Stabilization of Cytochrome c reductase using
Osmolytes
Thesis submitted in partial fulfillment of the requirements for
The degree of
Master of Technology
In
BIOTECHNOLOGY AND MEDICAL ENGINEERING
By
M. Archana
Roll No. 207BM202
Department of Biotechnology and Medical Engineering
National Institute of Technology
(Deemed University)
Rourkela-769008 (ORISSA)
May– 2009
Stabilization of Cytochrome c reductase using
Osmolytes
Thesis submitted in partial fulfillment of the requirements for
The degree of
Master of Technology
In
BIOTECHNOLOGY AND MEDICAL ENGINEERING
By
M. Archana
Roll No. 207BM202
Under the guidance of
Dr. Subhankar Paul
Department of Biotechnology and Medical Engineering
National Institute of Technology
(Deemed University)
Rourkela-769008 (ORISSA)
May – 2009
Dr. Subhankar Paul
Assistant Professor
Department of Biotechnology & Medical
Engineering
National Institute of Technology
Rourkela
Phone No: 0661-2462284
Email: spaul
Certificate
This is to certify that the thesis entitled “Stabilization of Cytochrome c reductase
using Osmolytes” by Miss M.Archana submitted to the National Institute of
Technology, Rourkela for the Degree of Master of Technology, is a record of
bonafide research work, carried out by her in the Department of Biotechnology and
Medical Engineering under my supervision. I believe that the thesis fulfils part of
the requirements for the award of master of Technology. The results embodied in
the thesis have not been submitted for the award of any other degree.
Dr. Subhankar Paul
(Assistant Professor)
Department of Biotechnology &Medical engineering
NIT Rourkela
Acknowledgement
I avail this opportunity to express my hereby indebtedness, deep gratitude and
sincere thanks to my guide, Dr. Subhankar Paul, Assistant Professor, Department
of Biotechnology and Medical Engineering for his in depth supervision and
guidance, constant encouragement and co-operative attitude for bringing out this
thesis work.
I extend my sincere thanks to Dr. Gyana Ranjan Sathpathy, Professor and Head of
the Department, Department of Biotechnology and Medical Engineering, N.I.T.
Rourkela for giving the kind permission and providing me the necessary laboratory
facilities to carry out this work.
I am also grateful to Prof. K.Pramanik, of Department of Biotechnology and
Medical Engineering, N.I.T., Rourkela for extending full help to utilize the
laboratory facilities in Dept of Biomedical Engineering.
Finally I extend my sincere thanks to Devendra Bramh Singh, Barnali Ashe and
Vishnu, Sravanthi, Tarangini, Kaleshwar, Ramakrishna, Chaitanya, Jeevan,
Ashutosh, Tulsi, Sahitya, Ramya , Nadeem, Srinivas and to all those who have
helped me during my dissertation work and have been involved directly or indirectly
in my endeavor.
And it goes without saying, that I am indebted to my parents Mr. Ranga Rao and
Mrs. Parvathi and Brother M. Aditya, whose patience, support and endurance
made completion of my course a reality.
M Archana
Roll No-207BM202
29
CONTENTS
ACKNOWLEDGEMENT
i
LIST OF TABLES
ii
LIST OF FIGURES
iii
ABBREVIATIONS
iv
ABSTRACT
v
Page No
1.
INTRODUCTION
1-3
2.
LITERATURE SURVEY
4-23
2.1
Proteins
4
2.2
Protein Folding
4
2.3
Protein Unfolding/Denaturation
6
2.4
2.5
2.3.1
Loss of Solubility
6
2.3.2
Increased Proteolysis
7
2.3.3
Loss of Biological Activity
7
2.3.4
Spectroscopic procedures
7
Causes of Denaturation
8
2.4.1
Thermal Denaturation
8
2.4.2
pH Denaturation
9
2.4.3
Changes in dielectric constant
10
2.4.4
Denaturation at interfaces
11
2.4.5
Ionic Strength
12
2.4.6
Chemical Denaturants
13
2.4.6.1 Urea
13
2.4.6.2 Guanidium Hydrochloride
13
Cytochrome c reductase
14
2.5.1
Role of cytochrome c in mitochondrial ETC
14
2.5.2
Role of cytochrome c in cancer
17
30
2.6
Osmolytes and their role in the stabilization of protein structure
21
2
MATERIALS AND METHODS
24-27
3.1
General
24
3.2
Chemicals
24
3.3
Glassware and Apparatus
24
3.4
Culture of Breast cancer Cell lines
24
3.5
Media Optimization.
25
3.6
Cell subculture
25
3.7
Viability test (Trypan blue staining)
25
3.8
SDS-PAGE Analysis for analysis of protein expression
25
3.9
Preparation of cell lysate
25
3.10
Determination of cyt-c reductase activity
26
3.11
Unfolding of breast cancer cell line cyt-c reductase by
chemical denaturants like urea and GdnHCl
3.12
Unfolding of breast cancer cell line cyt-c reductase
27
4
RESULTS
28-38
4.1
Cell Culture picture
28
4.2
Effect of temperature on enzymatic activity of cyt-c reductase
and its protection by different osmolytes like trehalose, DMSO,
glycerol and sucrose.
4.3
29
Effect of urea on denaturation of cyt-c reductase
and its protection by different osmolytes like sucrose,
trehalose, DMSO and glycerol.
4.4
32
Effect of GdnHCl on denaturation of cyt-c reductase
and its protection by different osmolytes like sucrose,
trehalose, DMSO, and glycerol.
4.5
35
Effect of GdnHCl, urea on denaturation of cyt-c reductase
and its protection by MgCl2
37
31
5
DISCUSSION
39-41
6
CONCLUSION
42-43
BIBLIOGRAPHY
44-47
MISCELLANEOUS
48-60
32
LIST OF TABLES
Page No
Table 1: List of Instruments used during the whole experiment their manufacturer
48
and function
Table 2: Percentage Residual activity of Cyt-c reductase at 60◦C
31
Table 3: Percentage Residual Activity of cyt-c reductase when it is denatured
at 1M Urea
34
Table 4: Percentage Residual Activity of cyt-c reductase when it is denatured
at 1M GdnHcl
37
Table 5: Percentage Relative Activity of cyt-c reductase under the application
of Temperature in the presence of trehalose at different molarities.
49
Table 6: Percentage Relative Activity of cyt-c reductase under the application
of Temperature in the presence of DMSO at different molarities.
49
Table 7: Percentage Relative Activity of cyt-c reductase at different temperatures
in the presence of glycerol
50
Table 8: Percentage Relative Activity of cyt-c reductase in the presence and
absence of sucrose at different temperatures.
50
Table 9: Percentage relative activity of cyt-c reductase in the presence and
absence of glycerol at different molarities of urea.
51
Table 10: Percentage Relative Activity of cyt-c reductase under the application
Of various Molarity of urea in the presence of urea and
sucrose at different molarities
52
Table 11: Percentage Relative Activity of cyt-c reductase under the application of
various Molarity of urea in the presence of urea and
33
Mgcl2 at different molarities
53
Table 12: Percentage Relative Activity of cyt-c reductase under the application
of various Molarity of Urea in the presence of Urea and
trehalose at different molarities.
54
Table 13: Percentage Relative Activity of cyt-c reductase under the application
of various Molarity of Urea in the presence of Urea and
DMSO at different molarities
55
Table 14: Percentage Relative Activity of cyt-c reductase under the application
of various molarities of GdnHcl in the presence of GdnHcl and
glycerol at different molarities
56
Table 15: Percentage Relative Activity of cyt-c reductase under the application
of various Molarity of GdnHcl in the presence of GdnHcl and
Mgcl2 at different molarities
57
Table 16: Percentage Relative Activity of cyt-c reductase under the application
of various Molarity of GdnHcl in the presence of GdnHcl and
sucrose at different molarities
58
Table 17: Percentage Relative Activity of cyt-c reductase under the application
of various Molarity of GdnHcl in the presence of GdnHcl and
trehalose at different molarities
59
Table 18: Percentage Relative Activity of cyt-c reductase under the application
of various Molarity of GdnHcl in the presence of GdnHcl and
DMSO at different molarities
60
34
LIST OF FIGURES
Page No
Fig.1 Representation of ETC
15
Fig.2 Regulation of apoptosis by the redox state of cytosolic cyt-c
20
Fig.3 MDA MB 231 cell pictures at 40x magnification
28
Fig.4 MDA MB 231 cell pictures at 10x magnification
28
Fig.5 Thermal Unfolding of breast cancer cellular cyt-c reductase.
29
Fig.5a: Thermal Unfolding of Breast cancer (MDA-MB231)
Cyt-c reductase in presence of different concentration of glycerol
30
Fig.5b: Thermal Unfolding of Breast cancer (MDA-MB231)
Cyt-c reductase in presence of different concentration of sucrose
30
Fig.5c: Thermal Unfolding of Breast cancer (MDA-MB231)
Cyt-c reductase in presence of different concentration of trehalose
30
Fig.5d: Thermal Unfolding of Breast cancer (MDA-MB231)
Cyt-c reductase in presence of different concentration of DMSO
Fig.6 Urea based unfolding of Cyt-c reductase
30
32
Fig.6a: Unfolding of breast cancer (MDA-MB231) cyt-c reductase
using urea in presence of different concentration of glycerol
33
Fig.6b: Unfolding of breast cancer (MDA-MB231) cyt-c reductase
using urea in presence of different concentration of sucrose
33
Fig.6c: Unfolding of breast cancer (MDA-MB231) cyt-c reductase
using urea in presence of different concentration of trehalose
33
Fig.6d: Unfolding of breast cancer (MDA-MB231) cyt-c reductase
using urea in presence of different concentration of DMSO
Fig.7: GdnHcl based unfolding of Cyt-c reductase
33
35
Fig.7a. Unfolding of Breast cancer (MDA-MB231) cyt-c reductase
using GdnHCl in presence of different concentration of glycerol
Fig.7b. Unfolding of Breast cancer (MDA-MB231) cyt-c reductase
35
35
using GdnHCl in presence of different concentration of sucrose
35
Fig.7c. Unfolding of Breast cancer (MDA-MB231) cyt-c reductase
using GdnHCl in presence of different concentration of trehalose
36
Fig.7d. Unfolding of Breast cancer (MDA-MB231) cyt-c reductase
using GdnHCl in presence of different concentration of DMSO
36
Fig.8. Unfolding of Breast cancer (MDA-MB231) cyt-c reductase using urea
in presence of different concentration of Mgcl2
37
Fig.9 Unfolding of Breast cancer (MDA-MB231) cyt-c reductase using
GdnHCl in presence of different concentration of Mgcl2
36
38
ABBREVIATIONS
SDS
Sodium dodecyl sulphate
PAGE
Poly acrylamide gel electrophoresis
GdnHCl
Guanidium Hydrochloride
cyt-c
Cytochrome c
DMSO
Dimethyl Sulphoxide
MgCl2
Magnesium Chloride
NADH
Nicotinamide adenine dinucleotide
DTT
Dithiothreitol
TMAO
Trimethylamine N-oxide
ATP
Adenosine triphosphate
U.V
Ultraviolet
KDa
Kilodalton
NaCl
Sodium chloride
CK
Creatine kinase
PEG
Poly ethylene glycol
FBS
Fetal bovine serum
EDTA
Ethylene Diamine Tetra acetic acid
NCCS
National Centre For Cell Science
SRL
Sisco Research Laboratories
PBS
Phosphate buffered saline
KH2PO4
Potassium Dihydrogen phosphate
37
Na2HPO4
Disodium hydrogen phosphate
KCl
Potassium chloride
STI
Soybean trypsin inhibitor
Rpm
Revolutions per minute
EGTA
Ethylene glycol tetra acetic acid
PMSF
Phenylmethanesulphonylfluoride
TMPD
Tetramethylphenylenediamine
GSH
Reduced glutathione
COX
Cytochrome oxidase
Cyt. cox
Cytochrome c oxidized form
Apaf-1
Apoptotic peptidase activating factor 1
Cyt. c red.
