electrophoresis in a pH gradient
based on their corresponding isoelectric prints (pI). A pH gradient gets
generated in the gel when an electric potential is applied across the gel.
At all values of pH other than pI, the proteins move to one of the electrodes dictated by the charges they possess. At the value of pI, protein
molecules do not carry any net charge, and hence they accumulate or focus
into a sharp band.
(A)
Low
pH
+
Figure 26.3
differences.
+
+
± +
−
±
Low pH
−
±
− ± High
− pH
−
+
+
(B)
High pH
−
Scheme depicting the separation of molecules by charge
Questions
(1) What is the principle of gel electrophoresis? Name the various gels
used in this method and specify their uses.
(2) What are the different types of gel electrophoresis and describe some
applications of this technique.
(3) Explain the terms: (i) DNA analysis (ii) Molecular cloning and (iii)
Genetic finger printing.
27
Uses of Analytical
Ultracentrifugation Methods
in the Analysis of
Biomolecules
27.1
Introduction
The analytical ultra centrifuge method is a versatile technique for the determination of the molecular weight and the hydrodynamic and thermodynamic properties of proteins or other macromolecules. Svedberg and Pedersen (1920) were the first workers to use ultracentrifuge for the study
of macromolecules. Further rapid growth in this area became possible
with advances in the field of molecular biology relating to manipulation
of structures of DNA and proteins.
27.2
Details of the Apparatus
In an analytical ultracentrifuge, a rotor must spin at an accurately controlled speed and temperature and also it must be possible to record the
concentration distribution of the sample at given times. High angular
velocities are necessary to achieve rapid sedimentation and minimize diffusion. Velocities of the order of 60,000 rpm are employed. To reduce frictional heating and minimize aerodynamic turbulence, the rotor is spun in
an evacuated chamber.
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_27
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Biophysical Chemistry
A schematic sketch of ultracentrifuge results is depicted below:
Absorbance
Solvent
meniscus
Sample
meniscus
Boundary
region
Plateau
0
Top
Bottom
Sample
reference
Figure 27.1 Schematic depiction of ultracentrifuge pattern. The sample
solution is placed in one sector and a sample of the solvent (in dialysis
equilibrium with the sample) is placed in the reference sector. The reference sector is filled slightly more than the sample sector so that the reference meniscus does not obscure the sample profile.
In ultracentrifuge cells, the sample is placed within a sector shaped
cavity sandwiched between two thick windows of optical grade quartz.
The cavity is produced within a centerpiece of aluminum alloy or reinforced epoxy. Sector shaped sample compartments are essential in velocity
work since sedimenting particles move along radial lines. Double sector
cells allow the user to take account of absorbing components in the solvent and to correct for the redistribution of solvent components especially
at high “g” values. A sample of the solution is placed in one sector and a
sample of the solvent is placed in the second sector (see Fig. 27.1). The
optical system measures the difference between the sample absorbance
and reference sector solution absorbance. Double sector cells also facilitate
measurements of differences in sedimentation coefficient and of diffusion
coefficients.
27.3
Detection Method and Data Collection
The data is collected as a set of concentration measurements at different
radial positions at a given time. Usually, the measurement of absorbance
of the sample at a given wavelength at fixed position of the cell is followed.
Uses of Analytical Ultracentrifugation Methods
411
Refractrometric methods are also used for obtaining concentration distributions as the sample solution has a greater refractive index than the
pure solvent. Two different optical systems are employed for this purpose:
(i) Schlieren system, and (ii) Rayleigh interference optic system.
In the Schlieren system, light passing through a region in the cell will
be deviated radically where the concentration (hence the refractive index)
is changing. This optical system converts the radial deviation of light into
a vertical displacement of an image at the camera. The displacement is
proportional to the concentration gradient. The Schlieren image is thus a
measure of the concentration gradient, dc/dr, as a function of radial distance. The change in concentration relative to that at some specific point in
the cell can be determined at any other point by integration of the Schlieren
profile.
27.4
Rayleigh Interference Optics
This technique depends on the fact that the velocity of light passing
through a region of higher refractive index decreases. Monochromatic light
passes through two fine parallel slits one below each sector of a double sector cell containing, respectively, a sample of a solution and a sample of solvent. Light waves emerging from the entrance slits and passing through
the two sectors undergo interference to yield a band of alternating light
and dark fringes.
Modern absorption optical systems have increased sensitivity and
wide wavelength range. It is possible to obtain absolute concentration at
any point. The increase means that samples may be examined at concentrations too dilute for Schlieren or interference optics. For example, measurements below 230 nm allow examination of concentrations 20 times more
dilute than can be studied with interference optics.
27.5
Applications
Molecular Weight Determination
To determine the molecular weight, several quantities are required: (i) data
of concentration distribution, (ii) density of solvent, (iii) partial specific volume of the solute (or more specifically, the specific density increment). Further to consider the effects of different solvents and temperatures on sedimentation behavior, (iv) the viscosity of the solvent and its dependence
412
Biophysical Chemistry
on temperature are also required. The partial specific volumes of macromolecular solutes may be calculated from knowledge of their composition
and the partial specific volumes of component residues.
Alternatively, it is possible to estimate both M and V̄ from the data
obtained using sedimentation equilibrium experiments. For some classes
of compounds, the variation in V̄ with composition is not large and an
average value of V̄ for Polysaccharides (0.61) not large and an average
value of V̄ for Polysaccharides (0.61), RNA (0.53) and DNA (0.55) have
been obtained.
Two additional basic experiments are performed with the ultracentrifuge i.e., sedimentation velocity and sedimentation equilibrium. In the
sedimentation velocity experiment, a uniform solution placed in the cell is
subjected to a high angular velocity to cause rapid sedimentations of the
solute to the cell bottom. This results in a depletion of the solute near the
meniscus and a sharp boundary is formed between the depleted region
and the uniform concentration of the sedimentating solute. From these
experiments, the sedimentation coefficient S, which depends on the mass
of the particles and the frictional coefficient can be obtained. Measurement
of the rate of spreading boundary yields the diffusion coefficient, D, of the
particles, which is given by
RT
D=
(27.1)
Nf
where R denotes the universal gas constant, T being the absolute temperature, N represents the Avogadro number and f is the frictional coefficient
which is linearly related to the viscosity of the medium and hydrodynamic
radius of the compound. An illustrative diagram of the movement of the
boundary in a sedimentation velocity experiment is given below.
Figure 27.2 Movement of the boundary in a sedimentation experiment.
Uses of Analytical Ultracentrifugation Methods
413
Movement of the boundary in a sedimentation velocity experiment
with a recombinant malaria antigen protein. As the boundary progresses
down the cell, the concentration of the plateau region decreases from radial
dilution and the boundary broadens from diffusion. The midpoint positions, rbud of the boundaries are shown. The ratio of the sedimentation to
diffusion coefficient gives the molecular weight M.
M=
S◦ RT
D ◦ (1 − V̄ρ)
(27.2)
where V̄ = partial specific volume, ρ= solvent density, S◦ = sedimentation
coefficient and D ◦ = diffusion coefficient. Multiple boundaries indicate
multiple sedimentating species.
27.6
Determination of Sedimentation
Coefficient
If the sedimenting boundary is sharp and symmetrical, the rates of movement of solute particles in the plateau region may be considered close to
the rate of movement of the mid points.
The velocity of the boundary increases gradually with the movement
of the boundary outwards.
S≡
U
dr /dt
= int2
2
w r
w r
(27.3)
Equation (27.3) can be written as
ln(rint /rm ) = Sw2 t
(27.4)
where rm is the radial position of the meniscus.
A plot of ln rint versus time (sec) yields a straight line whose slope is
2
Sw .
This plot is for the recombinant dehydratase domain protein. From the
slope of the graph, the sedimentation coefficient may be obtained. The sedimentation coefficient is dependent on the concentration, density of the solvent as well as its viscosity. Pure non-associating solutes show a decrease
in the sedimentation coefficient with increase of concentration. The concentration dependence of “S” may be given as
S=
S◦
(1 + k s C )
(27.5)
414
Biophysical Chemistry
1.88
ln r
1.84
1.80
20
40 60
Time (sec)
80
100
Figure 27.3 Estimation of the sedimentation coefficient.
where S◦ = the limiting sedimentation coefficient, k s = concentration
dependent coefficient, C = concentration of the substance. For globular
proteins k s ≈ 5 mlgm−1 .
In a sedimentation velocity experiment, the shape of the boundary is
subjected to different influences, some of which are
(i) Heterogeneity tending to spread out the boundary because different
species move with different velocities.
(ii) Diffusion also tends to spread the boundary.
(iii) Concentration dependence of sedimentation coefficient also leads to
self sharpening of boundaries.
Most solutes display significant boundary spreading due to diffusion.
When the concentration dependence of sedimentation coefficient is large,
such as in the case of rod shaped virus particles or DNA, the boundary
tends to sharpen itself overcoming the spreading due to diffusion.
In a study of antigen-antibody interactions, it was shown that with
absorption optics, a significant improvement to signal to noise ratio can be
made by use of dc/dt values at fixed radial positions in determining the
distribution of sedimentation coefficients.
27.7
Effect of Association on Sedimentation
Coefficient
When a macromolecule undergoes association, the molecular weight of the
particles increases and so “S” will increase with concentration.
In the case of a monomer-dimer association, only one asymmetric
boundary is produced. For monomer n, when n > 3, the boundary is
bimodal i.e., two boundaries may be observed.
Uses of Analytical Ultracentrifugation Methods
415
Figure 27.4 Concentration dependence of weight average “S” for DIP-α
chymotrypsin.
27.8
Active Enzyme Sedimentation
Band sedimentation is suitable for the study of sedimentation behavior of
enzyme activity in which a zone of enzyme solution is centrifuged through
a supporting solution containing chromogenic substrate. Enzyme activity
results in the migration of a moving boundary of product generated as the
enzyme band migrates down the cell.
27.9
Estimation of Diffusion Coefficients
An accurate estimate of diffusion coefficient is needed for the determination of molecular weight from sedimentation coefficient. The analytical
centrifuge can be used for measurement of diffusion coefficient. The best
way is to use a boundary cell to create an initial sharp boundary the spread
of which with time allows measurement of D. Under this condition, the
solvent is layered over the solution as the rotor reaches about 4000–6000
rpm. Scans of the cell contents at different times allow measurement of
the concentration in the plateau region (C p ) and the concentration gradient at the boundary (dc/dr )b by numerical differentiation of the data. If
the boundary is symmetrical, its position will be that of maximum concentration gradient and will occur at the point C = C p /2. The diffusion
coefficient is then given by 4π times the slope of the plot of [C p /(dc/dr )]2
against time in seconds. For human spectrin, by this method, D ≈ 3.91 ×
10−5 cm2 sec−1 . Since the diffusion coefficient is concentration dependent,
an extrapolated value of D ◦ , the limiting value as C → 0 must be determined.
416
27.10
Biophysical Chemistry
Estimation of Molar Mass
In sedimentation equilibrium experiments, a small volume of uniform solution is centrifuged at low angular velocities. As solute begins to sediment
towards cell bottom, its concentration at bottom increases and this process
of diffusion opposes the process of sedimentation. Measurement of concentrations at different points leads to the determination of molar mass
of the solute. When sedimentation equilibrium is reached, the diffusion
flow balances sedimentation flow at every point in the cell. For a nonassociating solute.
2RT
d ln C
M=
×
(27.6)
dr2
(1 − V̄ρ)ω 2
where M = molar mass of solute (in g/mol), ω = angular velocity of rotor
and C = concentration of the solute (in gm/l) at a radial distance “r” from
axis of rotation. A plot of log (concentration) vs. r2 for a solute at sedimentation equilibrium gives a linear graph whose slope is proportion to
molar mass. This method is applicable for determination of molar masses
of a wide range of solutes from a few hundreds to a few billions such as
viruses.