Cytochrome c reduced form
38
ABSTRACT
In the present investigation, we have used cytochrome c reductase (cyt-c) to understand its
unfolding process and also to study the protective ability of osmolytes on the unfolding process
of cyt- c reductase. Our study mainly aims at understanding the stabilization of cyt-c reductase
since its plays a major role in mitochondrial electron transport chain as well as in the apoptosis
of the cell. It catalyzes the conversion of cyt-c oxidized form to reduced form. Cyt- c reductase
was derived from breast cancer cell line MDA MB 231 and its unfolding process was studied
thermally and in the presence of chemical denaturants like urea and guanidium hydrochloride
(GdnHCl). The study was also carried out in presence of various osmolytes like glycerol,
dimethysulfoxide (DMSO), sucrose and trehalose. Salt like MgCl2 was also added to the cell
lysate to observe the effect of the conformational state of cyt- c reductase. The extent of
unfolding of the protein was expressed in terms of the biological activity retained by the enzyme
through measuring the change in the absorbance at 550nm due to the conversion of cy-c oxidized
form to reduced form. Results indicated that under the application of heat the enzyme started
unfolding at 40◦C and the enzyme showed an excess biological activity (150% ) till 50◦C and
then the activity decreased sharply and dropped to zero at 60◦C indicating the complete
denaturation of the enzyme. During thermal unfolding of the enzyme, 0.5M glycerol showed
100% activity at 60◦C whereas trehalose protected at 0.75M and 1M concentration and helped
cyt-c reductase to retain approximately 75-85% activity. At 80◦C the enzyme activity was lost
completely and the osmolytes also did not show any protection. During unfolding of cyt-c
reductase by urea, glycerol at 0.72M showed 102% activity at 0.5M urea concentration and at
1M urea concentration, sucrose at 0.25M, 0.5M retained 114% and 106% activity. Sucrose
showed protective effect only at 0.25M and 0.5M urea concentrations. Tehalose and DMSO in
the concentrations 0.25M, 0.5M, 0.75M, and 1M showed a protective effect against denaturation
by urea whereas the increasing concentrations of other osmolytes had a little protective effect on
activity of cyt-c reductase. During unfolding of cyt-c reductase by GdnHCl, at higher
concentrations of glycerol like at 4M, the enzyme was not denatured at 2M and rather 20%
enzyme activity was retained. Trehalose at 0.25M, 0.5M.0.75M and 1M concentrations showed
an enhanced activity from 20% to 40% at 2M GdnHCl concentration and maximum protection
by trehalose was observed at 0.125M GdnHCl concentration nearly up to 90%. Sucrose had
39
shown protective effect only at 1M concentration of GdnHCl. At 1M, 2M concentrations of
GdnHCl, 0.75M, 1M DMSO showed highest protection nearly by 80%. DMSO is showing
maximum protection at 2M concentration of GdnHCl where no other osmolytes had this much
protection as DMSO. Salt like MgCl2 were also used to observe its stabilizing effect on the
activity of cyt-c reductase.
Keywords: cytochrome c reductase; thermal Unfolding; guanidine hydrochloride; urea; sucrose;
glycerol; DMSO; trehalose.
Abbreviations: GdnHCl, guanidine hydrochloride; DMSO, dimethysulfoxide.
40
CHAPTER 1
INTRODUCTION
41
1. INTRODUCTION
Cytochrome c (cyt-c) reductase also known as ubiquinone cyt-c reductase, NADH
dehydrogenase, cytochrome bc1 complex (Complex III), has been determined by Johann
Deisenhofer and his colleagues. (21) It is the major enzyme involved in mitochondrial electron
transport chain. Under different conditions of denaturing stress it should be stable in order to
complete the process of mitochondrial electron transport chain for the supply of ATP to the cell.
So studying this enzyme under different stress is crucial. It catalyses the conversion of
cytochrome c oxidized form to reduced form in the presence of NADH as an acceptor.
Cytochrome c oxidized form has a major role in apoptosis. Cytochrome c reductase converts the
formed cyt-c oxidized form to reduced form and hence cannot escape mitochondrial membrane
and hence inhibits apoptotic pathway. In this way the enzyme regulate the apoptosis of cells in
various conditions depending upon the requirement. Therefore studies have been performed on
this enzyme in MDA MB 231 cell lines. In these cells the activity of the enzyme has been
measured and in general the activity of the enzyme is higher in cancer cells than to normal cells.
Activity of cyt-c reductase has been measured in the presence and absence of osmolytes under
denaturing conditions (heat, urea, GdnHCl) to study its unfolding process and protective ability
of osmolytes.
Denaturation of an enzyme is commonly defined as any noncovalent change in the
structure of a protein. This change may alter the secondary, tertiary or quaternary structure of the
molecules. The tertiary and quaternary structures of proteins are fragile and tend to come apart
under conditions less than optimal. This loss of shape, called denaturation, occurs as ionic and Hbonds break. Salty environments (excess Na+ and Cl-), acidic environments (too much H+), and
alkaline environments (too little H+) break ionic and H- bonds interfering with their electric
charges. Heat causes movement within molecules, which disturb their relatively weak bonds.
Once denaturated, most proteins will not re-form their original shape. (Proteins usually exposed
to unusual environments have tertiary and quaternary structures held by covalent bonds, between
the sulfur atoms of cysteine.) Enzymes are biomolecules that catalyze (i.e., increase the rates of)
chemical reactions. [1][2]. Enzyme stability is the major factor for many industrially important
enzymes. An industrial disadvantage of the most commercially used biocatalysts enzymes and
enzyme complexes - is their relatively low stability. Enzymes are generally globular proteins and
42
range from just 62 amino acid residues in size, for the monomer of 4-oxalocrotonate
tautomerase,[3] to over 2,500 residues in the animal fatty acid synthase. [4].
It has been reported that the stability and activity of the enzymes can be improved by
using osmolytes. Osmolytes are a series of different kinds of small molecules that can maintain
the correct conformation of protein by acting as molecular chaperons. Almost every functional
protein is the product of folding a disordered polypeptide into a specific three-dimensional
conformation. During the process of protein folding, various small compounds called “chemical
chaperones” play important roles in forming the correct protein conformation and protecting it
against thermal denaturation and aggregation [5-8]. These compounds including a variety of
polyols, sugars, polysaccharides, organic solvents, some amino acids and their derivatives seem
to improve the ability of cells to adapt to different metabolic insults due to the compounds’
stabilizing effects on the protein conformation [9–12]. Most of such compounds have no
substrate specificity, but some of them specifically stabilize certain proteins [13]. Several recent
reports about the function of osmolytes in the unfolding process of some proteins both in vivo
and in vitro have been published [14,15].Besides protecting the conformation of proteins,
osmolytes are known to stabilize proteins against aggregation. Protein aggregation is a frequently
observed phenomenon during protein unfolding or refolding, which was recognized mainly as
the result of nonspecific interactions between the hydrophobic regions of the polypeptide chains
[16]. Because preventing aggregation is important during protein purification, new approaches
based on “artificial chaperones” have been introduced to prevent aggregation by aiding protein
folding [17].Some osmolytes are known to stabilize proteins against aggregation; these are
recognized as protein folding helpers [18] and they are classified as ‘compatible osmolytes’,
including polyols and free amino acids, and the ‘counteracting osmolytes’ such as glycine
betaine and trimethylamine N-oxide (TMAO) [19]. Compatible osmolytes protect proteins that
are subjected to threatening conditions such as extreme temperature fluctuations, excessive
dryness or high salt environments [20], while the counteracting osmolytes protect cellular
proteins against urea inactivation [14]. Compatible and counteracting osmolytes may have
different mechanisms for protecting proteins because of the variety of the relevant environmental
stresses [20].
In our present investigation we have studied the unfolding characteristics of cyt-c
reductase under various conditions of heat, urea and GdnHCl and also the studies have been
43
performed on the protective ability of osmolytes like glycerol, sucrose, DMSO and trehalose on
the biological activity retained by the enzyme under these stress conditions. Salt like MgCl 2 was
also used to study its protective ability on the activity of cyt-c reductase.
OBJECTIVE
Our main aim of research was
 To study the unfolding process of cytochrome c reductase under conditions of denaturing
stress like heat, urea and GdnHCl in breast cancer cell line MDA MB 231 to understand
the conformational state of the enzyme.
 The work also involved the study of protective capability of osmolytes like trehalose,
DMSO, glycerol and sucrose on the unfolding of cyt-c reductase under denaturing
conditions of heat and in the presence of chemical denaturants like urea and GdnHCl.
 Salt like MgCl2 were also used to study its protective ability on the biological activity
retained by the enzyme under conditions of denaturation. (heat, urea, GdnHCl).
44
CHAPTER 2
LITERATURE SURVEY
45
2. Literature survey
2.1 Proteins
Proteins are natural polymer molecules consisting of amino acid units. The number of
amino acids in proteins may range from two to several thousand. Proteins are the basis for the
major structural components of animal and human tissue. They are very important molecules in
our cells. They are involved in virtually all cell functions. Each protein within the body has a
specific function. Some proteins are involved in structural support, while others are involved in
bodily movement, or in defense against germs. Proteins vary in structure as well as function.
They are constructed from a set of 20 amino acids and have distinct three-dimensional shapes.
They serve as enzymatic catalysts, are used as transport molecules (hemoglobin transports
oxygen) and storage molecules (iron is stored in the liver as a complex with the protein ferritin);
they are used in movement (proteins are the major component of muscles); they are needed for
mechanical support(skin and bone contain collagen-a fibrous protein); they mediate cell
responses (rhodopsin is a protein in the eye which is used for vision); antibody proteins are
needed for immune protection; control of growth and cell differentiation uses proteins
(hormones).
2.2 Protein Folding
Molecular assembly and molecular disassembly are essential procedures of life. The
synthesis of complex structures from a finite set of raw materials underlies the prodigious
complexity of life on earth. Control of molecular synthesis is achieved by enzymes, an elite class
of protein molecules. Enzymes know how to grab other molecules and break them apart or stick
them together, according to the very specific blueprint, contained in DNA molecules. The
importance of protein folding has been recognized for many years. Almost a half-century ago,
Linus Pauling discovered two quite simple, regular arrangements of amino acids. the α-helix and
the β-sheet protein. And in the early 1960s, Christian Anfinsen showed that the proteins actually
tie themselves: If proteins become unfolded, they fold back into proper shape of their own
accord; no shaper or folder is needed (23)
One of the most important results in understanding the process of protein folding was a
thought-provoking experiment that was carried out by Christian Anfinsen and colleagues in the
early 1960s. They investigated a protein called ribonuclease, which they isolated from the
46
pancreatic tissue of cattle. This enzyme, made up of 124 amino acids, cleaves any ribonucleic
acid (RNA) that could be harmful to the cell, such as truncated RNA that would not make a fully
operational protein. To do this although this was not known in Anfinsen’s time it briefly binds
RNA in a binding site and requires several sulphur-containing amino-acid cysteine residues in
the protein, which form bonds with each other (called disulphide bridges) and hold the protein
structure together.
Ribonuclease can be denatured by adding certain chemicals or by heat. The disulphide
bridges break and other forces of attraction between amino acids disappear, which makes the
enzyme collapse into a tangled, useless ball. In various studies, Anfinsen showed that this
denaturation process could be completely reversed by removing these denaturing chemicals or by
lowering the temperature. The ribonuclease then folds back to its natural functional state on its
own. So, Anfinsen concluded that the amino-acid sequence determines the shape of a protein, a
finding for which Anfinsen received the Nobel Prize in Chemistry in 1972.
Much interest is currently focused on the rapid and faithful folding of proteins from a
one-dimensional sequence of amino acids in a random coil, to a three-dimensional biologically
functional structure in the native state (24–28). Chemical and thermal denaturation of proteins
are standard techniques in protein biochemistry to determine protein folding and unfolding
equilibria and kinetics (26, 28, 29).A general view of protein folding is that it begins with
hydrophobic collapse, in which the random coil changes to a compact state, with the
hydrophobic groups in the interior region and polar groups at the surface interacting with the
surrounding water. The packing is not yet optimal, with hydrophobic groups somewhat free to
slide about in the interior of the globule, until residues are locked in place by the formation of
specific hydrogen bonds. These hydrogen bonds can be regarded as a sort of Velcro that locks
the various structural elements in the folded protein together. Once these interactions are
optimized, the native state is predominantly rigid with flexible hinges or loops at the surface—
the number and distribution of these depending on the particular protein.
Protein unfolding studies have a new urgency, as they begin to understand that the havoc
wrought by dementias such as Alzheimer's disease, and the threat posed by the spongiform
encephalopathy’s such as BSE, is a direct result of protein misfolding. If we can understand how
proteins fold, the puzzles of misfolding will be that much more tractable.
47
UK scientists have developed a more sensitive technique for studying how peptides unfold. They
expect that the method will be useful in fundamental research on protein structure and in
understanding conditions such as Alzheimer's disease. Ewan Blanch from the University of
Manchester and his colleagues used Raman spectroscopy to analyze protein model poly
(glutamic acid) as it unfolded in response to increasing pH. The scientists discovered two distinct
phases as poly (glutamic acid) goes from the helical to the disordered state. They speculate that
this is because the end and central helix regions have different thermodynamic stabilities. The
group combined normal and chiral Raman spectroscopies to examine the protein model. (30)
2.3 Protein Unfolding/Denaturation
Protein denaturation is commonly defined as any noncovalent change in the structure of a
protein. This change may alter the secondary, tertiary or quaternary structure of the molecules.
Since denaturation reactions are not strong enough to break the peptide bonds, the primary
structure (sequence of amino acids) remains the same after a denaturation process. Denaturation
disrupts the normal alpha-helix and beta sheets in a protein and uncoils it into a random shape.
Denaturation occurs because the bonding interactions responsible for the secondary
structure (hydrogen bonds to amides) and tertiary structure are disrupted. In tertiary structure
there are four types of bonding interactions between "side chains" including: hydrogen bonding,
salt bridges, disulfide bonds, and non-polar hydrophobic interactions which may be disrupted.
Therefore, a variety of reagents and conditions can cause denaturation (22) Denaturation is
largely dependent upon the method utilized to observe the protein molecule. Some methods can
detect very slight changes in structure while other requires rather large alterations in structure
before changes are observed. It includes the following methods.
2.3.1 Loss of Solubility
One of the oldest methods utilized to follow the course of denaturation was to measure
changes in solubility. The loss of solubility can be related to the loss of a great number of
desirable characteristics of the protein. When more sophisticated techniques are utilized many
changes in protein structure that eventually result in a loss of solubility can be detected. In these
cases the loss of solubility is more properly regarded as an effect of denaturation rather than as a
measure of denaturation.
48
2.3.2 Increased Proteolysis
It has been known that a variety of procedures that alter protein's structures make them
more susceptible to proteolysis. The rate and extent of proteolysis can be utilized as an indicator
of protein denaturation. In many cases, increases in proteolysis, like decreases in solubility, are
the result of many changes in protein structure. In a series of experiments on ribonuclease,
Burgess and Scheraga exposed this protein to a variety of combinations of pH and temperature.
The molecule was then mixed with one of three different proteases. Under conditions of mild
denaturation, they were able to observe which portions of the molecule were made susceptible to
proteolysis first. Increasingly harsh treatments exposed other portions of the molecule to the
action of the proteases. From these observations and knowledge of the tertiary structure of the
molecule, they were able to hypothesize a pathway for the thermal denaturation of ribonuclease.
This pathway was assumed to be the reverse of the pathway for protein folding, but there was no
evidence for this to be the case.
2.3.3 Loss of Biological Activity
Changes in the rate of the reaction, the affinity for substrate, pH optimum, temperature
optimum, specificity of reaction, etc., may be affected by denaturation of enzyme molecules.
Loss of enzymatic activity can be a very sensitive measure of denaturation as some assay
procedures are capable of detecting very low levels of product. In some cases the loss of activity
can be shown to occur only after some other changes in structure can be observed by other
procedures. There may technically, then be denaturation of the protein before loss of activity
occurs.
2.3.4 Spectroscopic Procedures
A variety of procedures have been developed that measure the interaction of
electromagnetic radiation with molecules. Some of these procedures have proven to be very
useful in the study of protein denaturation.
One such procedure is ultraviolet adsorption spectroscopy. This simply measures the wavelength
of and the amount of ultraviolet radiation absorbed by a molecule. In proteins, both the
wavelength and extent of absorption depend on the amino acids present and on their physical
environments. There are a large number of such groups in a protein molecule and thus its U.V.
49
spectrum quite often lacks detail. Under some circumstances however, these groups can absorb
at a low wavelength, generally in the U.V., and then emit light at a larger wavelength. This
process is known as fluorescence and is quite sensitive to the environment of the groups
involved.
Both ultraviolet and fluorescence spectroscopy have been utilized to follow changes in
the environments of various groups within protein molecules. Such changes in environment
reflect changes in protein structure and thus denaturation.
Out of all these methods we have selected spectroscopic method to observe the
denaturation of the enzyme by checking its biological activity.
2.4 Causes of Denaturation
Denaturation of enzyme occurs due to following reasons
2.4.1ThermalDenaturation
When proteins are exposed to increasing temperature, losses of solubility or enzymatic
activity occurs over a fairly narrow range. Depending upon the protein studied and the severity
of the heating, these changes may or may not be reversible.
As the temperature is increased, a number of bonds in the protein molecule are
weakened. The first affected are the long range interactions that are necessary for the presence of
tertiary structure. As these bonds are first weakened and are broken, the protein obtains a more
flexible structure and the groups are exposed to solvent. If heating ceases at this stage the protein
should be able to readily refold to the native structure. As heating continues, some of the
cooperative hydrogen bonds that stabilize helical structure will begin to break. As these bonds
are broken, water can interact with and form new hydrogen bonds with the amide nitrogen and
carbonyl oxygens of the peptide bonds. The presence of water further weakens nearby hydrogen
bonds by causing an increase in the effective dielectric constant near them. As the helical
structure is broken, hydrophobic groups are exposed to the solvent.
The effect of exposure of new hydrogen bonding groups and of hydrophobic groups is to
increase the amount of water bound by the protein molecules. The unfolding that occurs increase
the hydrodynamic radius of the molecule causing the viscosity of the solution to increase. The
net result will be an attempt by the protein to minimize its free energy by burying as many
hydrophobic groups while exposing as many polar groups as possible to the solvent. While this is
50
analogous to what occurred when the protein folded originally, it is happening at a much higher
temperature. This greatly weakens the short range interaction that initially direct protein folding
and the structures that occur will often be vastly different from the native protein.
Upon cooling, the structures obtained by the aggregated proteins may not be those of
lowest possible free energy, but kinetic barriers will prevent them from returning to the native
format. Any attempt to obtain the native structure would first require that the hydrophobic bonds
that caused the aggregation be broken. This would be energetically unfavorable and highly
unlikely. Only when all the intermolecular hydrophobic bonds were broken, could the protein
begin to refold as directed by the energy of short range interactions. The exposure of this large
number of hydrophobic groups to the solvent, however, presents a large energy barrier that make
such are folding kinetically unlikely.
Exposure of most proteins to high temperatures results in irreversible denaturation. Some
proteins, like caseins, however, contain little if any secondary structure and have managed to
remove their hydrophobic groups from contact with the solvent without the need for extensive
structure. This lack of secondary structure causes these proteins to be extremely resistant to
thermal denaturation.
The increased water binding noted in the early stages of denaturation may be retained
following hydrophobic aggregations. The loss of solubility that occurs will greatly reduce the
viscosity to a level below that of the native proteins. The effect of thermal denaturation on the
functional properties of specific proteins will be discussed in subsequent chapters.
2.4.2 pH Denaturation
Most proteins at physiological pH are above their isoelectric points and have a net
negative charge. When the pH is adjusted to the isoelectric point of the protein, its net charge
will be zero. Charge repulsions of similar molecules will be at minimum and many proteins will
precipitate. Even for proteins that remain in solution at their isoelectric points, this is usually the
pH of minimum solubility.
If the pH is lowered far below the isoelectric point, the protein will lose its negative and
contain only positive charges. The like charges will repel each other and prevent the protein from
aggregating as readily. In areas of large charge density, the intramolecular repulsion may be
great enough to cause unfolding of the protein. This will have an effect similar to that of mild
51
heat treatment on the protein structure. In some cases the unfolding may be extensive enough to
expose hydrophobic groups and cause irreversible aggregation. Until this occurs such unfolding
will be largely reversible.
Some proteins contain acid labile groups and even relatively mild acid treatment may
cause irreversible loss of function. This generally results from the breaking of specific covalent
bonds and thus should be considered separately from denaturation. Exposure to strong enough
acid at elevated temperatures will first release amide nitrogen from glutamine and asparagine
groups and eventually lead to hydrolysis of peptide bonds.
The effects of high pH are analogous to those of low pH. The proteins obtain a large
negative charge which can cause unfolding and even aggregation. The use of high pH to
solubilize and alter protein structure is very important to the formation of fibers from proteins of
plant origin
A number of reactions can cause chemical modification of proteins at alkaline pH's that
are commonly encountered in protein processing. Many of these involve cysteine residues.
Perhaps the most important are the base catalyzed beta eliminations of sulfur to yield
dehydroalanine which can react with lysine to form lysinoalanine. This result in a loss of
nutritive value of the protein and the products of the reaction may be toxic. Exposure of protein
molecules to high pH should be minimized as much as is possible. Exposure to very high pH at
elevated temperatures results in alkaline hydrolysis of peptide bonds
.
2.4.3 Changes in Dielectric Constant
The addition of a solvent that is miscible with water, but that is less polar will lower the
dielectric constant of the system. This will tend to increase the strength of all electrostatic
interactions between molecules that were in contact with water. Many of the protein hydrogen
bonds are effectively removed from the solvent and will not be affected. The presence of the less
polar solvent will also have the effect of weakening the hydrophobic bonds of the proteins. These
bonds depend upon an increase in the order of water when they are broken for their existence. As
there is less water in the system, this becomes less important and at some level of replacement,
these groups are at a lower energy level when in contact with the solvent.
The structure of the protein will be changed and hence, it will be denatured. The
reversibility of the process depends to a large extent on the nature of the non-polar solvent, the
52
extent of unfolding the temperature of the system and the rate of solvent removal. When large
amounts of the solvent are present, the protein will be largely unfolded with extensive exposure
of the hydrophobic groups. If the protein could be instantaneously transferred to pure water at
room temperature, the protein would most likely aggregate and precipitate. The sudden exposure
of the hydrophobic groups to water would cause them to try to remove themselves from the
aqueous phase as soon as possible. Even before the short range interactions could redirect the
folding of the protein aggregation would occur.
If the solvent exchange were slow, there would be a better chance that the hydrophobic
groups would be able to return to the interior of the molecule and prevent aggregation. If the
exchange occurred at low temperatures, the chances of regaining the native structure would be
even better. At low temperatures, the hydrophobic groups may in part be stable in the aqueous
phase or at least not as unstable. In this case, the removal of the solvent has little affected. When
the temperature is subsequently increased, the normal course of protein refolding can occur.
Solvent precipitation is often utilized as a means of purifying and concentrating enzymes. It is
extremely important that both the solvent and the protein solution be cold when they are mixed
and that the subsequent removal of the solvent be performed at reduced temperature. This helps
to insure the recovery of enzyme activity.
2.4.4 Denaturation at Interfaces
When proteins are exposed to either liquid-air or liquid-liquid interfaces, denaturation can
occur. As a liquid-liquid interface, the protein comes into contact with a hydrophobic
environment. If allowed to remain at this interface for a period of time proteins will tend to
unfold and place as many of their hydrophobic groups as possible in the non-aqueous layer while
maintaining as much charge as possible in the water layer.
To understand why protein unfolds at hydrophobic interfaces, it must be realized that the
tertiary structure of a protein is not rigid. There are continued fluctuations about an average
configuration. Any change in conformation that yields a higher energy state will spontaneously
go back to the state of lowest energy. As a part of this process, hydrophobic groups will
occasionally be positioned so that they have increased contact with the aqueous phase. When this
occurs, these groups will assume the configuration of lowest free energy and will be removed
from the water. If a hydrophobic group is exposed while a protein is in contact with a polar
53
solvent, these groups will find a state of lower energy exists if they enter into the solvent phase.
This will continue to occur until random fluctuations in protein structure can no longer yield a
configuration of lower free energy.
The amount of unfolding that occurs at such an interface will depend on how rigid the
three-dimensional protein structure is an on the number and location of hydrophobic groups in
the molecule. A flexible, non-cross linked protein will be able to unfold easier than will a highly
structured and cross linked one. If energy is applied to cause shear, the process will be
accelerated. The shear can cause the protein to unfold, thus exposing its hydrophobic groups to
the nonaqueous phase. It can also increase the interfacial area between the two phases and allow
more proteins to come into contact with the nonaqueous phase.
This unfolding is essentially non-reversible because of the large energy barriers. Even if
the phases should separate and the protein is forced into the aqueous phase the protein will not
regain its original structure. Rather an association of hydrophobic groups will cause the protein
to aggregate.
The same forces are in operation when a protein migrates to a liquid-air interface.