Sedimentation equilibrium in native solvents provides a method for
the determination of molar masses of oligomeric structures. When several
species with different molar masses are present, at sedimentation equilibrium, each will be distributed according to equation (27.6). Tangents to
ln C vs. r2 plot at various points give weight average molecular weight,
Mw , given by
M C + M2 C2
∑ Mi Ci
= 1 1
(27.7)
Mw =
C1 + C2
∑ Ci
where C1 and C2 denote two concentrations of the solute respectively.
Because of non-ideality due to size and charge of macromolecule, the
apparent molecular weight is concentration-dependent and may be
expressed as
M
Mapp =
(27.8)
1 + B2 MC
where B2 is referred to as the second virial coefficient. An extrapolated
value for M from the plot of Mapp vs. C gives a molar mass of 5.15 × 105
for aspertin.
For an association reaction 2A A2 at sedimentation equilibrium,
√
2 1 + 8KC
√
Mw = M1
1 + 1 + 8KC
(27.9)
Uses of Analytical Ultracentrifugation Methods
417
where Mw represents the weight average molar mass and K denotes the
equilibrium constant, C being the concentration of the solute.
From sedimentation equilibrium experiments, the apparent molar
mass, MW,app was found to vary with concentration for the self association
reaction of DNA binding protein. The variation is depicted in Figure 27.5.
Figure 27.5 Dependence of the apparent molar mass of DNA on concentration, studied at rotor velocity = 32000 rpm. The decrease at higher concentrations may be due to non-ideality.
27.11
Thermodynamic Parameters of
Association Reactions
The temperature dependence of the equilibrium constant of an association
reaction yields the enthalpy change from the relation.
ln K =
ΔS◦
ΔH ◦
−
R
RT
(27.10)
A plot of ln K vs. 1/T gives ΔS◦ from the intercept and ΔH ◦ from the slope
assuming that they are constant over the temperature range studied. The
change in CP is also given by
ΔCP =
∂ΔH ◦
T∂ΔS◦
=
∂T
∂T
(27.11)
The relative magnitudes of the thermodynamic parameters help in understanding the types of interaction.
418
27.12
Biophysical Chemistry
Sedimentation in Biological Environments
The crowded molecular conditions encountered in Vi V0 in cells and biological fluids may have dramatic effects on macromolecular interactions.
Non-ideal effects in concentrated solutions of macromolecules favor compact conformations and associated states.
The pyruvate dehydrogenase complex of Azotobactor vinelandii exists
in dilute solution as particles of sedimentation coefficient 17–20 S; in the
presence of 3% polyethyleneglycol, these particles aggregate to form 50–
60 S clusters.
The analytical ultracentrifuge is amenable to the study of crowding
effects. These effects may be studied in model systems in which the macromolecule of interest is in the presence of a high concentration of an inert
solute like sucrose, dextran or polyethylene glycol.
Sedimentation equilibrium measurements allow investigation of both
self interactions of macromolecules at high concentrations and the crowding effects of solutes like sucrose, or dextrin and conformation equilibrium
as a model of crowding processes in Vi V0 .
There is a method where by both sedimentation coefficient and molecular weight may be obtained from early stages of a sedimentation equilibrium experiment on the assumption that S is independent of C. This
method gave satisfactory results for the molar masses of proteins.
27.13
Density Gradient Sedimentation
Equilibrium
This method relies on the banding of a macromolecule within a gradient
of density at a point 1 − vρ = 0. With this method it is possible to measure
the buoyant density of a particle and to make analytical separations based
on differences in buoyant density.
In this experiment, a solution of the macromolecule in a proper concentration of density gradient solute (usually CrCl or Cs2 SO4 ) is centrifuged
until equilibrium is attained. Redistribution of the density gradient solute
leads to a density gradient from meniscus to the base and the macromolecules migrate to this position in the gradient. Knowledge of the initial concentration of the gradient material, its molar mass and v̄ and its
activity coefficient as a function of concentration permits the calculation of
equilibrium density and hence the buoyant density determination of the
macromolecule. This technique has been used for the analysis of nucleic
Uses of Analytical Ultracentrifugation Methods
419
acids. The method is sensitive to small density differences and has led to a
greater understanding of nucleic acid replication.
Differences in buoyant density may arise from differences in base composition of nucleic acids. Changes in buoyant density also arise from differences in solvation and ion binding.
The buoyant densities of proteins are a function of pH values; deprotonation of carboxyl groups, for example, leads to increased binding of
cs+ ions with an increase in buoyant density while deprotonation of lysine
residues leads to loss of binding of chloride ions and hence to an increase
in buoyant density.
The width of the band of macromolecules is a function of molecular
weight as well as the steepness of the local density gradient and the heterogeneity of the samples. For high density materials like DNA, the bands
may be very narrow.
Equilibrium density gradient sedimentation is useful in the examination of the assembly of complex macromolecular structures and also
for heteromolecular associations e.g., protein-nucleic acid and protein-lipid
interaction.
Questions
(1) Define the term sedimentation coefficient. How is it determined?
(2) Explain how the molar mass of a biomolecule is determined from sedimentation experiments.
(3) Explain how sedimentation principle is useful in the analysis of cell
and other biological fluids.
(4) Serum alumin has a sedimentation coefficient of 4.5S (1S= 10−13 sec).
Given the partial specific volume of the macro molecule (v̄) as 0.73 ml
gm−1 and its frictional coefficient as 1.15, and density of water, ρ = 1.0
gm ml−1 , calculate its molar mass.
(5) A protein has a molar mass of 1.5 × 105 Dalton (or amu) and its diffusion coefficient is 4.5 × 10−7 cm2 sec−1 . Given its partial specific volume as 0.73 cm3 gm−1 , calculate its sedimentation coefficient in the
units of Svedberg (temperature may be taken as 293 K.)
28
Ion Exchange
Chromatography and its
Applications in the
Separation of Biomolecules
28.1
Introduction
Ion Exchange Chromatography is one of the important adsorption techniques used in the separation of proteins, peptides, nucleic acids and related
biopolymers. The separation of these charged molecules of different molecular sizes is based on the formation of ionic bonds between charged groups
of biomolecules and an ion exchange support carrying the opposite charge.
Biomolecules exhibit different degrees of interaction with charged chromatography media due to their varying charge properties.
There are two mechanisms in this technique—(i) ion exchange due to
competitive ionic binding and (ii) ion exclusion due to repulsion between
similarly charged analyte ions and the ions fixed on the chromatographic
support.
28.2
Mechanism of Ion Exchange
In ion exchange chromatography there are mobile and stationary
phases. The mobile phase consists of an aqueous buffer system into which
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_28
421
422
Biophysical Chemistry
the mixture to be resolved is placed. The stationary phase consists of an
inert organic matrix having ionisable groups (fixed ions) that carry a displaceable oppositely charged ion. The ions in equilibrium between the
mobile and stationary phase may be cations or anions. The exchangeable matrix counter ions may be H+ , OH− , or singly charged alkali metal
ions, or doubly charged alkaline earth metal ions (Ca2+ , Mg2+ ) and multiply charged anions like SO24− , PO34− , etc. Cations are separated on cation
exchange resin column and anions on anion exchange resin column. Separation is based on the binding of analytes to positively or negatively
charged groups on the stationary phase and which are in equilibrium with
free counter ions in the mobile phase. This is depicted in Figure 28.1.
+++ −
++++
+++
++
––– ⊕
––––
–––
––
Negatively charged
analyte (anion)
Positively charged
analyte (cation)
(anion exchanger stationary
phase particle)
(cation exchanger stationary
phase particle)
Figure 28.1 Separation based on the binding of analytes to positively or
negatively charged groups on the stationary phase.
Ion exchange chromatography (I.E.C) involves separation of ionic and
polar analytes using chromatographic supports having ionic functional
groups that have charges opposite to that of analyte ions. The analyte
ions and other similarly charged ions of the eluent compete with the oppositely charged ionic functional group on the surface of the stationary phase.
In the case of exchanging ions (analytes and other ions in mobile phase)
which are cations (C+ ) the exchange reaction may be written as
SX CM
SXMC
(28.1)
In the above, the cation M+ of the eluent has replaced the analyte cation C+
bound to the anion X− which is fixed on the surface of the chromatographic
support(s).
In anion exchange chromatography, the exchanging ions are anions
and the reaction
S − X+ A− + B− ↔ S − X+ B− + A−
(28.2)
Ion Exchange Chromatography 423
The anion B− of the eluent has replaced the analyte A− ion bound to the
X+ ion on the surface of the stationary phase. Molecules vary considerably
in their charge properties and will exhibit different degrees of interaction
with charged chromatography support depending upon the differences in
their overall charge, charge density and surface charge distribution. The
net surface charge of all molecules with ionisable groups is highly pH
dependent.
28.3
Applications
This technique is often used for characterization of proteins in their native
or active form and for purification.
Proteins contain a variety of functionalities that can give rise to
differences in charge. The overall charge is dependent on the pH of the
Na⊕Cl–
−
SO3
⊕
−
⊕
SO3
−
SO3
−
−
SO3
SO3
⊕
⊕
−
SO3 Na⊕
−
⊕
SO3 Na
−
SO3
−
−
⊕
⊕
Na
SO3 Na⊕
⊕
Cl
Cl
−
⊕
⊕
⊕
⊕ Cl −
⊕ Cl −
−
SO3 Na⊕
⊕
Cl
⊕
−
⊕ Cl
⊕−
Cl
⊕ Cl −
Figure 28.2 Separation mechanism in ion exchange.
Cl
−
−
424
Biophysical Chemistry
surrounding solution and this in turn will affect the ion exchange method
that is used. The mobile phase must maintain a controlled pH throughout
the course of separation and so aqueous buffers are used as eluents. Figure
28.2 shows the separation mechanism in ion-exchange.
The technique of ion exchange is suitable for separating proteins with
differing isoelectric points. It is equally valuable in separating charged
iso forms of a single protein. In the important field of biopharmaceuticals, where proteins are manufactured through bioengineering and isolated from fermentation reactions, it is important to identify charged isoforms as these indicate a difference in primary structure of protein. A
difference in primary structure could indicate a change in glycosylation
or degradation path-way such as loss of C-terminal residues or amidation/deamidation. Ion exchange is used to separate and quantify charge
variants during the development process and also for quality control and
quality assurance during manufacture of biotherapeutics. With large
molecules such as monoclonal antibodies (mAbs) it is also important to
consider the size and structure of the molecule (mAbs are typically 150k
Daltons), particularly as the chromatographic interaction will occur only
with surface charges.
Figure 28.3 Schematic depiction of charged variants of monoclonal antibodies.
Ion Exchange Chromatography 425
Example 1: BioMAB column to identify C-terminal truncation
on heavy chains
Figure 28.4 Two examples illustrating the use of BioMAB column for
identification of c-terminal truncation on heavy chains.
Example 2:
Figure 28.5 High resolution of a mixture of proteins with a wide range of
isoelectric points.
426
28.4
Biophysical Chemistry
Purification of Adenovirus
Ion exchange chromatography offers a powerful method for adenoviral
fractionation because of its high capacity and resolution. I.E.C. exploits
the charge that proteins carry on their surface. Their net charge varies
with pH and the amino acids exposed in the protein surface. Adenoviral
capsids are highly anionic in nature, making anionic exchange ideal for
purifying them. Anion exchange resins carry positively charged groups
such as diethylamino ethyl (DEAE) or quaternary amino ethyl, which bind
anionic proteins which depends on pH. Elution may be accomplished by
changing pH to eliminate ionic interaction with the protein.