Hydrophobic groups tend to associate in the air and the protein unfolds. The presence of shear
causes to help unfold the protein and to introduce more air into the solution. Both of these effects
can be minimized by keeping the temperature low (to weaken hydrophobic bonds) and by
minimizing the interfacial area. If the interface is limited, then only a small amount of protein
will be able to denature. The presence of this denatured protein will serve as a barrier to further
denaturation. Proteins are often utilized in food products to stabilize emulsions or to incorporate
air. These cases will be examined in more detail when emulsions and foams are discussed.
2.4.5 Ionic Strength
Proteins are usually more soluble in dilute salt solutions than in pure water. The salts are
thought to associate with oppositely charge groups in the protein. This combination of charged
groups bonds more water than do the charged groups alone and protein hydration is increased.
With most proteins there is little change in solubility as more salt is added until some very high
salt content is reached. At very high levels of salt there is a competition between the ions and the
proteins for water of hydration.
54
When the salt concentration is high enough, the proteins will be sufficiently dehydrated
to lose solubility. Removal of the salt or dilution to a low enough concentration will usually
result in the recovery of native structure.
2.4.6 Chemical Denaturants
2.4.6.1 Urea
Urea is known to be a strong protein denaturant. Although it is widely used to unfold and study
proteins in the lab, the molecular mechanism of urea-induced protein unfolding still remains
unidentified. The denatured structures in both urea and at high temperature contained residual
native helical structure, whereas the β-structure was completely disrupted for the enzyme
chymotrysin. The average residence time for urea around hydrophilic groups was six times
greater than around hydrophobic residues and in all cases greater than the corresponding water
residence times. Water self-diffusion was reduced 40% in 8 M urea. Urea altered water structure
and dynamics, thereby diminishing the hydrophobic effect and encouraging Solvation of
hydrophobic groups. In addition, through urea's weakening of water structure, water became free
to compete with intraprotein interactions. Urea also interacted directly with polar residues and
the peptide backbone, thereby stabilizing nonnative conformations. These simulations suggest
that urea denatures proteins via both direct and indirect mechanisms. (38) There are two
mechanisms by which urea are thought to destabilize proteins. Urea may interact with backbone
peptide groups through the formation of hydrogen bonds. The crystal structure of the
diketopiperazine urea complex shows that the diketopiperazine molecule forms several hydrogen
bonds with urea molecules. Solvation of the diketopiperazine by urea is so extensive that there
are no hydrogen bonds between diketopiperazine molecules in the crystal (47)
2.4.6.2 GdnHCl
GdnHCl is an electrolyte with a pKa of about 11,which means that at pH values below
this the GdnHCl molecule will be present in a fully dissociated form, i.e., as Gdm+ and Cl-. The
presence of these ions would influence the stabilizing properties of proteins/enzymes. Mayr and
Schmid (31) studied the effect of GdnHCl and NaCl on the thermo stabilities of RNase T1.
Addition of 0.1-1 M NaCl or 0.1 M GdnHCl resulted in an increase in the Tm of RNase T1;
however, the enhancement in thermo stability for GdnHCl was significantly smaller than that for
55
NaCl. These observations were interpreted in terms of stabilization by cation (Na+ and Gdm+)
binding to the negatively charged moieties of the RNase T1 molecule. (42)
Oscar d. Monera et (1994) al have studied to address the question of whether or not urea and
guanidine hydrochloride (GdnHCl) give the same estimates of the stability of a particular
protein. They previously suspected that the estimates of protein stability from GdnHCl and urea
denaturation data might differ depending on the electrostatic interactions stabilizing the proteins.
Therefore, 4 coiled-coil analogs were designed, where the number of intrachain and interchain
electrostatic attractions (A) was systematically changed to repulsions (R): 20A, 15ASR,
lOAIOR, and 20R. The GdnHCl denaturation data showed that the 4 coiled-coil analogs, which
had electrostatic interactions ranging from 20 attractions to 20 repulsions, had very similar
[GdnHCl]1/2,values (average of =3.5 M) and, as well, their
G, values were very close to 0 (0.2
kcal/mol). In contrast, urea denaturation showed that the values proportionately decreased with
the stepwise change from 20 electrostatic attractions to 20 repulsions (20A, 7.4 M; 15ASR, 5.4
M; IOAIOR, 3.2 M; and 20R, 1.4 M), and the
G, values correspondingly increased with the
increasing differences in electrostatic interactions (20A - 15A5R, 1.5 kcal/mol; 20A - IOAIOR,
3.7 kcal/mol; and 20A - 20R, 5.8 kcal/mol). These results indicate that the ionic nature of
GdnHCl masks electrostatic interactions in these model proteins, a phenomenon that was absent
when the uncharged urea was used. Thus, GdnHCl and urea denaturations may give vastly
different estimates of protein stability, depending on how important electrostatic interactions are
to the protein.(43) This indicates that mechanisms of GdnHCl and urea are different in
stabilizing a protein structure. Several studies performed on different enzymes also show that
mechanisms of urea and GdnHCl based denaturation are different Studies performed on Glucose
oxidase reveals that the mechanism of urea and GdnHCl based denaturation are different and
urea-induced unfolding of GOD was a two-state process with dissociation and unfolding of the
native dimeric enzyme molecule occurring in a single step. On the contrary, the GdnHCl-induced
unfolding of GOD was a multiphasic process with stabilization of a conformation more compact
than the native enzyme at low GdnHCl concentrations and dissociation along with unfolding of
enzyme at higher concentrations of GdnHCl. Comparative studies on GOD using GdnHCl and
NaCl demonstrated that binding of the Gdm+ cation to the enzyme results in stabilization of the
compact dimeric intermediate of the enzyme at low GdnHCl concentrations. (48)
56
2.5 Cytochrome c reductase
2.5.1 Role of cytochrome c reductase in mitochondrial ETC
Cells of almost all eukaryotes (animals, plants, fungi, algae, protozoa – in other words,
the living things except bacteria, archaea, and a few protists) contain intracellular organelles
called mitochondria, which produce ATP. Energy sources such as glucose are initially
metabolized in the cytoplasm. The products are imported into mitochondria. Mitochondria
continue the process of catabolism using metabolic pathways including the Krebs cycle, fatty
acid oxidation, and amino acid oxidation.
The end result of these pathways is the production of two kinds of energy-rich electron
donors, NADH and succinate. Electrons from these donors are passed through an electron
transport chain to oxygen, which is reduced to water. This is a multi-step redox process that
occurs on the mitochondrial inner membrane. The enzymes that catalyze these reactions have the
remarkable ability to simultaneously create a proton gradient across the membrane, producing a
thermodynamically unlikely high-energy state with the potential to do work. Although electron
transport occurs with great efficiency, a small percentage of electrons are prematurely leaked to
oxygen, resulting in the formation of the toxic free-radical superoxide.
The similarity between intracellular mitochondria and free-living bacteria is striking. The
known structural, functional, and DNA similarities between mitochondria and bacteria provide
strong evidence that mitochondria evolved from intracellular bacterial symbionts.
57
Fig 1 Stylized representation of the ETC. Energy obtained through the transfer of electrons (black
arrows) down the ETC is used to pump protons (red arrows) from the mitochondrial matrix into
the intermembrane space, creating an electrochemical proton gradient across the mitochondrial
inner membrane (IMM) called ΔΨ. This electrochemical proton gradient allows ATP synthase
(ATP-ase) to use the flow of H+ through the enzyme back into the matrix to generate ATP from
adenosine diphosphate (ADP) and inorganic phosphate. Complex I (NADH coenzyme Q reductase;
labeled I) accepts electrons from the Krebs cycle electron carrier nicotinamide adenine dinucleotide
(NADH), and passes them to coenzyme Q (ubiquinone; labeled UQ), which also receives electrons
from complex II (succinate dehydrogenase; labeled II). UQ passes electrons to complex III
(cytochrome bc1 complex; labeled III), which passes them to cytochrome c (cyt c). Cyt c passes
electrons to Complex IV (cytochrome c oxidase; labeled IV), which uses the electrons and hydrogen
ions to reduce molecular oxygen to water.
Four membrane-bound complexes have been identified in mitochondria. Each is an
extremely complex transmembrane structure that is embedded in the inner membrane. Three of
them are proton pumps. The structures are electrically connected by lipid-soluble electron
carriers and water-soluble electron carriers. The overall electron transport chain
NADH → Complex I → Q → Complex III → cytochrome c → Complex IV → O2
↑
Complex II
58
Complex I
Complex I (NADH dehydrogenase, also called NADH:ubiquinone oxidoreductase; EC
1.6.5.3) removes two electrons from NADH and transfers them to a lipid-soluble carrier,
ubiquinone (Q). The reduced product, ubiquinol (QH2) is free to diffuse within the membrane. At
the same time, Complex I moves four protons (H+) across the membrane, producing a proton
gradient. Complex I is one of the main sites at which premature electron leakage to oxygen
occurs, thus being one of main sites of production of a harmful free radical called superoxide.
The pathway of electrons occurs as follows:
NADH is oxidized to NAD+, reducing Flavin mononucleotide to FMNH2 in one twoelectron step. The next electron carrier is a Fe-S cluster, which can only accept one electron at a
time to reduce the ferric ion into a ferrous ion. In a convenient manner, FMNH2 can be oxidized
in only two one-electron steps, through a semiquinone intermediate. The electron thus travels
from the FMNH2 to the Fe-S cluster, then from the Fe-S cluster to the oxidized Q to give the
free-radical (semiquinone) form of Q. This happens again to reduce the semiquinone form to the
ubiquinol form, QH2. During this process, four protons are translocated across the inner
mitochondrial membrane, from the matrix to the intermembrane space. This creates a proton
gradient that will be later used to generate ATP through oxidative phosphorylation.
Complex II
Complex II (succinate dehydrogenase; EC 1.3.5.1) is not a proton pump. It serves to
funnel additional electrons into the quinone pool (Q) by removing electrons from succinate and
transferring them (via FAD) to Q. Complex II consists of four protein subunits:
SDHA,SDHB,SDHC, and SDHD. Other electron donors (e.g., fatty acids and glycerol 3phosphate) also funnel electrons into Q (via FAD), again without producing a proton gradient.
Complex III
Complex III (cytochrome bc1 complex; EC 1.10.2.2) removes in a stepwise fashion two
electrons from QH2 at the Qo site and sequentially transfers them to two molecules of
cytochrome c, a water-soluble electron carrier located within the intermembrane space. The two
other electrons are sequentially passed across the protein to the Qi site where quinone part of
ubiquinone is reduced to quinol. A proton gradient is formed because it takes 2 quinol (4H+4e-)
oxidations at the Qo site to form one quinol (2H+2e-) at the Qi site. (in total 6 protons: 2 protons
59
reduce quinone to quinol and 4 protons are released from 2 ubiquinol). The bc1 complex does
NOT 'pump' protons; it helps build the proton gradient by an asymmetric absorption/release of
protons.
When electron transfer is hindered (by a high membrane potential, point mutations or
respiratory inhibitors such as antimycin A), Complex III may leak electrons to oxygen resulting
in the formation of superoxide, a highly-toxic species, which is thought to contribute to the
pathology of a number of diseases, including aging.
Complex IV
Complex IV (cytochrome c oxidase; EC 1.9.3.1) removes four electrons from four
molecules of cytochrome c and transfers them to molecular oxygen (O2), producing two
molecules of water (H2O). At the same time, it moves four protons across the membrane,
producing a proton gradient. (Wikipedia).
2.5.2 Role of cytochrome c reductase in cancer
Cytochrome c is a respiratory protein located on the surface of the mitochondrial inner
membrane facing the intermembrane space, and plays a role in the transfer of electrons from the
cytochrome bc1 complex to cytochrome c oxidase .This protein is encoded- by a nuclear gene
and translated on cytosolic ribosomes as apocytochrome c. Apocytochrome c is subsequently
translocated into mitochondria where a haem group is attached covalently to form
holocytochrome c .Cytochrome
c is bound to an acidic component, hinge protein, of the
cytochrome bc1 complex on the inner membrane. Thus, cytochrome c is located on the surface of
the inner membrane via the hinge protein, suggesting that the hinge protein contributes to the
location of cytochrome c. A report by Okazaki M, Ishibashi Y, Asoh S, and Ohta S suggests that
overexpression of mitochondrial hinge protein, a cytochrome c-binding protein, and accelerates
apoptosis by enhancing the release of cytochrome c from mitochondria. The release of
cytochrome c is regulated by the hinge protein of the respiratory chain. (41)
Cytochrome c reductase also known as ubiquinone cyt- c reductase, NADH
dehydrogenase, and cytochrome bc1 complex (Complex III), has been determined by Johann
Deisenhofer and his colleagues. (Deisenhofer was a co-recipient of the Nobel Prize in Chemistry
for his work on the structure of a photosynthetic reaction center). Complex III is present in the
60
mitochondria of all animals and all aerobic eukaryotes and the inner membranes of most
eubacteria. Mutations in Complex III cause exercise intolerance as well as multisystem disorders.
The complex is a dimer, with each monomer consisting of 11 protein subunits and 2165
amino acid residues monomer mass, 248 kDa. The dimeric structure is pear-shaped and consists
of a large domain that extends 75Å into the mitochondrial matrix, a transmembrane domain
consisting of 13 transmembrane α-helices in each monomer and a small domain that extends 38
Å into the intermembrane space. Most of the Rieske protein (a Fe-S protein named for its
discoverer) is mobile in the crystal, and Deisenhofer has postulated that mobility of this subunit
could be required for electron transfer in the function of this complex. (39).
It catalyses the conversion of cytochrome c oxidized form to reduced form in the
presence of NADH as an acceptor. Cytochrome c oxidized form has a major role in apoptosis.
Cytochrome c reductase converts the cyt-c oxidized form to reduced form. Cytochrome c exists
in interconvertible reduced (haem Fe2+) or oxidized (haem Fe3+) forms. The structures of these
two forms are similar but there are significant differences leading to different physical properties
of compressibility, stability, solvent accessibility, radius of gyration and maximum linear
dimension. The reduced form of cytochrome c also binds less to anions, and binds less tightly to
negatively charged membranes. Because the reduced and oxidized forms of cytochrome c have
different physical and biochemical properties, one may ask whether they are equally capable of
activating the apoptosome. However, there were two reports apparently showing that the redox
state of cytochrome c was not important for caspase activation through the apoptosome, shortly
after the discovery of the role of cytochrome c in apoptosis. However, it has found that according
to Borutaite V, Brown G.C while homogenates of healthy cells reduce added cytochrome c,
those of apoptotic cells oxidize cytochrome c probably due to the cytochrome c oxidase of the
cytochrome c-permeable apoptotic mitochondria. In this way the enzyme regulate the apoptosis
of cells in various conditions depending upon the requirement. Therefore studying its unfolding
characteristics is crucial under stress as it is also an important enzyme in mitochondrial
respiration. (40) Later evidences suggests that redox state is important for apoptosis Pan et al.
found that addition of oxidized cytochrome c to cell extracts induced apoptotic activity
(measured by nuclear fragmentation), whereas addition of reduced cytochrome c had no effect.
Furthermore, addition of cytochrome c reductase completely blocked the ability of the added
oxidized cytochrome c to induce apoptotic activity. This activity was also inhibited by ascorbate,
61
glutathione, cysteine and N-acetyl-cysteine, which are all capable of reducing cytochrome c.
This work indicated that reduced cytochrome c was incapable of inducing apoptosis (at least at
the concentrations, time and ionic strength used).Suto et al. found that addition of oxidized
cytochrome c to a cytosolic extract resulted in processing and activation of both caspase 9 and
caspase 3, whereas addition of reduced cytochrome c had no effect on either processing or
activation of either caspase. They also found that addition of glutathione or cysteine to reduce the
cytochrome c inhibited the ability of added oxidized cytochrome c to activate the caspases. (40)
This enzyme has been mainly selected due to its dual role in apoptosis and mitochondrial
electron transport chain. The activity of cytochrome c reductase is more in cancer cells whereas
its activity gets reduced in apoptotic cells.
Fig.2 Regulation of apoptosis by the redox state of cytosolic cytochrome c. Cytochrome c is oxidized
by mitochondrial cytochrome oxidase (COX) and in this oxidized form (Cyt. cox) it binds to Apaf-1
forming the apoptosome which activates pro-caspase-9 leading to apoptosis. Cytosolic cytochrome c
can be reduced (Cyt. cred.) by various reductants which include superoxide, ascorbate, reduced
glutathione (GSH), some chemicals such as tetramethylphenylenediamine (TMPD) and reducing
enzymes (cytochromes b5, P450, NOS, neuroglobin, cytosolic NAD(P)H oxidases). This reduced
cytochrome c cannot activate the apoptosome, and therefore does not promote apoptosis. (40)
62
Accumulating evidence suggests that the balance between reductive and oxidative
pathways in cells determines the redox steady-state of cytochrome c and through this may
regulate caspase activation by the apoptosome. Apoptosis mediates programmed cell death, host
defence and some pathology. Regulation of apoptosis is also important to the development of
cancer and its treatment. Indeed there is evidence that activation of caspase 9 by cytochrome c is
blocked in some cancer cells. Therefore, understanding how apoptosis is regulated at various
levels may have important clinical implications. (41)
Enzyme stability can be improved by using several osmolytes. Many reports have been
published on the osmolytes showing protection against denaturation of protein. The results
presented in this work shown by Maryam Mehrabi et al (2008) shows the effects of osmolytes,
including sucrose, sorbitol and proline on the remaining activity of firefly luciferase were
measured. Heat inactivation studies showed that these osmolytes maintain the remaining activity
of enzyme and increase activation energy of thermal unfolding reaction. Fluorescence and
circular dichroism (CD) experiments showed changes in secondary and tertiary structure of
firefly luciferase, in the presence of sucrose, sorbitol and proline. The unfolding curves of
luciferase (obtained by far-UVCD spectra), indicated an irreversible thermal denaturation and
raising of the midpoint of the unfolding transition temperature(Tm) in the presence of osmolytes.
(31)
Rajesh Mishra et al (2005) also showed that increasing concentrations of polyols increase
protein stability in general, the refolding yields for CS decreased at higher polyol concentrations,
with erythritol even reducing the folding yields at all the concentrations studied. Among the
various polyols used, glycerol was the most effective in enhancing CS refolding yield and a
complete recovery of the enzyme activity was obtained at 7M glycerol and 10 g/ml protein
concentrations, a result superior to the action of molecular chaperones GroEL/ES in vitro. A
good correlation between the refolding yields and the suppression of protein aggregation by
glycerol was observed, with no aggregation detected at 7M concentration. The polyols prevented
the aggregation of CS depending on the number of hydroxyl groups in them. Stopped-flow
fluorescence kinetics experiments suggested that polyols including glycerol act much early in the
refolding process as no fast and slow phases were detectable. (32)
Unfolding studies on cyt-c reductase were not reported until now so it was interesting to study its
unfolding process and also the effects of osmolytes on its unfolding.
63
2.6 Osmolytes and their role in the stabilization of protein structure
Natural selection is believed to be an unforgiving and relentless force in the evolution of
life on earth. An organism that cannot adapt to a changing environment or an environment
hostile to cell functions is at risk as a species. So it is important to understand the mechanisms
used by plants, animals, and microorganisms in adapting to environments in the biosphere that
would ordinarily denature proteins or otherwise cause disruption of life-giving cellular processes.
These hostile environments involve such stresses as extremes of temperature, cellular
dehydration, desiccation, high extracellular salt environments, and even the presence of
denaturing concentrations of urea inside cells (33). It has been recognized for some time that
many plants, animals, and microorganisms that have adapted to environmental extremes also
accumulate significant intracellular concentrations of small organic molecules (33–35). From
these (and other) observations comes the hypothesis that these small organic molecules, called
osmolytes, have the ability to protect the cellular components against denaturing environmental
stresses (33–36). In this chapter, we seek to understand the molecular-level phenomena involving
proteins and the naturally occurring osmolytes that result in the stabilization of proteins against
denaturation stresses. In our present study we have observed the effects of glycerol, sucrose,
trehalose, and DMSO on cyt-c reductase.
Fan-Guo Meng et al (2004) has studied the ability of glycerol as protective agent against
environmental stress on creatine kinase. In their study, the effect of glycerol on protection of the
model enzyme creatine kinase (CK) against heat stress was investigated by a combination of
spectroscopic method and thermodynamic analysis. Glycerol could prevent CK from thermal
inactivation and aggregation in a concentration-dependent manner. The spectroscopic
measurement suggested that the protective effect of glycerol was a result of enhancing the
structural stability of native CK. A further thermodynamic analysis using the activated-complex
theory suggested that the effect of glycerol on preventing CK against aggregation was consistent
with those previously established mechanisms in reversible systems. The osmophobic effect of
glycerol, which preferentially raised the free energy of the activated complex, shifted the
equilibrium between the native state and the activated complex in favor of the native state. A
comparison of the inactivation rate and the denaturation rate suggested that the protection of
enzyme activity by glycerol should be attributed to the enhancement of the structural stability of
64
the whole protein rather than the flexible active site. (44) Trehalose, a non-reducing disaccharide
of glucose (α-Dglucopyranosyl- α-D-glucopyranoside), is synthesized and accumulated by
several organisms as a stress protectant under distinct conditions, as high temperature and low
water activities. Trehalose is considered to have an important role in the survival of these
organisms, stabilizing membranes and proteins in the face of stress. Indeed, this sugar has been
described to act as the best stabilizer of structure and function of several macromolecules
submitted to different stress conditions. Although the molecular mechanism by which trehalose
promotes these stabilization effects has not yet been elucidated, it was proposed that trehalose,
more efficiently than other sugars, is totally excluded from the hydration shell of the proteins
studied in a phenomenon called preferential exclusion. In this phenomenon, the protein becomes
preferentially hydrated, i.e., preferentially surrounded by water molecules, but the apparent
volume of protein decreases, leading to a more stable protein configuration. Several osmolytes
besides trehalose are preferentially excluded from the vicinity of a protein and serve to stabilize
protein structure. These osmolytes include sorbitol, sucrose, PEGs and glycerol All these
osmolytes present different effectiveness of exclusion and protection, however trehalose have
been described as the most effective in both phenomena. This allows the growth of the myth that
trehalose presents special properties for stabilization of biomaterials. However the “special
properties” could be explained by the findings that the hydrated radius of trehalose is larger than
of the other sugars tested. We have proposed that having a larger hydrated radius, trehalose
occupies a larger volume and this fact is responsible for the strong stabilization effects. This
hypothesis was confirmed by adding glycerol in a concentration that occupies the same volume
that trehalose does, and protection of yeast soluble enzymes against 50ºC incubation was
coincident. Besides their protective properties, when enzymes are in the presence of these
compounds they usually have an altered catalytic activity which always includes inhibition of
catalytic rate. In this way, protection has always been correlated with transitory inhibition of
enzymatic rate of catalysis (45)
65
66
CHAPTER 3
Materials & methods
3. MATERIALS AND METHODS
3.1. General
This chapter describes materials used and outlines the experimental design for culture of
breast cancer cell line MDA MB 231 and denaturation study of cyt-c reductase enzyme in
presence of various osmolytes like glycerol, sucrose, trehalose, DMSO and salts like MgCl2. It
also gives an overview of the viability testing.
67
3.2. Chemicals
Pure and analytical grade chemicals were used in all experiments including media
preparation for growth of cells. Fetal bovine serum (FBS), penicillin-streptomycin, and trypsinEDTA were purchased from (Himedia, Mumbai, India). The human breast cancer cell line
MDA-MB-231 was purchased from National Centre for Cell Science (NCCS), Pune, India. Co2
incubator (Shel lab), Laminar hood (NUAIRE- NU205SM-DR), T-25 flasks (Orange Scientific),
centrifuge (Remi-C30BL), Sonicator, water bath -LAUDA Ecoline-staredition RE-104.
Lysozyme, DMSO, trehalose, glycerol was purchased from Himedia, India. MgCl2 was
purchased from Merck, India and sucrose from Nice chemicals. Cytochrome c was purchased
from Sisco Research Laboratories (SRL) and β- NADH (DPNH, Disodium salt) was purchased
from Himedia.
3.3. Glassware and Apparatus
All glass wares (conical flasks, measuring cylinders, beakers, Petri plates and test tubes
etc.) are purchased from M/s Bhattacharya & Co. Ltd (Kolkata, India) under the name Borosil.
3.4. Culture of Breast cancer Cell lines
The human breast cancer cell line MDA MB 231 was purchased from National Centre for
Cell Science (NCCS), Pune, India. The human breast cancer cell line MDA MB 231 were
routinely maintained in a Leibovitz L-15 media supplemented with 10% fetal bovine serum, 2
mM glutamine, penicillin (100 mg/mL), and streptomycin (100 mg/mL, final concentration) in a
humidified chamber at 37°C in 5% CO2 / 95% O2. Cells were grown in T-25 flasks to yield
5x106 cells/ml. Cells were trypsinized (0.25% Trysin –EDTA solution), washed with phosphate
buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4.7H2O, 1.4 mM KH2PO4,
pH 7.4), resuspended in a Leibovitz’s L-15 medium and pooled.