28.5
Separation of Membrane Phospholipids
Biological membranes are anionic and hence are amenable to anionic
exchange chromatography. Membrane phospholipids are either neutral
or anionic (such as phosphatidic phosphalidic acid, phosphalidylserine,
phosphatidylinositol, phosphatidylglycerol). All membranes have varying degrees of negative charge density and will bind to anion exchange
columns. The higher the −ve charge density, the tighter the membrane
binding. Membrane fractions are released from the column with either
increasing retention time in the presence of a fixed anion (normally Cl−
ions) concentration or by adding solutions of increased anion concentrations. The poorest binding membrane fraction has the lowest negative
charge density and is the first to be released. The most negative membrane
fraction is the tightest bound and is the last to be released.
28.6
Separation of Soyabean Proteins
Soyabean contains mainly 11S and 7S globulins. For separating them by
High pressure ion exchange chromatography, the binary gradient approach
was used with the mobile phase being a borate buffer of pH 9.0. The mixed
proteins were eluted with the same buffer with the gradient starting with
an isocratic step at zero percent B (buffer) for 2.5 min and 0 to 70% B in
14 minutes. The detection was UV spectroscopy at 254 nm. The stationary
phase employed was an anion exchange perfusion column POROS HQ/10
packed with cross linked poly styrene divinyl benzene beads.
Ion Exchange Chromatography 28.7
427
Choice of Column Media
In all chromatographic techniques, there is a range of columns to choose
from. The first consideration is “should it be anion or cation exchange”.
Also, there is a choice of strong or weak ion-exchange. In most circumstances, it is best to start with a strong ion exchange column. Weak ionexchangers can then be used to provide a difference in selectivity. The functional group in a strong cation exchange column is sulfonic acid resulting
in the stationary phase being −vely charged. In an anion exchange column, however, the functional group is a quaternary amine group which is
+vely charged. Strong ion exchange columns, therefore, have the widest
operating range. Weak ion exchange sorbents (such as carboxylic acids in
weak cation exchangers and amines in weak anion exchangers) are more
strongly affected by the mobile phase conditions. The functionalities are
not dissimilar to the charged groups on proteins and the degree of charge
can be influenced by ionic strength as well as pH of the mobile phase. This
can result in a change in resolution that may be subtly controlled and optimized through careful choice of operating conditions.
Questions
(1) Discuss mechanistic aspects of ion exchange chromatography.
(2) Explain the application of this method in (i) characterization of proteins
(ii) purification of adenovirus.
(3) Briefly explain the application of this method in (i) separation of membrane phospholipids and (ii) separation of soy bean proteins.
29
Surface Enhanced Raman
Scattering and its
Bioanalytical Applications
29.1
Introduction
The phenomenon of Raman scattering arises as a result of interaction of
electromagnetic radiation with matter which results in the alteration of the
frequency of the incident radiation. Raman spectroscopy has grown into a
highly sensitive technique to probe the structural details of complex structures of biomolecules. However, its applications became restricted due to
low scattering cross section achieved in this technique.
Surface Enhanced Raman scattering (SERS) effect deals with the gigantic amplification of the weak Raman scattering intensity by molecules in
the presence of a nanostructured metallic surface.
The SERS enhancement factor may be defined as the ratio of Raman
signals obtained from a given number of molecules in the presence of the
metal nanostructure to that in the absence of the nanostructure and this
ratio depends largely on the size and morphology of the nanostructures.
Generally, this enhancement factor is around 106 .
29.2
Principle of SERS
Fleischmann and his co-workers at the university of Southampton carried
out Raman spectroscopic study of the adsorbed molecules of a compound
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_29
429
430
Biophysical Chemistry
on a roughened metal electrode surface in 1974 and obtained high intensity
scattering signals. They attributed the enhancement in Raman scattering
intensity to an increase in the surface area of the electrode by the roughening method adopted. The following diagram explains the principle of
Raman and Rayleigh scattering.
Figure 29.1 Electromagnetic and chemical enhancement mechanism.
The technique is so sensitive that even a single molecule can be
detected in addition to various electrochemical processes.
The dominant contributor to SERS processes is the electromagnetic
enhancement mechanism. The enhancement results from the amplification of the light by the excitation of localized surface plasmon resonances
(LSPRS). This light concentration occurs preferentially in the crevices, gaps
of the plasmonic materials which are generally coinage or noble metals
(like Cu, Ag, Au) with nano scale features. Reproducible and robust structures that strongly enhance the electromagnetic field are preferred in SERS.
Another mechanism involved in signal enhancement is chemical
enhancement which involves charge transfer mechanism where the
excitation wave length is resonant with the metal molecule charge transfer
electronic states. The total SERS enhancement factor is the product of electromagnetic and chemical enhancement mechanisms. The enhancement
factor may approach 1010 to 1011 for highly optimized surfaces.
Figure 29.2 SERS enhancement and enhancement mechanism.
Surface Enhanced Raman Scattering 29.3
431
Experimental Aspects
The first parameter to be considered is the choice of enhancing substrate.
Substrates range in structure from nano rods to three dimensional colloidal
solutions with tunable Plasmon resonances and a range of enhancement
factors. The largest enhancements are found in a few nanometers closest
to the substrate surface.
The second parameter to consider is a proper excitation source. It
should enable efficient excitation of the Plasmon source. A laser tuned
to the peak of Plasmon resonance for a substrate gives maximum enhancement of the signal. Following excitation of the Plasmon resonance and generation of SERS signal, the detection process is identical to normal Raman
experiments. A long pass filter is used to reflect or absorb any Rayleigh
scattering while allowing for the transmission of Raman signal and a spectrograph and detector are used to image Raman spectra across a wide spectral region.
The third factor, one must consider, is the choice of plasmonic materials. The success of SERS is very much dependent on the interaction
between absorbed molecules and the surface of plasmonic nanostructures
which are often substrates of Au, Ag, or Cu. All the three metals have
LSPRS (light supported Plasmon resonances) that cover most of visible and
near infrared range where most Raman experiments are carried. The diagram below gives the wave length ranges of the three metals.
Cu
Au
Ag
200
300
400
500
600
700
800
900
1000 1100 1200
Wave length (nm)
Figure 29.3 Metals exhibiting SERS and their wave length ranges.
In SERS molecules are attached to a nanostructure. SERS is not much
of a “surface effect” but a “nanostructure effect”. Figure 29.4 shows another
perspective of a SERS experiment.
The nanostructure is an aggregate of spherical metal colloidal particles. In ’normal’ RS, the total Stokes Raman signal (P RS (vS )) is proportional to the Raman cross section σ R
f ree , the excitation laser intensity I ( v L )
432
Biophysical Chemistry
Figure 29.4 Schematic depiction of SERS for spherical aggregates.
and the number of molecules in the chosen volume N.
P RS (vS ) = N × σ R
f ree × I ( v L )
(29.1)
To determine the SERS stokes power PSERS (vS ), equation (29.1) has to be
modified to describe the specific effects of the metal nanostructures. Two
effects need to be considered:
(i) the scattering occurs in the enhanced local optical fields of the metallic nanostructures (i.e., the electromagnetic field enhancement),
(ii) a molecule in contact with a metal nanostructure exhibits a “new
Raman process” at a cross section larger than the Raman cross section of a free molecule (chemical or electronic enhancement)
PSERS (vS ) = N σ(Rads) | A(v L )|2 | A(vS )|2 I (v L )
(29.2)
In the equation (29.2), A(v L ) and A(vS ) represent enhancement facR is the
tors for the Laser and for the Raman scattered field, σods
increased cross section of the new Raman process of the adsorbed
molecule and N is the number of molecules involved in the SERS
process which may be smaller than the number of molecules in the
probed volume N.
The concept of electromagnetic SERS enhancement is illustrated in the following diagram (Fig. 29.5).
E M = E0 + ESp
(29.3)
Surface Enhanced Raman Scattering 433
Molecule
d
r
ε = ε ′ + iε ″
Metal sphere
Figure 29.5 Field of a point dipole.
( E = Field of a point dipole at the center of the sphere)
ESp = r3
ε − ε0
1
E0 ×
ε + 2ε 0
(r + d )3
(29.4)
The metallic nanostructure is a small sphere with complex dielectric constant ε D in a surrounding medium of dielectric constant ε v . The diameter
of the sphere 2r is small compared with the wave length of light.
A molecule in the vicinity of the sphere is exposed to the field E M
which is the superposition of the incoming field E0 and the field of a dipole
ESp induced in the metal sphere. The field enhancement factor is the ratio
of the field at the position of the molecule and the incoming field
Field enhancement factor = A(v) =
EM (v)
ε − ε0
=
E0 (v)
ε + 2ε 0
r
(r + d )
3
(29.5)
A(v) is quite strong when the real part of ε(v) is equal to −2ε 0 . Further,
for strong electromagnetic enhancement, the imaginary part of dielectric
constant must be small. These conditions meet the resonant excitation of
surface plasmons of the metal sphere.
29.4
Applications of SERS
SERS finds applications in biophysical/biochemical and biomedical fields.
Many SERS experiments have been conducted on amino acids, peptides,
purine and pyrimidine bases and also on large molecules like proteins,
DNA and RNA. Intrinsically-colored molecules like chlorophyll and other
pigments were also studied by SERS. SERS was also employed to detect
and characterize biomolecules. It has also been used to monitor transport
through membranes. SERS has been shown to discriminate between the
movement of different molecules across a membrane.
(1) Detection and identification of micro-organisms: SERS spectroscopy
is a valuable tool for identifying bacteria based on their vibrational
spectrum. This method has the advantage of needing only a small
434
Biophysical Chemistry
amount of the material because of its high sensitivity. The SERS spectrum of the bacterium Escherichia coli on which colloidal silver particles were deposited, was first reported.
SERS spectra of E.Coli K12 in contact with silver and gold colloidal
particle have been obtained.
(2) SERS study of neurotransmitters: The detection, identification and
measurement of concentration changes of neurotransmitters like glutamate in central nervous system is very important in neurochemistry.
SERS spectra of different neurotransmitters have been measured on silver colloidal particles in water. As an example, the SERS spectra of
dopamine in silver colloidal solution is shown below:
Figure 29.6 SERS of dopamine in silver colloidal solution. Spectrum was
obtained in 50 ms using 100 mW NIR excitation (NIR: near infrared SERS).
Norepinephrine which has a similar structure to dopamine shows clear
differences in SERS spectra. Such differences are due to small differences in the adsorption behavior of these molecules. SERS can thus
show improved sensitivity and selectivity between structurally similar
molecules. It is interesting to note that glutamate has been detected in
microdialysis samples of rat brains in 0.4 − 0.5 × 10−6 M concentration.
(3) Immunoassays employing SERS:
(a) SERS has been first employed in an immunoassay of thyroid stimulating hormone (TSH). In this experiment, SERS-active substrate
was silver film which was coated with anti TSH-bodies and then
incubated with TSH. A second anti-TSH labelled with p-dimethyl
amino benzene was added to bind the TSH. The SERS signal of the
label was used to quantitatively determine TSH.
Surface Enhanced Raman Scattering 435
(b) Detection of antigens by SERS: Colloidal gold particles supported on
gold surface serve as SERS-active substrate. Antigens from solution are captured by immobilized antibodies on the gold surface.
Gold nanoparticles labeled with specific antibodies and a specific
reporter bind to the captured antigen. By immobilizing different
antibodies and using specific reporters, the presence of different
antigens can be detected by the characteristic SERS spectrum of the
specific reporter molecules. In Figure 29.7, SERS spectra of three
types reporter labeled immune gold colloids are shown along with
the “empty” spectrum of commercial gold colloids conjugated with
goat anti rat IgG.