3.5. Media Optimization
The effect of various temperatures for optimum culture growth and compatibility of cell
line with different antibiotic (Penicillin, Streptomycin and neomycin) concentration was studied.
The optimized parameters are further considered for contamination free happy culture.
68
3.6. Sub culturing
Stock solution of Growth medium, Phosphate-buffered saline (PBS) (Ca2+/Mg2+ free),
Trypsin solution (0.05% (w/w) tissue-culture-grade trypsin, 0.53 mM EDTA; was prepared and
sub culturing was done in T-25 flask. Medium was removed from mother culture T-flask. Then
cells were washed with 5 ml of PBS. 3 ml trypsin was added. Cells were Incubate at 37°C for 3
minute. When the cells have rounded up and are coming off the plate, resuspend in 5 ml of serum
containing medium and wash cells by centrifugation at 800 rpm. Resuspend in 5 ml medium. If
using serum-free media, the trypsin should be neutralized with 1 ml of a 1 mg/ml solution of
soybean trypsin inhibitor (STI).
3.7. Trypan Blue Staining
Cells were mixed with trypan blue solution in 1:1 ratio. Trypan Blue solution is prepared
at a concentration of 0.8 mM with PBS (Store at room temperature, Stable for 1 month). Dead
cells stain blue, while live cells exclude trypan blue.
3.8. SDS-PAGE Analysis
The proteins present in the given sample are checked by performing sds-page analysis.
The proteins which are being over expressed occur in the form a thick band which is clearly
visible. The mol.wt of this protein can be determined by the markers which we run along with
the sample during the gel electrophoresis.
3.9. Preparation of cell lysate
Cultured cells were taken out by scraper from T-flask and centrifuged at 4000 rpm for 15
minute. Cell pellets were resuspended in Lysis buffer (50mM Tris buffer pH-7.5, 1mM EDTA,
1mM EGTA,1% Triton X-100, and 0.1mM PMSF). Normally ratios of cell wet weight to buffer
volume of 1:1 to 1:4 are used. Sonicate the cell suspension with 10 short burst of 10 sec followed
by intervals of 30 sec for cooling. The sonicated cells are taken in a centrifuge tube and
centrifuged at 10,000 rpm for 30min.The supernatant is discarded and the pellet is again
resuspended in sonication buffer. Then total cell lysates was centrifuged and supernatant was
collected for enzyme analysis.
3.10 Determination of cyt-c reductase activity
69
Cyt-c reductase activity was measured spectrophotometrically at 300C (Systronics 2203
double beam spectrophotometer) by recording the change in absorbance at 550 nm (46) due to
the conversion of cyt-c oxidized form to the reduced form in the presence of NADH as electron
acceptor. In the solution containing 0.1ml of NADH, 0.1ml of cyt-c, and 0.1ml of cell lysates
was added to 2.7ml PBS buffer. Enzyme activity was calculated using the following formula
Units/mg enzyme = [∆A 5 50nm/min (Test) - ∆A550nm / min (Blank)]
(21.0) (mg enzyme/ml RM)
21.0 = ∆ millimolar extinction coefficient between oxidized and reduced cytochrome c at pH 7.4
RM = Reaction Mixture (ml)
3.11. Unfolding of breast cancer cell line cyt-c reductase by chemical denaturants like urea
and GdnHCl
Unfolding of cyt-c reductase was done by urea (0M to 8M) and GdnHCl (0M to 6M) in
the presence of different molar concentration of trehalose (0.25M, 0.50M, 0.75M, 1.0M) sucrose
(0.25M, 0.50M, 0.75M, 1.0M), glycerol (0.18M, 0.36M, 0.57M, 1M, 2M, 3M), dimethyl
sulfoxide (DMSO) (0.25M, 0.50M, 0.75M, 1.0M), and MgCl2(2M,4M, 6M, 8M, 15M, 20M,
25M). The reaction mixtures were incubated for 30 minutes and then enzyme activity was
assayed .All the solutions were prepared in 50mM phosphate buffer of pH 7.4.
3.12. Unfolding of breast cancer cell line cyt-c reductase by Temperature
The enzymatic activity retained by cyt-c reductase after exposure to various temperatures
(40°C, 50°C, 60°C, 70°C, 80°C, 90°C, 100°C) and in presence of various concentration of
trehalose (0.25M, 0.50M, 0.75M, 1.0M) ,sucrose (0.25M, 0.50M, 0.75M, 1.0M), glycerol
(0.18M, 0.36M, 0.57M, 1M, 2M, 3M), dimethyl sulfoxide (DMSO) (0.25M, 0.50M, 0.75M,
1.0M), to 100 µl of enzyme extract, pH 7.4 (phosphate buffer) was studied. The reaction
70
mixtures were incubated at 30°C, and after 30 minutes the enzyme activity was assayed. All the
solutions were prepared in 50mM phosphate buffer of pH 7.4.
CHAPTER 4
71
Results
72
4. RESULTS
4.1 Cell culture
Breast cancer cell line MDA MB 231 was cultured in Leibovitz L-15 media with 10% fetal
bovine serum and 1% antibiotic solution. Cells were allowed to grow for a time period of 24hrs
and the picture was obtained by observing the T-25 flask under phase contrast microscope with
100X and 40X magnification.
40X
10X
Fig.3 MDA MB 231 cell lines incubated in
Fig.4 MDA MB 231 cell lines incubated in
Leibovitz L-15 media with 10% fetal bovine
Leibovitz L-15 media with 10% fetal bovine
serum for one day and the picture was taken
serum for one day and the picture was taken
under phase contrast microscope (Nikon) with
under phase contrast microscope (Nikon) with
canon power shot G9 camera at a resolution of
canon power shot G9 camera at a resolution of
40X magnification
10X magnification
.
73
In the following experiments inactivation of enzyme was performed by using various
denaturants like heat, GdnHCl and urea. Enzyme inactivation studies was performed by using
various concentrations of denaturants and protection of enzyme activity have been studied using
various osmolytes like glycerol, sucrose, DMSO, trehalose and MgCl2 .Enzyme activity has been
measured by incubating the enzyme for 30min at different temperatures and the % relative
activity of enzyme was measured keeping the enzyme activity at 30ºC as a control.
4.2 Effect of temperature on enzymatic activity of cyt-c reductase and its protection
by different osmolytes like trehalose, DMSO, glycerol, and sucrose.
The unfolding of reductase induced by temperature was studied. The enzymatic activities of cytc reductase after exposure at different temperatures(40ºC, 50ºC, 60ºC, 70ºC, 80ºC, 90ºC,
100ºC)and in presence of various osmolytes like glycerol, sucrose, trehalose and DMSO were
shown in Fig.5a to Fig.5d. It clearly showed that the enzyme retained the activity till higher
temperature like 50ºC. Beyond that the enzyme unfolds rapidly by the increase in temperature of
10 ºC from 50ºC - 60ºC and at 60ºC it gets denatured completely.
Fig.5 showed the unfolding curve of cyt-c reductase with temperature. The Figure clearly
showed that the enzyme lost its activity at higher temperature beyond 60ºC indicated complete
denaturation of the enzyme. When cyt-c reductase was unfolded thermally in presence of various
osmolytes of different concentrations, it was observed that a fair level of protection was provided
by all the osmolytes like sucrose, glycerol, DMSO and trehalose (Fig.5a to 5d).
%Relative Activity of cyt-c reductase
Temperature based unfolding of cyt-c reductase
120
100
80
60
40
20
0
30
40
50
60
70
Temperature
1
80
90
Fig.5 Thermal Unfolding of breast cancer cellular cyt-c reductase. The cell lysates was incubated in water
bath at different temperature for 30 minutes and simultaneously the activity assay was carried out to
0M Glycerol
0.25M Glycerol
0.5M Glycerol
1M Glycerol
2M Glycerol
3M Glycerol
5M Glycerol
120
100
80
%Relative Activity of cyt-c reductase
%Relative Activity of cyt-c reductase
estimate the residual biological activity of cyt-c reductase.
60
40
20
0
30
40
50
60
70
80
90
120
0M Sucrose
0.25M Sucrose
0.5M Sucrose
0.75M Sucrose
1M Sucrose
1.5M Sucrose
100
80
60
40
20
0
30c
40c
0
50c
60c
70c
80c
90c
Temperature(c)
Temperature( c)
Fig 5a
Fig 5b
120
100
%Relative activity of cyt-c reductase
%Relative Activity of cyt-c reductase
140
0M Trehalose
0.25M Trehalose
0.5M Trehalose
0.75M Trehalose
1M Trehalose
140
80
60
40
20
0
-20
30
40
50
60
70
80
90
0
0M DMSO
0.25M DMSO
0.5M DMSO
1M DMSO
1.5M DMSO
120
100
80
60
40
20
0
-20
30
40
50
60
70
80
90
Temperature
Temperature( c)
Fig 5c
Fig 5d
Thermal Unfolding of Breast cancer (MDA MB 231) cyt-c reductase in presence of different
concentration of (a)glycerol; (b) sucrose; (c) trehalose; (d) DMSO. Different concentration of urea was
added to the cell lysates already containing various concentrations of different osmolytes were
maintained.
2
The Table2 below shows the % residual enzyme activity measurements in presence of various
osmolytes of different concentrations at 60ºC.
Table 2 Percentage Residual activity of cyt-c reductase at 60 ºC
Osmolytes
Concentration
Glycerol
Sucrose
Trehalose
DMSO
0
0
0
0
0
0.25
66.73
39.76
59.76
59.76
0.5
100
39.76
69.76
63.72
0.75
-
39.76
75.58
63.72
1
41.6
59.76
83.72
63.72
1.25
-
-
-
-
1.5
-
59.76
-
-
2
50.58
-
-
-
3
0
-
-
-
4
66.73
-
-
-
5
66.73
-
-
-
6
0
-
-
-
(M)
When cyt-c reductase was unfolded by temperature in presence of various osmolytes of different
concentrations it was observed that trehalose and DMSO showed good level of protection and
glycerol and sucrose has inhibitory effect on the enzyme activity at lower temperatures. But they
also provided thermal protection above 50ºC and it was continued up to 80ºC and further no
protection was observed above 80ºC indicated the complete denaturation of the enzyme.
In table 2 it can be seen that glycerol stabilizes cyt-c reductase maximally because 0.5M glycerol
showed 100% activity at 60ºC whereas next best is trehalose which at 0.75M and 1M helped cytc reductase to retain activity from 75-85% .At 80ºC the enzyme activity is completely lost and
the osmolytes also had no protection in retaining the activity of the enzyme.
3
4.3 Effect of urea on denaturation of cyt-c reductase and its protection by different
% Relative Cyt-C reductase activity
osmolytes like Sucrose, Trehalose, DMSO and Glycerol.
160
140
%Relative enzyme activity of cyt-c
reductase in the presence of urea
120
100
80
60
40
20
0
-20
0
2
4
6
8
Conc of urea(M)
Fig.6 Unfolding of breast cancer (MDA MB 231) cyt-c reductase using urea. Different concentration of
urea was added to the cell lysates already containing various concentrations of different osmolytes and
incubated for 30 min followed by the estimation of the residual biological activity of cyt-c reductase.
Urea induced unfolding of cyt-c reductase was studied in breast cancer cell line MDA MB 231.
Fig 6 showed the results of incubation of cyt-c reductase in the presence of different
concentrations of urea. After 30min of incubation the relative activity of the samples was
examined.