Figure 29.7 (A) Scheme of an immuno assay system using two different SERS Labels. (B) SERS signatures of three types of reporter labeled
immuno gold colloid (a) MBA/goat anti-rat IgG, (b) NT/goat antirat IgG,
(c) TP/goat anti rabbit IgG and (d) goat anti rat IgG.
436
Biophysical Chemistry
Antibodies immobilized on a solid substrate bind antigen (mouse IgG)
which in turn binds to a second antibody labeled with peroxidase.
When thee immuno complexes are reacted with o-phenylenediamine,
azoaniline is formed. The reaction product is adsorbed on colloidal
particles which results in a strong SERS spectrum of azoaniline. The
signal strength is proportional to the concentration of antigen. The
method of colloidal silver SERS has been successfully applied to detect
and to quantify prostaglandin H synthase-1 and 2 (PGSH-1 and 2) in
normal human hepatocytes and human hepatocellular carcinoma
(HepGz) cells.
(4) Probing of DNA and genes by SERS: Quantification of DNA and
its fragments during polymerase chain reaction is important. A SERS
based method for monitoring the concentration of double stranded (ds)
DNA amplified by PCR was proposed. In this method, a DNA intercalator, 4’, 6-diamidino 2-phenylindole dihydrochloride (DAPI) was used
to complex with ds-DNA. DAPI gives a strong SERS signal in colloidal
silver solution.
SERS labels offer some advantages for multiplex screening and simultaneous detection of different sequences in hybridization of DNA and
its fragments. The use of SERS label in detecting the DNA hybridization is shown in Figure 29.7.
SERS has been applied in hybridization experiments for detecting
p(dA)18 . The oligonucleotides have been attached on a nitrocellulose
surface. Nucleic acid fragments consisting of 18 deoxy ribonucleotide
oligomers of Thymine, p(dT)18 , have been tagged with cresyl fast violet (CFV) as the SERS label. The hybridized oligomers were deposited
on a SERS-active silver substrate. A strong SERS peak was observed
from the labeled p(dT)18 .
The effectiveness of the SERS gene technique has also been demonstrated in experiments dealing with human immunodeficiency virus
(HIV) gag gene sequence. In experiments involving nucleic acids, structural changes such as denaturation were shown to occur when colloidal
silver particles were used in SERS experiments. SERS on electrochemically etched (or roughened) silver surfaces was applied to detect a single mismatch in a ds-DNA fragment (290 base pairs) without the use of
a label molecule. This observation agrees with SERS spectra measured
for native and denatured DNA in which the appearance of strong adenine ring line confirmed denaturation. The target nucleic acids were
applied in concentrations ranging from 10−5 to 10−8 mol lit−1 ) but the
Surface Enhanced Raman Scattering 437
most interesting aspects of SERS on nucleic acids may be ascribed to
the highly sensitive and single molecule capabilities of the method.
The large non-resonant surface enhanced Raman cross sections provided by the strong field enhancement of colloidal clusters was
exploited in single molecule experiments. The nucleotide bases show
well distinguished SERS spectra. Thus, after cleaving single native
nucleotides from a DNA or RNA strand into a medium of colloidal
clusters detection and identification of single native nucleotides is quite
possible because of the unique SERS spectra of their bases. The SERC
spectra of nucleotide bases based on the idea of rapid sequencer base
are shown in Figure 29.8.
Figure 29.8 Cleavage of single nucleotides and attachment to colloidal
silver or gold clusters.
(5) Studies inside living human cells using SERS: Colloidal particles of
silver were incorporated inside the cells and SERS was applied to monitor intracellular distribution of drug in the whole cell and to study the
anti-tumour drugs/nucleic acid complexes. These experiments show
SERS spectra of the drug/DNA complexes represented by Raman lines
of the drugs but no SERS spectra of the native cell constituents were
detected.
Strong SERS spectra attributed to dimethyl crocetin (DMCRT) in a living HL60 cell in contact with gold film showing weak SERS bands (due
to phenylalanine and amide I and III bands) were detected.
SERS mapping over a cell monolayer (intestinal epithelial cells HT29)
with 1 μm lateral resolution shows different Raman spectra at all places
which indicates inhomogeneous chemical constitution of the cells. The
438
Biophysical Chemistry
Raman lines may be assigned to native chemical constituents in the
cell’s nucleus and cytoplasm such as DNA, RNA, phenyl alanine, tyrosine etc. The evidence that cells are alive after treatment with colloidal
gold comes from inspection of the cultures. The cultures show that cells
incubated with colloidal gold are growing and there is no evidence of
cell rounding or cell detachment from the growth surface when compared with monolayers.
The above experiments and observations demonstrate the feasibility of
measuring SERS of native constituents in a single cell using colloidal
gold particles as SERS-active nanostructures and generating a surface
enhanced Raman image inside a living cell.
(6) Detection of bacteria original biomolecules: SERS is well suited for
detection of bacteria from molecular to cellular level due to its sensitivity and selectivity. A new type of SERS chip, consisting of sandwich
graphene (G)-AgNP–silicon nanohybrid has been developed which
could achieve both molecular and cellular analysis in different samples. The chip could realize sensitive and accurate quantification of
ATP with LOD of 1 pM and can also simultaneously capture, discriminate and inactivate the bacteria. By combining SERS with a microfluidic chip using nanoparticle clusters as labels, a universal platform for
sensitive and specific detection of pathogen and antigens was established. LOD’s were 1 pgm/ml for Entamoeba histolytica antigen EHI
115350.
Questions
(1) The intensity in Raman shift experiments for a 10−5 M solute of silver
citrate in presence of sodium chloride is 11627 × 10−5 and that in the
absence of the same is 8095 × 10−5 . Calculate the SERS enhancement
factor (EF).
Solution:
EF =
11627 × 10−5
= 1.436
8095 × 10−5
(2) Outline the principle of surface enhanced Raman scattering with special reference to the contributing factors to SERS processes.
(3) Discuss some applications of this method in biophysical biomedical
fields.
Surface Enhanced Raman Scattering 439
(4) How is this technique useful in (i) probing of DNA and genes (ii) detection of bacteria in biological molecules.
(5) The incident Raman radiation in a Raman scattering experiment
occurs at 104 nm and the scattered light has a wave length of 105 nm.
Calculate the Raman shift in wave numbers (v̄).
(6) The Raman shift in the DNA analysis of phosphate bond occurs at 785
cm−1 . What wave length does it correspond to when expressed in nm.
30
Mass Spectrometry and its
Applications in the Analysis
of Biomolecules
30.1
Introduction
Mass spectrometry is a very important analytical technique employed to
quantitatively determine known materials and to identify unknown compounds within a sample. It is also used to elucidate the structure and
chemical properties of different molecules. This technique is basically concerned with studies of the effect of ionization of energy on molecules. The
actual process involves the conversion of the sample into gaseous ions,
with or without fragmentation, which are then characterized by their mass
to charge (m/z) ratios and relative abundances. Thus chemical reactions
are considered in gas phase in which sample molecules are consumed during formation of ionic and neutral species.
30.2
Principle of the Technique
Multiple ions from the sample under consideration are generated in a mass
spectrometer and it then separates them according to their specific mass to
charge ratio (m/z) and then records the relative abundances of each ion
type. The first step in the mass spectrometric analysis of any compound is
the production of gas phase ions of the compound by electron ionization.
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_30
441
442
Biophysical Chemistry
The molecular ion formed undergoes fragmentation and the primary
product formed from this ion undergoes further fragmentation. The ions
are separated in the mass spectrometer according to their m/z ratio and
are detected in proportion to their abundance. A mass spectrum of the
molecule is thus produced and displayed as a plot of ion abundance vs.
m/z ratio. The ions provide information concerning the nature and structure or the precursor molecule. In the spectrum of a pure compound, the
molecular ion appears at the highest value of m/z, followed by ions of containing heavier isotopes and gives the molecular mass of the compound.
30.3
Basic Components of a Mass Spectrometer
There are three major components in a mass spectrometer:
(i) Ion source: for producing gaseous ions from the substance under
consideration.
(ii) Analyser: for resolving the ions into their characteristic mass components according to their m/z ratio.
(iii) Detector system: for detecting the ions and recording the relative
abundance of each of the resolved ions.
Further, a sample introduction system is necessary to introduce the samples to the ion source under high vacuum requirements (10−6 to 10−8 mm
of Hg). For manipulating the data, a computer is required too.
The block diagram of a mass spectrometer is shown in the Figure 30.1
given below:
Figure 30.1 Components of a mass spectrometer.
30.4
Ionisation Methods in Mass Spectrometry
There are many types of ionization methods adopted in mass spectrometry. The classical methods that chemists are familiar with are: (i) electron
impact (EI) and fast atom bombardment (FAB). More modern methods
Mass Spectrometry and its Applications in the Analysis of Biomolecules
443
such as atmospheric pressure chemical ionization (APCI), electro spray
ionization (ESI), matrix assisted laser desorption ionization (MALDI) have
now come into vogue. Both ESI and MALDI have greatly advanced our
ability to analyse thermally labile molecules by providing an efficient
means of generating intact gas phase ions. Moreover, both methods have
been used to gain molecular weight information on biological samples
with great speed, accuracy and sensitivity. ESI generates ions directly from
solution (usually an aqueous or aqueous-organic solvent mixture) by creating a fine spray of charged droplets in the presence of a strong electric field.
Subsequent vapourisation of charged droplets results in the formation of
gaseous ions.
Gas phase ions are generated by the laser vaporization of a solid matrixanalyte mixture in which the matrix (usually a small crystalline organic
compound) strongly absorbs the laser radiation and acts as a receptacle
for energy deposition. The concentrated energy deposition results in the
vaporization and ionization of both matrix and analyte ions.
30.5
Applications
Both methods (ESI and MALDI) give mass accuracy of 1 part in 10,000
for proteins with molar masses less than 30 to 40 kD (i.e., 30,000 to 40,000
amu) and with a reduced mass accuracy for larger proteins. Proteins with
molar masses upto 100 kD (i.e., 1,00,000 amu) can be analysed at picomole
(10−12 mole) sensitivities to give simple mass spectra corresponding to
intact molecule. Accurate measurements of molar mass of biopolymers
are necessary for this analytical technique. A technique with high accuracy is MALDI time of flight (TOF) mass spectrometry. This technique
provides the mass of a protein with on accuracy of 0.01%. In MALDI, proteins introduced as solid or in solution are converted into intact, naked
ionised molecules in the gas phase. Subsequently in the mass analyser, the
m/z ratios of the naked protein molecule ions are determined.
When the measured mass of a protein agrees with that calculated from
the gene sequence, one can conclude that the gene sequence is correct.
An intense production of intact naked ionized protein molecules can
be achieved when small amounts of proteins embedded in a solid matrix
are bombarded with intense, short bursts or pulse of focused UV laser light
usually 337 nm from a N2 laser. The solid matrix consists of low molar
mass organic molecules that strongly absorb the UV irradiation.
444
30.6
Biophysical Chemistry
Analysis of Glycoproteins
Peptides and oligonucleotides are composed of linear head to tail combinations of different amino acids and nucleotides. But oligosaccharides contain many isobaric monosaccharides that not only can be linked through
different hydroxyl groups but can also form complex branching patterns.
It is not enough to determine monosaccharide composition and sequence
analysis to determine detailed primary structure. MALDI-TOF-MS has
been introduced to study the ionization of large peptides and proteins. A
combination of HPLC or Capillary Electrophoresis (CE) with MS peptide
mapping of protein is ideal for evaluating the presence of modifications.