4
%Relative Activity of cyt-c reductase
%Relative Activity of cyt-c reductase
160
0M Glycerol
0.18M Glycerol
0.36M Glycerol
0.54M Glycerol
0.72M Glycerol
1M Glycerol
2M Glycerol
3M Glycerol
140
120
100
80
60
40
20
0
0
2
4
6
160
120
100
80
60
40
20
0
-20
8
0
Conc of Urea(M)
%Relative Activity of cyt-c reductase
%Relative Activity of cyt-c reductase
120
4
6
8
Fig 6b
0M Trehalose
0.25M Trehalose
0.5M Trehalose
0.75M Trehalose
1M Trehalose
140
2
Conc of Urea(M)
Fig 6a
160
0M Sucrose
0.25M Sucrose
0.5M Sucrose
1M Sucrose
1.5M Sucrose
140
100
80
60
40
20
0
-20
Urea(M)0M 0.25M0.5M 1M 2M 3M 4M 5M 6M 7M 8M
--
Conc of Urea(M)
160
0M DMSO
0.25M DMSO
0.5M DMSO
0.75M DMSO
1M DMSO
140
120
100
80
60
40
20
0
-20
Urea(M)0M 0.25M0.5M 1M 2M 3M 4M 5M 6M 7M 8M
--
Conc of Urea(M)
Fig 6c
Fig 6d
Unfolding of breast cancer (MDA-MB231) cyt-c reductase using urea in presence of different
concentration of (a) glycerol (b) sucrose(c) trehalose (d) DMSO. Different concentration of urea was
added to the cell lysates already containing various concentrations of different osmolytes and incubated
for 30 min followed by the estimation of the residual biological activity of cyt-c reductase.
Cyt-c reductase activity was measured in the presence and absence of several osmolytes
to observe the protective ability of osmolytes like glycerol, sucrose, DMSO, and trehalose. Urea
at different concentrations from 0.5M to 7M was used to unfold the enzyme. Urea based
unfolding shows that at 2M concentration of urea the enzyme showed a higher activity than its
5
native form. This may be due to the dissociation of dimeric form to individual monomers. Later
at an increased concentration of urea the activity decreased and finally lost its activity at 7M
concentration of urea. When cyt-c reductase was unfolded then a sudden drop of activity from
100% to 50% was found at 0.25M urea. This decrease of activity was continued up to 1M.The
activity was again dropped rapidly at 3M urea concentration where the activity was
approximately 37%.Further increase of urea concentration denatured the enzyme gradually till
6M urea conc. And suddenly it dropped to zero at 7M.This whole phenomenon showed a super
active state at 2M conc. Increasing molarities of trehalose and DMSO had a protective function
against denaturation by urea whereas in other osmolytes the increasing concentrations of
osmolytes had a little effect on enzyme activity.
Table 3. Percentage Residual Activity of cyt-c reductase when it is denatured at 1M Urea
Osmolyte
Concentration
(M)
Glycerol
Sucrose
Trehalose
DMSO
0
48.2
48.2
44.8
44.8
0.25
10.5
114.16
59.76
59.76
0.5
89.15
106.6
59.76
59.76
0.75
96.38
17.8
63.72
67.44
1
50.16
83.3
67.44
75.69
1.25
-
-
-
-
1.5
-
-
-
-
2
49.67
-
-
-
3
50.12
-
-
-
6
4.4 Effect of GdnHCl on denaturation of cyt-c reductase and its protection by
%Relative Activity of cyt-c reductase
different osmolytes like Sucrose, Trehalose, DMSO, and Glycerol.
100
GnHcl based unfolding of cyt-c reductase
80
60
40
20
0
0
1
2
3
4
5
6
Conc of GnHcl(M)
Fig.7 Unfolding of breast cancer (MDA MB 231) cyt-c reductase using GdnHCl .Different concentration
of GdnHCl was added to the cell lysates already containing various concentrations of different osmolytes
and incubated for 30 min followed by the estimation of the residual biological activity of cyt-c reductase.
GdnHCl based denaturation of the enzyme has showed that it had maximum activity at lower
concentrations of denaturant but it was completely denatured at 2M concentration of GdnHCl.
The native activity of enzyme was decreased by 20% as the concentration of GdnHCl has been
increased. At 1M GdnHCl the enzyme started losing its activity nearly by 60% and at 2M its
activity was completely lost.
100
0M Glycerol
1M Glycerol
2M Glycerol
3M Glycerol
4M Glycerol
80
%Relative Activity of cytc reductase
%Relative Activity of cyt-c reductase
100
60
40
20
0
0
1
2
3
4
5
6
0M Sucrose
0.5M Sucrose
0.75M Sucrose
1M Sucrose
80
60
40
20
0
0
Conc of GdnHCl(M)
1
2
3
4
Conc of GdnHCl(M)
7
5
6
Fig 7a
0M Trehalose
0.25M Trehalose
0.5M Trehalose
0.75M Trehalose
1M Trehalose
80
100
%Relative Activity of cyt-c reductase
100
%Relative Activity of cyt-c reductase
Fig 7b
60
40
20
0
0
1
2
3
4
5
6
0M DMSO
0.25M DMSO
0.5M DMSO
0.75M DMSO
1M DMSO
80
60
40
20
0
0
1
Conc of GdnHCl(M)
2
3
4
5
6
Conc of GdnHCl(M)
Fig 7c
Fig 7d
Unfolding of breast cancer (MDA MB 231) cyt-c reductase using GdnHCl in presence of different
concentration of (a) glycerol (b) sucrose (c) trehalose (d) DMSO. Different concentration of GdnHCl was
added to the cell lysates already containing various concentrations of different osmolytes and incubated
for 30 min followed by the estimation of the residual biological activity of cyt-c reductase.
Cyt-c reductase activity was measured in the presence and absence of several osmolytes to
observe the protective ability of osmolytes like glycerol, sucrose, DMSO, and trehalose. GdnHCl
at different concentrations from 0.25M to 4M was used to unfold the enzyme. GdnHCl based
unfolding has showed that no super active state was found unlike urea and thermal denaturation.
GdnHCl based unfolding of cyt-c reductase have showed that the activity was decreased by
nearly 20% at 0.25M which further decreased by 20% at an increasing concentrations of
GdnHCl. The enzyme completely lost its activity at 2M GdnHCl.
8
Table 4. Percentage Residual Activity of cyt-c reductase when it is denatured at 1M
GdnHcl
Osmolytes
Concentration
Glycerol
Sucrose
Trehalose
DMSO
0
59.76
48.2
44.8
44.8
0.25
10.5
114.16
59.76
59.76
0.5
89.15
106.6
59.76
59.76
0.75
96.38
17.8
63.72
67.44
1
39.76
83.3
67.44
75.69
1.25
-
-
-
-
1.5
-
-
-
-
2
39.76
-
-
-
3
59.76
-
-
-
(M)
4.5 Effect of GdnHCl, Urea on denaturation of cyt-c reductase and its protection by
MgCl2
%Relative Activity of cyt-c reductase
200
0mM Mgcl2
2mM Mgcl2
5mM Mgcl2
10mM Mgcl2
15mM Mgcl2
25mM Mgcl2
180
160
140
120
100
80
60
40
20
0
Urea Conc.
0M 0.25M 0.5M
1M
2M
3M
4M
Conc of Urea(M)
Fig 8
9
5M
6M
--
Fig.8. Unfolding of Breast cancer (MDA MB 231) cyt-c reductase using urea in presence of different
concentration of MgCl2. Different concentration of urea was added to the cell lysates already containing
various concentrations of Mgcl2 and incubated for 30 min followed by the estimation of the residual
biological activity of cyt-c reductase.
%Relative Actviity of cyt-c reductase
100
0mM MgCl2
5mM MgCl2
10mM MgCl2
15mM MgCl2
80
60
40
20
0
0
1
2
3
4
5
6
Conc of GdnHCl(M)
Fig 9
Fig.9. Unfolding of breast cancer (MDA MB 231) cyt-c reductase using GdnHCl in presence of different
concentration of MgCl2.Different concentration of GdnHCl was added to the cell lysates already
containing various concentrations of Mgcl2 and incubated for 30 min followed by the estimation of the
residual biological activity of cyt-c reductase.
The effect of GdnHCl and urea on the activity of native and presence of salts like Mgcl2 were
studied and the residual activities of the enzyme are showed in the fig 8 and 9.The enzyme
activity was measured by incubating the enzyme in different concentrations of Mgcl2.Enzyme
showed higher activity in the presence of urea at 2M concentration.
10
CHAPTER 5
DISCUSSION
11
DISCUSSION
In the present investigation, the unfolding of 550Kda dimeric cytochrome c (cyt-c)
reductase from breast cancer cell line MDA MB 231.The enzyme was unfolded thermally and in
presence of chemical denaturants like urea and guanidium hydrochloride (GdnHCl). The study
was also carried out in presence of various osmolytes like glycerol, dimethysulfoxide (DMSO),
sucrose and trehalose. Salt like MgCl2 was also added to the cell lysate and unfolding study was
performed. The extent of unfolding of the protein was expressed in terms of biological activity
retained by the enzyme. Cytochrome c reductase activity was measured by observing the change
in the absorbance at 550 nm due to the conversion of cyt-c oxidized form to reduced form. The
human breast cancer cell line MDA MB 231 was routinely maintained in a Leibovitz L-15 media
supplemented with 10% fetal bovine serum. Cells were grown, trypsinized and pooled. Cell
lysate was prepared by sonication.
While unfolding thermally, it was found that the enzyme started unfolding at 40ºC and
showed a increased biological activity till 50ºC followed by the sharp decrease of the activity.
Complete lost of the activity was observed at 60ºC which also indicated the complete
denaturation of the enzyme. It was found that the enzyme reached to a super active state (120%
activity) at 50ºC. When cyt-c reductase was unfolded thermally in presence of various osmolytes
of different concentrations, it was observed that trehalose and DMSO showed fair level of
protection and glycerol and sucrose showed inhibitory effect on the protection enzyme activity at
lower temperatures. But they also provided thermal protection at 60ºC and above. For all the
cases the super active intermediate state of the enzyme was appeared. In table 1 it can be seen
that glycerol stabilizes cyt-c reductase maximally because 0.5M glycerol showed 100% activity
at 60ºC whereas trehalose protected at 0.75M and 1M and helped cyt-c reductase to retain
approximately 75-85% activity. At 80ºC the enzyme activity was lost completely and the
osmolytes also had no protection.
When the cell lysate containing cyt-c reductase was added with different concentration of
urea, a sudden drop of enzyme activity from 100% to 50% was found at 0.25M urea. This
decrease of activity was continued up to 1M of urea concentration. The enzyme showed a rapid
increase of activity till 2M urea concentration where it showed a maximum 150% activity
12
followed by a rapid drop of activity till 3M urea concentration where the activity was
approximately 37%. Further increase of urea concentration brought a further reduction of
enzyme activity and at 7M urea concentration the activity became zero indicates complete
unfolding. The result also indicated that at 2M the enzyme reached to a super active state where
probably the dimeric enzyme converted into monomeric one and this monomer perhaps showed
a efficient way to catalyze the reaction and hence the enhanced activity of the enzyme was
found.
When the similar kind of work was carried out in presence of osmolytes, different level
of protection against unfolding was observed by them. Up to 1M glycerol, DMSO and trehalose
concentration and the lower concentration of sucrose like 0.25M, 0.5M showed a fair level of
protection of activity from denaturation.. Although at 2M urea, super active state was appeared
in the presence of all these osmolytes (except sucrose), the activity at the super active state was
lower than the activity achieved by the enzyme in absence of any osmolyte. This protection was
particularly evident in presence of 0.72M glycerol, 1M DMSO, 1M trehalose at higher
concentration of urea concentration (5, 6 and 7M). In contrast, the sucrose did not show any
protection beyond 4M urea concentration at 0.5M urea, glycerol (0.72M) showed 102% activity
and at 1M urea, presence of 0.25M and 0.5M sucrose helped the enzyme retained 114% and
106% activity. Sucrose showed protective effect only at 0.25M and 0.5M concentrations.
Increasing molarities of trehalose and DMSO were consistent with more protection against
denaturation by urea whereas the increasing concentrations of other osmolytes had a little effect
on enzyme activity.
From the results it was found that during thermal and urea-based denaturation of enzyme,
super active state was found at 50ºC and 2M urea concentration, respectively. Surprisingly, no
such super active state was observed during GdnHCl based unfolding of cyt-c reductase. This
also indicated that the mechanism of action of urea and GdnHCl are different. In thermal
unfolding, the enzyme started unfolding at 50ºC and it lost its activity completely at 60ºC. Urea
based unfolding of the enzyme indicated that higher concentration of urea was required to cause
complete loss of its activity.
Urea at 7M concentration caused complete denaturation of the enzyme whereas 2M
GdnHCl caused complete denaturation of enzyme. The native activity of enzyme decreased by
20% as the concentration of GdnHCl was increased. At 1M GdnHCl the enzyme lost its activity
13
nearly by 60% and at 2M its activity was completely lost indicative of complete unfolding of the
enzyme. Cyt-c reductase activity was measured in the presence and absence of several osmolytes
to see their protective ability. GdnHCl at different concentrations ranges from 0-6M was used to
unfold the enzyme. GdnHCl based unfolding showed no super active state unlike urea and
thermal denaturation. GdnHCl based unfolding of cyt-c reductase showed that the activity was
decreased by nearly 20% at 0.25M and then there was no loss of activity till 0.5M. After 0.5M
GdnHCl concentration there was rapid drop of the activity till 2M where the complete unfolding
was observed which was evident by measuring an activity of zero.