Because FAB techniques cannot be applied to biomolecules of mass
greater than 6000, ESI methods are applied to most biomolecules.
30.7
ESI of Equine Apomyoglobin
As a result of electrospray process a molecule is charged by one or multiple
H+ and/or Na+ ions. This result appears as charge clusters i.e., m/z peaks
which represents the same molecule, but with different charges, the pattern
can be deconvoluted to the Mr which corresponds to the m/z peak with
a single charge. The deconvolution pattern of m/z peaks of equine apo
myoglobin to the m/z values of ∼ 17, 000 is shown below.
16950.584
2.5
M15+
M16+ 1130.7
2.0
M14+
1211.5
M17+
998.1
1.5
1.0
1304.7 1413.6
943.1
849.1
0.5
800
900
16930 16950 16970
1000
1100
1200
1300
1400
Figure 30.2 ESI of equine apomyoglobin.
In a native protein, fewer basic residues become exposed and charged
in the ESI process than in the corresponding denatured (unfolded protein).
Mass Spectrometry and its Applications in the Analysis of Biomolecules
ESI
Native protein
+
+
+
+
+
445
+
+
+ +
+ + + + + +
+
+
+
++
+++++
+++
m/z
+
Denatured
protein
ESI
+
+
+
+
m/z
Figure 30.3 Distribution of charged states characteristic of native and
denatured proteins.
30.8
Analysis of Phosphoproteins
Phosphoproteins have an important role in many intracellular processes
including signal transduction and regulation of cell division. The recombinant protein of alpha catalytic subunit of CAMP-dependent protein kinase,
which was isolated as a mixture of molecular species containing same peptide chain (but differing from each other in degree of phosphorylation
at specific residues) was subjected to HPLC-electro spray MS. The molar
masses of three isoforms of the protein were determined after desalting
them in a form suitable for electrospray MS analysis. The isoforms differed
from one another by the mass of a single - PO3 H group.
30.9
Protein Ladder Sequencing
The protein ladder sequencing method relies on the capabilities of matrix
assisted laser desorption MS to measure the molar masses of proteins and
peptides. The sequence defined fragments of a polypeptide chain have
been analysed by using MALDI in a single operation as a protein ladder.
The identity of a particular amino acid (based on the distinctive mass of
each genetically coded amino acid) was established by the mass differences
between consecutive peaks. The family of fragments found defines the
sequence of amino acids in the original peptide chain.
446
30.10
Biophysical Chemistry
Specific Examples of Biomolecules
Electron impact (EI) or chemical ionization (CI) are not useful to bring into
vapour phase, the biomolecules, because of their high molar masses. Ionisation techniques such as fast atom bombardment (FAB), secondary ion
mass spectrometry (SIMS), ESI and MALDI are required for this purpose.
The general range of application of different MS methods is presented in
Table 30.1. Because of the large molecular weights and the large number of
atoms in the formula it is important to be aware of the effects of isotopes
that contribute to the satellite peak pattern.
Table 30.1 General range of applications of different MS methods.
Ionisation
method
Detection limit
(picomole)
Fast atom
bombardment
Electrospray
(also with TOF)
MALDI
1–50
Common
application
range of mass
6000
Precision (%)
0.01–0.10
< 1, 30, 000
0.01
0.001–0.01
< 3, 00, 000
0.05
0.05
Native hmb
Denatured hmb
20+
Denatured apo Mb
20+
A
Denatured hmb
Native mb
11+
20+
B
Time
Denatured apo Mb
C
600
800
1000
1200
1400
1600
1800
2000
m/Z
Figure 30.4 Denaturation of myoglobin in an acidic environment.
Mass Spectrometry and its Applications in the Analysis of Biomolecules
447
The denaturation process of the protein, myoglobin in an acidic environment can be followed with time and which results in the loss of heme
factor. The resulting mass spectra are shown in Figure 30.4.
30.11
Peptides, Proteins and Polynucleotides
Fragmentation of positive ions of peptides and proteins once created by
FAB, ESI or MALDI can occur spontaneously and be detected by separating the ions. It can also be induced by tandem mass spectrometry in which
ions are made to undergo collisions with inert gas molecules like Ar at
low pressure in an ion trap. Peptides and proteins tend to fragment near
the amide bond. The fragmentation pattern is shown in Figure 30.5 and
Figure 30.6.
Figure 30.5 ESI of cytochrome-C and glucagon.
Figure 30.6 Fragmentation pattern of protonated peptide in FAB/MS.
448
Biophysical Chemistry
A
100
C
T
A
G
C
C
A
T
G
G
C
A
T
G
90
472.4
70
50
425.1
531.6
[M – 11H]
608.0
30
709.1
386.3
10
0
[M – 6H]6–
300
350
400
450
500
550
600
650
700
750
800
Figure 30.7 Mass spectrometry of nucleotide structures.
The bases in the structures of DNA and RNA are Adenine, guanine,
cytosine, thymine (in DNA) and Uracil instead of Thymine in RNA. The
MS of nucleotide structures is shown Figure 30.7.
30.12
Polysaccharides
Mass spectrometry of polysaccharides is more complex than that of proteins and polynucleotides. The pattern of MS is shown in Figure 30.8.
RO
RO
RO
O
OR
CH3 O O
OR
O
O
NHAC
273
B1
O
O
OR
O
500
561 576
B2
RO
RO
O
RO
OR
m/Z
OR
O
OR
1100
848
B3
1136
B4
1322
Figure 30.8 Pattern of mass spectrum in polysaccharides.
RO
RO
B5
1424
1500
1484
Mass Spectrometry and its Applications in the Analysis of Biomolecules
449
450
Biophysical Chemistry
Questions
(1) Draw a neat sketch of a mass spectrometer indicating the various parts.
(2) What are the different types of ionisation methods in mass spectrometry? Illustrate the electron spray ionisation method with a sketch of
E.S.I. spectrum.
(3) Outline the applications of mass spectrometry in understanding the
structure of proteins, peptides and polysaccharides.
31
X-Ray Studies in the
Elucidation of Structure of
Biomolecules
31.1
Introduction
X-ray diffraction can be used to determine the crystalline structure and
lattice parameters of a crystal. The information so obtained can be used to
identify the material since each metallic element has a unique combination
of lattice structure and parameter.
When an X-ray beam is directed at a metallic crystal, the beam strikes
the atoms and produces two types X-rays called white X-rays and characteristic X-rays. The characteristic X-rays, which are of interest, are caused
by the ejection of an electron from the inner shell of an atom hit by the incident X-ray. When an outer shell electron moves to fill the space created in
the inner shell, energy is emitted in the form of a X-ray photon.
By applying Bragg’s law, it is possible to determine the crystal parameters from its characteristic X-ray pattern.
Sketch of an X-ray Diffractometer
A schematic diagram of an X-ray diffractometer is given below (Fig. 31.1).
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5_31
451
452
Biophysical Chemistry
Figure 31.1 Schematic diagram of an X-ray diffractometer.
31.2
Bragg’s Law
The X-rays that strike a crystal have a wave length of about the same wave
length as the space between the atoms in crystal lattice. If each plane acts as
a surface which is struck by the incident X-ray beam, the beam is reflected
in some cases and not reflected in other cases. In the case of reflection, the
beams exiting the crystals are in phase and act to reinforce each other. This
occurs when the incident beam hits the parallel planes at certain angles
known as Bragg’s angle θ. In the non reflecting case, the waves leaving
the crystal are out of phase and cancel each other. The reflection of X-rays
from the planes of a crystal is shown in Figure 31.2.
Figure 31.2 Reflection of X-rays in ( hkl ) planes of a crystal.
If the geometry of the reflected beam is examined (Fig. 31.3), the relationship between Bragg’s angle, the wavelength λ of the X-rays and the
interplanar spacing, d, can be found.
X-Ray Studies in the Elucidation of Structure of Biomolecules Incident
x-rays
453
Reflected
x-rays
O
θ
θ
M
θ
2θ
N
d
P
(hkl) planes
Figure 31.3 Diffraction angle of X-rays.
The arbitrary planes of atoms whose indices are ( hkl ) and which has
interplanar spacing “d” are shown in Figure 31.3. If the X-rays entering the
crystal are in phase at OM and those reflected are in phase at ON, then the
distance MPN must be qual to an integral number of wave lengths, nλ.
It may be noted that MP = PN = d sin θ. Therefore for reflected X-ray
beams
nλ = 2d sin θ
(31.1)
where n = 1, 2, 3, . . ..
In the powder method of X-ray diffraction, the material under consideration is placed in the camera as shown in Figure 31.4.
θ
θ1 2
Monochromatic
x-ray beam
θ4
θ5
Beam entrance
S
Film
Film holder
Poly crystalline sample
Beam exit
S1
S2
S3
S4
S5
Figure 31.4 (a) X-ray camera and (b) X-ray film showing diffraction lines.
454
Biophysical Chemistry
The specimen is placed at the center of camera with the film around
in circle. When a monochromatic beam of X-rays is directed at the specimen diffraction takes place and characteristic X-rays are emitted in conical
sections that intersect the film and expose it at different arcs. These arcs
are seen as lines in the flattened film (Fig. 31.4). The Bragg angle of 90◦
corresponds to the distance between beam exit and entrance of the X-ray
film. The Bragg angle of each characteristic line on the film can be found
by using the ratio.
Si
θ
= i
(31.2)
Sn
θn
where Si = distance from the exit to the line of interest, Sn = distance from
the exit to the entrance, θi = Bragg angle of the line, θn = Bragg angle 90◦
from the exit to the entrance. After all the Bragg angles have been found, it
is possible to determine the crystal structure of the sample by considering
its geometry.
For pure metals in a cubic structure
dhkl = √
a
h2 + k 2 + l 2
(31.3)
where dhkl = interplanar distance between ( hkl ) planes, “a” is the lattice
parameter and h, k, l are Miller indices of the planes. Substituting eqn.
(31.3) into equation (31.1), we get
λ= √
2a sin θ
h2 + k 2 + l 2
(31.4)
Let Q2 = h2 + k2 + l 2 and then
λ2 =
4a2 sin2 θ
Q2
(31.5)
Or,
Q2 λ2
= sin2 θ
4a2
Since λ2 /4a2 is a constant (C), equation (31.6) may be written as
Q2 C = sin2 θ
(31.6)
(31.7)
From eqn. (31.7), it is seen that the squares of the sines of the angles that
result in a diffraction peak (line on the film) occur in a certain ratio of whole
numbers and this arises due to the structure factor of the lattice. These
ratios can be used to determine the crystal structure.
X-Ray Studies in the Elucidation of Structure of Biomolecules 455
Figure 31.5 Lines corresponding to planes of different cubic structures.
Figure 31.5 shows the lines corresponding to the planes which diffract
X-rays for cubic lattice structures. Once the crystal structure has been
found, the lattice parameter can be found from
λ
a= √
2 c
Note that
c=
(see eqn. 31.6)
sin2 θ
Q2
(31.8)
(31.9)
From the lattice parameter and the lattice structure, it is possible to identify
the metal from the corresponding standard table of crystal structures.
Generally, an X-ray diffraction experiment is done in two parts
(1) From a set of given data, determine the crystal structure and lattice
parameter which will allow identification of the material.
(2) From the camera and the strip of film, estimate true Bragg angles,
crystal structure and lattice parameter.
The data are tabulated as follows:
456
31.3
Biophysical Chemistry
Structural Determination of Proteins
The first step in crystallographic studies is the production of single crystals. This is achieved by slowly bringing the sample from a state of supersaturation to the crystalline state. The crystallization process is affected
by sample saturation, concentration of precipitants, ionic strength pH and
buffer concentration.
The X-ray diffraction experiments are next carried out using MIR (Multiple Isomorphous Replacement) or MAD (Multiple Wavelength Anomalous Dispersion) methods, the election density equation given
ρ( x, y, z) =
1
NV
h
k
l
∑ ∑ ∑ f (s)ρ[−2πi (hx+ky+lz] e−iα
(31.10)
−h −k −l
is solved and an image for the molecular transform can be completed. In
eqn. (31.10), V = Volume of the asymmetric cell, N = number of molecules
in volume V. The electron density is only a rough outline of the crystallized sample and requires further interpretation using graphical and computational methods. Structural refinement uses geometrical factors such as
proper bond lengths, bond and tetrahedral angles, planarity and backbone
angles as guides.
31.4
X-ray Structures of Haemoglobin
and Myoglobin
Haemoglobin and myoglobin, the first proteins for which 3-dimension
structures were determined at high resolution, play a crucial role in oxygen
transport and storage in muscle. This class of proteins consist entirely of
α-helices. In Myoglobin, there are eight helices of which two are oriented
such that they form a V-shaped pocket. In the holoenzyme the pocket contains haem group (prosthetic group or cofactors) a large heterocyclic ring
containing four pyrrole rings. The center of haem is occupied by a Fe2+
cation.
The histidine residue adjacent to the iron is important in mediating the
non-cooperative binding of oxygen to the protein. In haemoglobin a similar arrangement of residues can be found in the haem binding pocket.
However, oxygen binding and release is allosterically regulated by the
pseudo-tetrameric arrangement (a dimer of dimers, d2 β 2 ) of the individual protein subunits.
X-Ray Studies in the Elucidation of Structure of Biomolecules 457
Figure 31.6 X-ray structure of Myoglobin obtained at high resolution.
31.5
Photosynthetic Reaction Centers
Photosynthetic reaction centers (RC) are important catalysts in the photosynthetic process, and they are important for this process in the biosphere.
It is necessary to note that the conversion of light energy to chemical energy
is a prerequisite for all higher life forms on earth. RC’s are large multiprotein complexes in the outer membranes of plants and bacteria. The X-ray
structure of the reaction centre is the first structure of an integral membrane protein determined at high resolution. There are four protein chains,
the H, L and M subunits and cytochrome C. The H chain has one transmembrane helix, while the L and M chains have five each. The cytochrome
C subunit has no membrane spanning helix and it is anchored by proteins L and M. From the crystal structure, one can see how the photosynthetically active components bacteriochlorophyll, quinone and the haem
groups are arranged.
The spatial arrangement of these chromophores reveals the path and
the order of the electron transfer steps.
31.6
Ribosomal Subunit
Ribosomes are large molecular assemblies (Cytoplasmic organelles) consisting of complexes of proteins and in eukaryotes upto four RNA
458
Biophysical Chemistry
molecules. A large (50S) and a small (30S) subunit are loaded onto a mRNA
molecule to mediate the translation of the genetic message into a specific
sequence of amino acids or a polypeptide chain. The ribosomal proteins
play only a subordinate architectural role and do not directly participate
in the peptidyl transferase activity of the ribosome. X-ray crystallography
provides proof that the ribosome is a ribozyme (catalytic RNA).
Questions
(1) Give the schematic diagram of an X-ray diffractometer and indicate its
parts.
(2) Show the reflection of X-rays in (hkl) planes of a crystal. Write down
the relation between the diffraction angle and wavelength of X-rays.
(3) Describe briefly the structures of Haemoglobin and myoglobin determined by X-ray diffraction technique.
(4) X-rays of wavelength 1.545Å impinge on a (111) plane of a silver lattice.
If the reflection from this plane occurs at 2θ = 38.5◦ , calculate its lattice
parameter.
32
CRISPR-CAS-9, A Method for
Genome Editing
32.1
Introduction to CRISPR-CAS-9
It is a gene editing mechanism derived from a primordial immune system in bacteria called “Clustered regularly interspersed short palindromic
repeats” (CRISPR). The synthetic guide RNA (created by Emmanuelle Charpentier and Jennifer Doudna) is complementary to a target DNA sequence,
directs the CAS-9 enzyme (see Fig. 32.1) to a specified location for DNA
cutting. Some applications require an additional DNA template to fill in
the cut. It allows the scientists to cut any strand of DNA. Since its creation
in 2012, diverse experiments have been carried out using CRISPR to alter
Figure 32.1 Parts of a bacterial immune system: Genomic DNA, CAS-9,
target sequence and guide RNA.
© The Author(s) 2023
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Biophysical Chemistry
DNA in organisms across the tree of life, mushrooms, tomatoes as well as
tumours.
32.2
Genesis of the Discovery
The two scientists were inspired by a strange and little studied bacterial
immune system. It is known that bacteria too have viral infections just like
people do, and some bacteria used an enzyme called CAS-9 to chop up
invading viruses and store molecular mugshots of them to quickly attack
any repeat invaders. In 2011, these scientists worked out the details of how
two bacterial RNA molecules called trace RNA and CrRNA controlled this
process.
They synthesized a new molecule called the single guide RNA which
combines essential features of the two bacterial RNA’s to direct CAS-9 to
a specific site in DNA for cutting. Their method is cheaper, faster and easier to use than previous gene-editing tools which are complex and costly.
Today, scientists can readily order CAS-9 and guide RNA’s customized to
target a specific sequence of DNA. CRISPR opened up gene editing to the
masses.
But CRISPR’s ease of access has its negative aspects. For example, in
2018, a Chinese scientist used CRISPR to edit two human embryos that
were carried to term. The announcement of the first gene edited babies
shook the world. The experiment crossed an ethical red line in the minds
of many gene editing scientists. Even though CRISPR’s applications are in
the realm of biology, the technique requires sophisticated and very complex chemistry. With new treatments for human genetic diseases in needy
patients, the era of human genome editing has began.
32.3
Details on Enzyme “CAS-9”
This acts as a pair of molecular scissors that can cut the two strands of
DNA at a specific location in the genome so that the bits of DNA can be
added or removed. The scaffold part binds to DNA and the predesigned
“sequenced guides CAS-9” to the right part of the genome.
Its main function is to cut DNA and there by alter a cell’s genome.
More scientifically, “CAS-9” is a dual RNA-guided endonuclease enzyme
associated with CRISPR adaptive immune system in streptococcus pyogenes. It cleaves foreign nucleic acids bearing sequence complementary to
the RNA loaded into the enzyme during bacterial adaptive immunity.
CRISPR-CAS-9, A Method for Genome Editing 32.4
461
CAS-9 Mechanism
The key step in editing an organism’s genome is selective targeting of a
specific sequence of DNA. Two biological micromolecules, the CAS-9 proteins and guideRNA, interact to form a complex that can identify target
sequences with high selectivity. The CAS-9 protein is responsible for locating and cleaning target DNA, both in natural and artificial (CRISPR/CAS
systems). The CAS-9 protein has six domains, REC-I, REC-II, Bridge helix,
PAM interacting, HNH and RuvC. The REC-I domain is the largest and is
responsible for binding guide RNA. The role of REC-II is not well understood.
The arginine-rich bridge helix is crucial for initiating cleavage activity upon binding of target DNA. The PAM interacting domain confers
PAM specificity and is therefore responsible for initiating binding to target
DNA. The HNH and RuvC domains are nuclease domains that cut singlestranded DNA. They are highly homologous to HNH and RuvC domains
found in other proteins.
REC I
Bridge
helix
REC II
vC
HNH
Ru
M
PA
ng
cti
ra
nte
i
Figure 32.2 The six domains of CAS-9.
The “CAS-9” protein remains inactive in the absence of guide RNA. In
engineered crisper systems, guide RNA is comprised of a single strand of
RNA that forms a T-shaped molecule which is comprised of one tetra loop
and two or three stem loops. The guide RNA is engineered to have a 5’
end that is complimentary to the target DNA sequence.
462
Biophysical Chemistry
Target complimentary region
Tetraloop
Stem loop 1
Stem loop 2
Stem loop 3
Figure 32.3 Single strand of RNA forming a T-shaped molecule comprising of one tetra-loop and stem-loops (1, 2 or 3).
This artificial guide RNA binds to the CAS-9 protein and upon binding, induces a conformational change in the protein (see Fig. 32.4). The
conformational change converts the inactive protein into its active form.
The mechanism of conformational change is not fully understood but it is
hypothesized that either steric interactions or weak binding between protein side chains and RNA bases may induce the change.
Figure 32.4 CAS-9 complex (inactive) and target complimentary region of
guide RNA.
Figure 32.5 CAS-9/guide RNA and target DNA leading to CAS-9/guide RNA complex bound to target DNA.
CRISPR-CAS-9, A Method for Genome Editing 463
464
Biophysical Chemistry
Once the CAS-9 protein is activated, it stochastically searches for target DNA by binding with sequences that matches its protospacer adjacent
motif (PAM) sequence. A PAM is a two or three base sequence located
within one nucleotide down stream of the region complementary to the
guide RNA. PAMs have been identified in all CRISPR systems, and the specific nucleotides that define PAMS are specific to the particular category of
CRISPR system. The PAM in Streptococcus pyogenes is 5t’-NGG-3. When
the “CAS-9” protein finds potential target sequence with the appropriate
PAM, the protein will melt the bases immediately upstream of the PAM
and pair them with the complementary region on the guide RNA. If the
complementary region and the target region pair properly, the RuvC and
HNH nuclease domains will cut the target DNA after the third nucleotide
base upstream of the PAM (Fig. 32.5).
32.5
Target DNA Binding and Cleavage by CAS-9
(1) CAS-9 scans potential target DNA for the appropriate CAM (Stars), (2)
When the protein finds PAM, the protein-guide RNA complex will melt
the bases immediately upstream of the PAM and pair them with the target complimentary region of the guide RNA, and (3) If the complimentary
region and the target region pair properly, the RuvC and HNH domains
will cut the target DNA after the third nucleotide base upstream of the
PAM.
Questions
(1) Explain the CAS-9 mechanism of editing an organism’s genome.
(2) By means of a sketch, show the six domains of CAS-9.
(3) How does the enzyme CAS-9 cleave the target DNA?
Appendix A
Donnan Membrane Potential
In this Appendix, the equations pertaining to Donnan membrane potential
will be derived for a polyion possessing a net negative charge.
The Donnan membrane potential refers to the transport of all ions
(except chosen macroions) across a semi-permeable membrane. For brevity,
the macro polyion is assumed to possess a net negative charge (magnitude
of charge = z).
Consider an electrolyte designated as MX with anions ( X − ) and
cations ( M+ ) being allowed to diffuse across the membrane. The polyanions ( Macro −z ) can however not diffuse across the membrane and is initially present in the compartment ‘a’. Hence the polyanion ( Macro −z ) provides excess negative charges in compartment a whereas the anions X −
enter the right and the cations M+ towards the left as shown in the figure.
a
b
cations
anions
poly anion
Figure A.1. Schematic depiction of the Donnan membrane potential when
the polyanion is impermeable across the membrane. The anions move
to the right side while the cations move to the left till the equilibrium is
established.
© The Author(s) 2023
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Biophysical Chemistry
When the diffusion and migration effects balance each other, a potential difference Δϕ arises. Eventually, an equilibrium is reached in which a
voltage difference (Δϕ) is balanced by diffusive forces. The accumulation
of cations in the compartment ‘a’ and anions in compartment ‘b’ leads to
the negative values of the interfacial potential.
However, the Nernst equation provides the potential difference as a
function of the concentration. For an 1:1 electrolyte,
RT [ M+ ]b
ln
F
[ M+ ]a
RT [ X − ]b
ln −
Δϕ =
F
[X ]a
Δϕ = −
(A.1)
(A.2)
At equilibrium,
[ M+ ]b [ X − ] a = [ M+ ] a [ X − ]b
(A.3)
If the concentration of anions and cations in the two compartment obeys
the above equation, it indicates the existence of the Donnan membrane
potential.
The principle of electroneutrality indicates that in the compartment
designated as ‘a’
[ M+ ] a − [ X − ] a − z[ Macro −z ] = 0
(A.4)
Similarly in the compartment ‘b’, electroneutrality dictates that
[ M+ ]b = [ X − ]b
(A.5)
Furthermore, the concentrations of cations and anions are equal in this
case. Hence the salt concentration cb can be introduced in stead of the
individual concentrations.
The cationic and anionic concentrations can then be written using the
salt concentration. Hence
cb
= e− FΔϕ/RT
[ M+ ]a
cb
= e FΔϕ/RT
[X− ]a
(A.6)
(A.7)
The above two equations are reminiscent of the Boltzmann distribution.
Employing these equations in conjunction with eqn. (A.4), it is possible to
Appendix A: Donnan Membrane Potential 467
obtain the following equation in terms of the interfacial potential difference
Δϕ
cb e FΔϕ/RT − cb e− FΔϕ/RT − z[ Macro −z ] = 0
(A.8)
The above equation can be rewritten as
e2FΔϕ/RT −
z[ Macro −z ] FΔϕ/RT
e
−1 = 0
cb
(A.9)
Upon solving the quadratic equation and using the physically realistic positive root, the Donnan membrane potential follows as a function of the system parameters.
e FΔϕ/RT =
=
z[ Macro −z ]cb
z[ Macro −z ]
+
2cb
z[ Macro −z ] 2
cb
2
+4
z[ Macro −z ]
2cb
(A.10)
2
+1
(A.11)
The Donnan membrane potential can now be estimated using the two concentrations [ Macro −z ] and the bulk concentration of the salt (cb ). The
Donnan membrane potential alters the ionic concentrations in a significant manner. This is not the case for the Nernst equation wherein the
ionic concentrations get altered to a minor extent for changes in the potential. It is well known that when the concentration ratio changes ten times,
the potential is altered by about 59 millivolts, according to the Nernst
equation.
Appendix B
Nernst Planck Equation
The three conventional transport processes governing any physicochemical system are: (i) diffusion; (ii) migration and (iii) convection. The
corresponding driving forces are chemical potential gradient, electrical
potential gradient, and non-uniformity in velocities. Among these, diffusion and migration processes of charged species are more extensively
pursued on account of their immense applicability in diverse fields. The
phenomenological equations governing diffusion are Fick’s first and second laws while the migration effects can be described using Ohm’s law or
non-linear current-potential equations.
It is of interest to enquire the governing equations pertaining to the
simultaneous occurrence of diffusion and migration. Although there are
a hierarchy of equations for describing diffusion and migration together,
the simplest ones are the Nernst–Planck equation and Donnan equation.
A few salient features of Nernst Planck equation are provided below on
account of its importance in estimating potential differences in membranes.
The Donnan membrane potential has been described briefly in Appendix A.
The classical Fick’s first law of diffusion for the one-dimensional flux
( Ji ) of the species i is represented as
Ji = − Di
∂ci
∂x
(B.1)
Since the driving force for migration is the electrical potential gradient, it
can be added to the flux thereby yielding the Nernst–Planck equation as:
z F ∂Δφ
∂ci
(B.2)
+ i ci
Jion = − Di
∂x
RT ∂x
© The Author(s) 2023
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Biophysical Chemistry
for the ith ionic species. Di denotes the diffusion coefficient of i across
the region of transport, Δφ being the potential difference, ∂x being the
infinitesimal distance of transport.
The above equation is valid for one-dimensional transport. It is easy
to recognize the two different contributions to the overall flux. In biophysical chemistry, the Nernst–Planck equation describing the movement
of charged species under concentration and electrical field gradients is
of immense importance due to its usefulness in computing the diffusion
potentials and ion transport across membranes. We emphasize the following limitations of the above equation viz. (i) neglect of activity coefficients;
(ii) non-inclusion of interionic interactions and (iii) validity for dilute solutions.
B.1
Nernst–Planck Equation from
Onsager’s Linear Flux-Force Relation
According to the linear flux-force formalism of Onsager, the ionic flux can
be represented as
Ji = Liv ( Xi − Xv )
(B.3)
where Xi and Xv denote the driving forces at a site occupied by the ion and
at vacant site respectively. Liv denotes the Onsager’s phenomenological
coefficient.
However, the driving force is the gradient of electrochemical potential
and hence
∂
Xi = − (μiel )
∂x
where μiel is the electrochemical potential of the species with charge zi
given by
μiel = μ0,ch
+ RT ln ci + zi FΔφ
(B.4)
i
and μ0,ch
is the standard chemical potential of i. The driving force now
i
becomes
z F dΔφ
1 dci
(B.5)
Xi = − RT
+ i
ci dx
RT dx
Similarly,
− RT dcv
,
(B.6)
cv dx
since the vacant sites are neutral. Consequently, the ionic flux equation
becomes
1 dci
zi F dΔφ
1
+
+
Jion = − Liv RT
ci
cv dx
RT dx
Xv =
Appendix B: Nernst Planck Equation 471
For dilute solutions, the concentration of vacant sites is large (i.e., cv → ∞).
Hence
z F dΔφ
1 dci
(B.7)
Jion = − Liv RT
+ i
ci dx
RT dx
The Onsager’s coefficient is related to the diffusion coefficient in the following manner:
Liv = ci Div /RT
(B.8)
For emphasizing the diffusion of ions through vacant sites within a lattice
model framework, the subscript v is introduced for the diffusion coefficient
and is not different from the conventional diffusion coefficient.
Substituting equation (B.8) in equation (B.7),
Jion = − Div
z F dΔφ
dci
+ i ci
dx
RT dx
(B.9)
which is one-dimensional Nernst–Planck equation. Employing the above,
the diffusion potential can be deduced from the respective transference
numbers of ions.
The current density arising from the transport of ions of charge z is
Current = i = zFJion
The above description is made simple but in reality, the estimation of the
steady state ionic flux as well as the time-dependence of ionic concentrations requires the numerical solution of the spatio-temporal diffusion
migration equations with realistic boundary conditions, especially when
the two and three dimensional transport is considered.
Appendix C
Goldman-Hodgkin-Katz
Equation
This equation is employed in cell membrane physiology to determine the
potential across a cell’s membrane taking into consideration all the ions
that are permeant through the membrane. Considering K+ , Na+ and Cl−
ions inside and outside of a membrane, the membrane potential can be
derived as
Em =
p [K+ ]out − pNa [Na+ ]out + pCl [Cl− ]int
RT
ln K +
zF
pK[K ]in + pNa[Na+ ]in + pCl[Cl− ]out
where p s denote permeability coefficients of the respective ions, K+
,
(out)
, Cl−
represent the concentrations of the ions indicated outside
Na+
(out)
(out)
the cell membrane and the “in” terms related to the concentrations of the
ions inside the membrane.
Although the above equation pertains to monovalent cations and
anions, its generalization to other valencies is straight forward.
Questions
(1) Calculate the membrane potential from the GHK equation for the skeletal muscle of a toad at 298K under the following conditions.
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
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Biophysical Chemistry
Ions
Na+
K+
Cl−
Permeability
factor
(cm/sec)
5 × 10−8
5 × 10−6
5 × 10−6
Intracellular
concentration (in
10−3 moles/litre)
10
150
5
Extracellular
concentration (in
10−3 moles/litre)
150
5
125
Solution:
pK [K+ ]ex + pNa [Na+ ]ex + pCl [Cl− ]in
pK [K+ ]in + pNa [Na+ ]in + pCl [Cl− ]ex
25 × 10−9 + 750 × 10−11 + 25 × 10−6
= 0.0591 log
750 × 10−9 + 50 × 10−11 + 625 × 10−6
RT
ln
Em =
F
= −0.081 V
(2) The electrical mobility of the Li+ in water is 5 × 10−4 cm2 /sec-V at
25◦ C; the corresponding values of K+ and Br− are 7 × 10−4 and 8 ×
10−4 . Using the Goldman equation, estimate the electrode potential
across a film separating a 100-mM KBr solution from a 100-mM LiBr
solution.
Appendix D
Salient Aspects of COVID-19
D.1
Introduction
Many countries all over the world are currently grappling with a virus
which has presumably developed as an out break in China and referred
as severe acute respiratory syndrome (SARS). The World Health Organization (WHO) identified it as SARS-CoV-2 as it is a common virus known
as corona virus that infects the nose, respiratory tract and the sinuses of
a person. It is currently referred as COVID-19 and was first reported in
December, 2019.
D.2
Composition of the COVID-19 Virus
Corona viruses contain Ribonucleic acid (RNA) in their core which is akin
to Deoxyribonucleic acid (DNA). The RNA acts as a molecular messenger which enables the formation and production of proteins which are
required for the other parts of the virus. The following diagram provides
a rough sketch of its structure.
Figure D.1. Schematic depiction of Corona virus.
© The Author(s) 2023
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Biophysical Chemistry
The viral envelope encapsulates or covers the RNA genome thus
protecting the virus when it is outside its host i.e., the cell. The outer
envelope constitutes a layer of lipids consisting of a long chain of saturated fatty acids such as palmitic acid (CH2 (CH2 )14 COOH), or stearic acid
CH3 (CH2 )16 COOH. It is possible that unsaturated fatty acids such as oleic
acid (CH3 (CH2 )7 CH = CH(CH2 )7 COOH) or linolenic acid also may form
part of the layer of lipids.
These lipid layers serve two purposes:
(i) they serve as an anchor to the structural proteins required by the
virus to infect the cells in the body (see the small closed circles in
the diagram).
(ii) the bulky projections protruding outside the cell are known as spike
proteins which act as hooks for the virus to attach themselves to host
cells and promote infections.
A common observation is that corona viruses do not thrive or reproduce
outside of a host cell.
D.3
Symptoms and Other Effects of COVID-19
The main symptoms of a person afflicted with this virus include: (i) fever,
(ii) cough with shortness of breath, (iii) fatigue, (iv) body aches, (v) running
nose and (vi) loss of smell or taste.
The virus can lead to other more serious conditions like respiratory
failure, heart and liver problems, septic shock and death. The virus can
infect the immune system with proteins, known as cytokines and are thus
inimical to the body. The disease spreads when a sick person sneezes or
coughs. It is also possible that the virus can get into the body by touching
a surface or a part of the body like mouth or the nose as it can live for
several hours.
D.4
Remedial Measures
There are several ways by which one can get rid of the virus viz.
(1) Washing or sanitizing hands regularly. It is also necessary to disinfect
the surface, one is likely to touch or come in contact with.
(2) By following social distancing i.e., keeping away from others by about
six feet or wearing a face mask.
Appendix C: Salient Aspects of COVID-19 477
(3) It is possible to prevent infection by getting vaccinated with a suitable
vaccine.
– Details of vaccines available till date: Currently, the vaccine developed by the Pfizer pharmaceutic company, known as Pfizer BioNTech COVID-19 vaccines is used for people 16 years or older. Apart
from this, the vaccines developed by Moderna, Johnson and Johnson
pharmaceutical companies are used. Usually two does of the vaccine
are given to a person to compact the virus or its variants.
– Vaccines based on Ribonucleic acid (RNA): The vaccines, developed by Pfizer BioNTech and Moderna generated messenger RNA
(or mRNA) which works by instructing the cells to create a covid2 spike protein that stimulates the body to make antibodies. An
unwanted development in using mRNA vaccines is that they triggered a response resulting in the breakdown of mRNA.
However, Katalin Kariko at the University of Pennsylvania USA (and
also at BioNTech, Mainz, Germany) and Drew Weissman also at
University of Pennsylvania showed that replacing one type of molecule
in mRNA, namely Uridine (C9 H12 N2 O6 ) by a similar one Pseudouridine
(C9 H12 N2 O6 ) avoids the immune reactions.
Notes and Bibliography
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– Figures 1.1–1.5 were adapted from M.G. Greenwood News-Medical Life
Sciences (2014).
– Figures 1.6–1.14 Open access book available: Download for free at
https://openstax.org/details/books/anatomy-and-physiology.
– Figures 1.17–1.25 are from NPTEL Biotechnology course-module 3.
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lucidly illustrated in W. Bustin Mereer., Technical report 640, the living
cell as an open thermodynamic system, Bacteria and Irreversible Thermodynamics, Department of Army, Fort Detrick, Maryland, May 1971.
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C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
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Biophysical Chemistry
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in CRC Handbook of Chemistry and Physics (84th Edition CRC Press
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The structures of MA1 and TA1 and all electrochemical data are from
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Miguel de la Guardia, Trends in Analytical Chemistry, 107, 1 (2018).
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– A detailed account has been provided in Kim Gail Bioprocess Engineering Woodhead Publishing Limited (2013).
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discussed in G.E. Briggs and J.B.S. Haldane, Biochemical Journal, 19, 338
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Chapter 12
– A comprehensive account of all electrochemical techniques has been
given in Cynthia Zoski Handbook of Electrochemistry Elsevier (2007).
– The mathematical treatment of different electrochemical techniques is
lucidly discussed in Alan J. Bard and Larry Faulkner Electrochemical
Methods–Fundamentals and Applications John Wiley & Sons (2001).
– An overview of electroanalytical techniques is given in Joseph Wang
Analytical Electrochemistry John Wiley & Sons (2006).
Notes and Bibliography 483
Chapter 13
– Figures 13.1 to 13.4 adapted from (Yijun Tang, Xiangqun Zeng and Jennifer Liang, J. Chem. Ed., 87, 742 (2010) are reproduced with permission
from the American Chemical Society.
Chapter 14
– Sazia Iftekhar, Susan T. Ovbude and David S. Hage, Frontiers in Chemistry, 7, Article 673 (2019).
Chapter 15
– Figures 15.1 to 15.10 are adapted from Beckmann Coulter. Introduction
to capillary electrophoresis- Handbook (open access).
Chapter 16
– J. Lu., NMR in Biomedical research, Materials and Methods, 3, 170 (2013).
Chapter 17
– Indra D Sahu, Robert M McCarrick, Kaylee R Troxel, Rongfu Zhang,
Hubbell J Smith, Megan M Dunagan, Max S Swartz, Prashant V Rajan,
Brett M Kroncke, Charles R Sanders and Gary A Lorigan, Biochemistry,
52, 6627 (2013).
Chapter 18
– Xiwei Zheng, Zhao Li, Sandya Beeram, Maria Podariu, Ryan Matsuda,
Erika L. Pfaunmiller, Christopher J. White II, NaTasha Carter and David
S Hage, J. Chromatography, B968, 49 (2014).
– Claire Vallance, An Introduction to Chemical Kinetics Morgan and Claypool publishers (2007).
– H. Gutheard et al., Ann. Rev. Biochem., 40, 315 (1971).
– Xiwei Zheng, Cong Bi, Zhao Li, Maria Podariu and David S. Hage, J. of
Pharm and Biomed Analysis, 113, 163 (2015).
Chapter 19
– S.L. Frien, E.S. Lewis and A. Weinberger (Eds), Techniques of organic
chemistry. Vol. III, Part 2, Wiley (Interscience) (1963).
– G. Czerlinski Ali and M. Eigen, Z. Electrochem., 63, 652 (1959).
484
Biophysical Chemistry
– C. Kalidas, Chemical Kinetic Methods, New Age International Publishers, New Delhi (2002).
Chapter 20
– J.M. Sturtevant, Ann. Rev. Phys. Chem., 38, 468 (1987).
– J.E. Ladbury and B.Z. Chowdury, Chem. Biol., 3, 791 (1996).
Chapter 21
– J.S. Davis and H. Gutfreund, FEBS Letters, 72, 2 (1976).
Chapter 22
– S.M. Kelly and N.C. Price, Current Protein and Peptide Science, 1, 349
(2000).
Chapter 23
– Figure 23.1 reproduced with permission from (Thomas Hofelich, Lars
Wadsö, Allan L. Smith et al., Journal of Chemical Education, 78, 1083 (2001).
Copyright 2001 American Chemical Society.
Chapter 24
– R. Chang, Physical chemistry for the biosciences, University Science, New
York (2005).
Chapter 25
– E.E. Conn and P.K. Stumpf, Outlines of Biochemistry, Fourth Edition John
Wiley & Sons (1976).
– H.B.F. Dixon, Biochemical Education, 13, 181 (1985).
Chapter 26
– Figures 26.1 to 26.3 are adapted from Pulimamidi Rabindra Reddy and
Nomula Raju Gel Electrophoresis and its Applications Intech open
access (2012).
Chapter 27
– G. Rabston, in Beckman Coulter (Life Sciences) Analytical Ultracentrifuge (open access) (2013).
Notes and Bibliography 485
Chapter 28
– Figure 28.1 adapted from O.B. Acikara Ion exchange chromatography,
Intech open access (2012).
Chapter 29
– Figures 29.1 and 29.2 adapted from Ujit Sur, Surface Enhanced Raman
Scattering (2017) Intech Open access.
Chapter 30
– Thomas P. E. Hollenbeck, Gary Siuzdak and Robert D. Blackledge,
J. Forensic. Sci., 44, 783 (1999).
Chapter 31
– Figure 31.9 adapted from Yunbeom Lee, Jong Goo Kim, Sang Jin Lee,
Srinivasan Muniyappan, Tae Wu Kim, Hosung Ki, Hanui Kim, Junbeom
Jo, So Ri Yun, Hyosub Lee, Kyung Won Lee, Seong Ok Kim, Marco Cammarata and Hyotcherl Ihee, Nature Communications, 12, 3677 (2021).
Chapter 32
– Figures 32.1 to 32.5 adapted from Cavanagh and Garrity, “CRISPR Mechanism”, CRISPR/Cas9, Tufts University (2014).
https://sites.tufts.edu/crispr/
Index
Active transport 17
Affinity Chromatography 287
Aldopentoses 66
Allosteric enzymes 197
Amperometric biosensors 266
Analysis of glycoproteins 444
Analysis of Lipids 92
Analytical Ultracentrifugation 409
Antibodies 153
Antiport 21
ATP hydrolysis 250
Basic components of a mass
spectrometer 442
Basics of NMR 314
Bifunctional oligomeric enzymes
202
Biochemical function of Biotin 230
Boltzmann Distribution 35
Capillary zone electrophoresis,
303
CAS-9 Mechanism 461
Cellulose 73, 290
Chiral Recognition 309
chymotrypsin 284
Circular Dichroism 361
Circular Dichroism using
synchrotron radiation 364
Classes of peptides 130
Classification of Amino Acids 115
Classification of Proteins 145
Co-enzymes 217
Cofactors in enzyme catalysis 198
Competitive inhibition 187
Composition of Proteins 143
Conformational changes in
proteins 368
Continuous culture of bacteria 43
Coupling of reactions 54
CRISPR-CAS-9 459
Denaturation of Proteins 153
Deoxyribonucleic acid 475
Distribution of Lipids 99
Donnan Membrane Potential 465
Double site-directed spin labeling
323
Effect of cofactors and ligands on
circular dichroism 365
Electro osmosis 302
Electro-reduction of adenine 168
Electrochemical biosensors 265
© The Author(s) 2023
C. Kalidas and M. V. Sangaranarayanan, Biophysical Chemistry,
https://doi.org/10.1007/978-3-031-37682-5
487
488
Biophysical Chemistry
Electron nuclear double resonance
spectroscopy 329
electrophoresis 302
Endoplastic Reticulum 7
Enzymatic sensing of glucose 267
Enzymatic sensing of urea 271
Enzyme “CAS-9” 460
Eukaryotic cells 9
Examples of nucleotides 166
Examples of Proteins 151
Exocytosis 23
Immunoassays employing SERS
434
Impedimetric biosensors 273
Ion Exchange Chromatography
421
Ionisation methods in Mass
spectrometry 442
Isoelectric focusing 303
Isothermal Calorimetry 375
Isothermal Heat Flow Calorimeter
376
Facilitated diffusion 18
Fick’s diffusion laws 469
Flash photolysis 351
Flavin Adenine Dinucleotides 228
Flow Methods 335
Joule Heating 303
Gel filtration 395
Gel filtration of hemoglobin 399
Geometric Isomerism 91
Gibbs free energy 43
Glycolipids 107
Glycolytic enzymes 201
Glycosides 68
Goldman-Hadgkin-Katz Equation
473
Linear laws 45
Lineweaver-Burke Plot 186
Lipid Bi-layers 105
Lipoproteins 98
Helix-Coil Transitions 154
Hemoglobin 152
High performance affinity
chromatography 288
High phosphoryl capacity of ATP
247
Immobilisation of enzymes 209
Immobilization by N-hydroxy
succinimide method 293
Ketohexoses 67
Kinetics of Helix-Coil
Transformation 154
Magic angle spinning 320
Mass spectrometry 441
Membrane Proteins 155
Metal complexation studies 358
Metal-Chelate Affinity
Chromatography 295
Metalloflavoproteins 228
Methodology 389
Micellar electrokinetic capillary
chromatography 303
Michaelis–Menten constant 185
Michaelis-Menten constants for
enzymatic biosensors 270
Mitochondria 6
Molecular aggregation 40
Molecular cloning 405
INDEX Molecular Weight Determination
411
Multi-substrate enzyme reactions
188
Multienzyme complexes 203
Mutational Studies 383
Myosin assembly 357
Negative feed back inhibition 196
Nernst Planck Equation 469
NMR in biomedical research 314
NMR in protein structure
determination 315
non-competitive inhibition 193
Non-enzymatic sensing of glucose
271
Non-equilibrium thermodynamics
in microbiology 44
Nucleic acid chromophores 368
Oligosaccharides 70
Osmotic effects 37
Oxygen Evolving Complex 332
Passive transport 17
Phase transitions of phospholipids
392
Phenomenological coefficients
53
Phospholipid Bilayer 22
Polysaccharides 72
Potentiometric sensors 272
Pressure Jump Relaxation 355
Prokaryotic cells 8
Protein ladder sequencing 445
Protein structure determination
using solid state NMR 323
Purification of Adenovirus 426
489
Reaction of horse radish
peroxidase with H2 O2 212
Reactions of Amino Acids 120
Reactions of the TCA cycle 253
Redox potential data on water
soluble B-Vitamins 227
Relation between Co-enzymes and
Vitamins 220
Ribosomes 9
Separation of Membrane
Phospholipids 426
spectrophotometry 346
Step gradient elution method 293
Surface Plasmon Resonance 279
Symport 25
Target DNA Binding 464
Temperature jump 345
Thermal denaturation of proteins
390
Thermochemistry of
Carbohydrates 74
Transport across cell membrane 17
Turn Over Rates 208
Unimolecular Reactions 337
Vitamin B-6 group 232
Voltammetric biosensors 272
Water soluble vitamins 221
Waxes 105
X-ray Diffractometer 451
Zeta potential data 241