Glycerol at lower concentrations showed lower protective effect and the activity was
increased by 10%. But at higher concentrations of glycerol like at 4M, the enzyme was not
denatured at 2M and rather 20% enzyme activity was retained. Trehalose at 0.25M, 0.5M.0.75,
1M concentrations showed an enhanced activity from 20% to 40% at 2M GdnHCl concentration
and maximum protection by trehalose was observed at 0.125M GdnHCl concentration nearly to
90%. Among all osmolytes studied highest protection was found by DMSO at 1M concentration.
But at 2M GdnHCl concentration, DMSO with 0.75 and 1 M concentration showed highest
protection and this was nearly to 80%. DMSO showed maximum protection at 2M concentration
of GdnHCl where no other osmolytes has this much protection as DMSO.
While in urea based unfolding process the presence of MgCl2 showed an increase in
enzyme activity to an increase of 300% activity at 0.5M concentration. At higher concentrations
of denaturant i.e. at 3M and 4M, MgCl2 showed maximum protection ranging from 5mM to
25mM concentration. In GdnHCl based unfolding of enzyme, MgCl2 showed protection at only
1M concentration of denaturant. It indicates that the GdnHCl is more powerful denaturant and
salts like MgCl2 is unable to retain the enzyme activity at higher concentrations of GdnHCl. At
0.125M GdnHCl, MgCl2 showed maximum protection. It indicated that salts can induce
stabilizing effects only at lower denaturant concentrations. At 0.25M urea concentration MgCl2
showed maximum protection. This also indicated that effects caused by both the denaturants are
different.
14
CHAPTER 6
CONCLUSIONS
15
CONCLUSIONS
The whole investigation gave us a better understanding about the conformational behavior of the
enzyme cyt-c reductase under various stress conditions like high temperature, urea and GdnHCl.
Since it is an important enzyme in mitochondrial electron transport chain and plays a major role
in the apoptosis of cells and also the apoptosis is directly correlated with cancer cell death and
hence therapy, to gain knowledge regarding its conformational nature and stabilization under
stress condition is very essential. The following conclusions were made from our experimental
results that were obtained during unfolding of cyt-c reductase in MDA MB 231 breast cancer cell
line.
 During thermal and urea based unfolding of cyt-c reductase, a super active state of the
enzyme was observed but no such super active state was found during GdnHCl based
unfolding of the enzyme. This probably indicates that during unfolding of cyt-c
reductase, the enzyme which is normally a dimer, may go to a monomeric state after
being dissociated by heat and urea. This super active state showed a higher activity than
the native dimeric form in the presence of elevated temperature (i.e. at 50ºC) and at 2M
urea concentration.
 The enzyme was completely unfolded at a urea and GdnHCl concentration of 7M and 2
M, respectively. This proved that GdnHCl is a much stronger denaturant than urea.
 The nature of unfolding by urea and GdnHCl was not similar since the super active state
of the enzyme was not found in GdnHCl-based unfolding but it was appeared during
urea-induced denaturation process and this proved the fact that the mechanism of action
of urea and GdnHCl are different.
 Cyt-c reductase in the presence of elevated temperatures i.e. at 60ºC lost its activity
completely which indicates the complete unfolding of the enzyme and its temperature
sensitivity.
 Addition of various osmolytes like glycerol, sucrose, trehalose and DMSO provides fair
level of protection to cyt-c reductase against thermal stress although at lower temperature
16
glycerol and sucrose exhibit little inhibitory effect but all of their presence delays the
denaturation points of the enzyme to a maximum of 20ºC.
 While unfolding cyt-c reductase by urea in presence of all four osmolytes, no clear super
active state was found which indicates that the unfolding mechanism could be altered by
the presence of the mentioned osmolytes. Moreover, osmolytes presence delays the
unfolding end point to 8M urea concentration instead of 7M.
 When the enzyme was deactivated by GdnHCl in the presence of osmolytes, fair level of
resistance against unfolding was observed. Among all osmolytes, 1M DMSO provided
maximum protection at 2M GdnHCl concentration since it retained almost 90%
biological activity of the enzyme. Even though DMSO and other osmolytes showed good
protection, the enzyme showed complete loss of activity at 3M GdnHCl which indicate
the strong nature of its action.
 When MgCl2 was used as additive during the unfolding process of the enzyme by urea
and GdnHCl, all concentration of MgCl2 ranges 2-20mM showed good protection against
deactivation.
17
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21
MISCELLANEOUS
Table 1: List of Instruments used during the whole experiment their make and function
Instruments
Manufacturer
Function
Humidified chamber
Shel-lab
cell culture
Vertical Autoclave
Test Master
Sterilization
Analytical Balance
Precisa XR205SM-DR
Weight Measurement
Laminar airflow
NUAIRE- NU205SM-DR
Aseptic Environment
pH meter
Systronics
Measurement of pH
Ultra Low Temperature freezer
Remi-RQFP 265
Preservation of cultures
Preparation of the stock
Ultra pure water system
Millipore
solution, throughout the
experiment etc.
Spectrophotometer(UV/Vis)
Systronics 2203 Double
Protein concentration
beam
estimation
Cell biomass
Incubator shaker
Remi-CIS24BL
Ultra Centrifuge
Remi-C30BL
Collection of pellet
T-25 flasks
Orange Scientific
Cell culture
Water bath
LAUDA Ecoline-staredition
RE-104
Vertex Mixer
Genie
Magnetic stirrer
Spint
Agar &SDS-PAGE unit
ELECTROPAGE-100
22
production
Heat treatment
Proper mixing of
reagent
Mixing
Running of protein
sample
Results
Table 4: Percentage Relative Activity of cyt-c reductase under the application of
Temperature in the presence of Trehalose at different molarities.
40
0M
Trehalose
100
100
0.25M
Trehalose
100
100
0.5M
Trehalose
100
103.4
0.75M
Trehalose
100
103.4
1M
Trehalose
100
123.25
50
118.6
126.74
126.74
127.74
131.3
60
0
59.76
69.76
75.58
83.72
70
0
45.81
47.79
49.76
19.76
80
0
0
0
0
0
90
0
0
0
0
0
Temp
30
Table 5: Percentage Relative Activity of cyt-c reductase under the application of
Temperature in the presence of DMSO at different molarities.
Temp
0M DMSO
0.25M
DMSO
100
0.5M DMSO
100
0.75M
DMSO
89.53
1M DMSO
30
100
40
100
83.72
87.67
95.34
95.34
50
118.6
126.74
126.74
125.5
125.5
60
0
59.76
63.72
63.72
63.72
70
0
39.76
39.76
39.76
19.76
80
0
0
0
0
0
90
0
0
0
0
0
23
85.69
Table 6: Percentage Relative Activity of cyt-c reductase at different temperatures in the
presence of glycerol
Temperature 0M
(ºC)
gly
30
100
0.25M 0.5M
gly
gly
100
100
1M
gly
80
2M
gly
80
3M
gly
80
4M
gly
80
5M gly
6M gly
80
80
40
100
66.73
83.65
110
83.65
83.65
66.73
66.73
0
50
118.6
100
100
50.58
100
110
100
100
0
60
0
66.73
100
41.6
50.58
0
66.73
66.73
0
70
0
33.6
33.6
66.73
66.73
0
0
0
0
80
0
0
0
0
0
0
0
0
0
90
0
0
0
0
0
0
0
0
0
Table 7: Percentage Relative Activity of cyt-c reductase in the presence and absence of
sucrose at different temperatures.
Sucrose
0M
Sucrose
0.25M
Sucrose
0.5M
Sucrose
0.75M
Sucrose
1M
Sucrose
1.5M
Sucrose
30c
100
100
100
100
100
100
40c
100
34.8
39.88
39.76
84.2
85.6
50c
118.6
100
59.76
100
100
100
60c
0
39.76
39.76
39.76
59.76
59.76
70c
0
2.08
19.76
0
0
0
80c
0
0
0
0
0
0
90c
0
0
0
0
0
0
24
Table 8: Percentage relative activity of cyt-c reductase in the presence and absence of
glycerol at different molarities of Urea.
Molarity 0M
0.18M
(Urea)
glycerol glycerol
0
100
100
0.36M
0.57M
0.72M
glycerol glycerol glycerol
100
100
90
1M
2M
glycerol glycerol
80
80
3M
glycerol
80
0.5
51.8
53
60.24
57.83
102.7
48.2
44.8
45.78
1
48.2
10.35
10.5
89.15
96.38
50.16
49.67
50.12
1.5
44.8
45.78
75.9
48.19
55.42
55.42
55.42
55.42
2
150
23.6
23.6
48.19
55.42
69.97
45.45
24.09
3
37.83
16.49
16.49
44.5
44.57
57.83
40.2
15.6
4
27.71
22.06
22.06
37.8
48.19
28.64
28.46
24.09
5
24.09
10.4
10.4
27.7
37.35
22.9
24.09
24.1
6
20.48
11.78
12
30.12
34.93
19.01
20.48
20.5
7
0
2
3
10
14
3
3
3
8
0
0
0
0
0
0
0
0
25
Table 9: Percentage Relative Activity of cyt-c reductase under the application of various
molarities of Urea in the presence of urea and sucrose at different molarities
Molarity,
Urea
0M suc
0.25M suc
0.5M suc
0.75Msuc
1M suc
0
100
100
100
100
100
1
48.2
114.16
106.6
17.8
83.3
2
150
85.83
108.3
12.5
100
3
37.83
13.3
23.3
42.83
14.16
4
27.71
14.16
15
4.16
12
5
24.09
7.33
6.5
2.858
4.16
6
20.48
2.58
2.3
5.75
4.16
7
2
0
0
0
0
8
0
0
0
0
0
26
Table 10: Percentage Relative Activity of cyt-c reductase under the application of various
molarities of Urea in the presence of urea and Mgcl2 at different molarities.
0mM
Mgcl2
100
2mM
MgCL2
100
5mM
MgCL2
100
10mM
MgCL2
100
15mM
MgCL2
100
25mM
MgCL2
100
0.25M
51.8
100
100
100
100
100
0.5M
48.2
177.9
79.76
59.76
59.76
59.76
1M
44.8
39.76
79.76
59.76
79.76
79.76
2M
150
59.76
79.76
79.76
90
100
3M
37.83
79.76
100
137.14
100
100
4M
27.71
19.76
39.76
120
100
79.76
5M
24.09
59.76
59.76
59.76
59.76
59.76
6M
24.48
39.76
39.76
59.76
59.76
59.76
7M
0
19
19
29
29
29
8M
0
0
0
0
0
0
Urea Conc.
0M
27
Table 11: Percentage Relative Activity of cyt-c reductase under the application of various
molarities of Urea in the presence of Urea and Trehalose at different molarities.
0M
100
0.25M
Trehalose
100
0.25M
51.8
63.72
63.72
65.11
69.76
0.5M
48.2
61.74
63.72
67.44
69.76
1M
44.8
59.76
59.76
63.72
67.44
2M
150
89.53
89.53
100
100
3M
37.83
63.72
63.72
63.72
63.72
4M
27.71
39.76
39.76
39.76
39.76
5M
24.09
39.76
39.76
39.76
39.76
6M
20.48
0
0
19.76
19.76
7M
4
0
0
3
3
8M
0
0
0
0
0
Urea(M)
0M Trehalose
28
0.5M
Trehalose
100
0.75M
Trehalose
100
1M
Trehalose
100
Table 12: Percentage Relative Activity of cyt-c reductase under the application of various
molarities of Urea in the presence of Urea and DMSO at different molarities.
Urea(M)
0M DMSO
0.25M DMSO
0.5M DMSO
0.75M DMSO
1M DMSO
0M
100
100
100
89.53
86
0.25M
51.8
63.72
67.44
71.74
75.74
0.5M
48.2
67.44
75.69
80.23
58.6
1M
44.8
59.76
59.76
67.44
75.69
2M
150
100
89.53
95.58
100
3M
37.83
63.72
63.72
75.69
75.69
4M
27.71
39.76
43.83
47.79
51.86
5M
24.09
39.76
43.83
45.81
47.79
6M
20.48
0
0
0
0
7M
3
0
0
0
0
8M
0
0
0
0
0
29
Table 13: Percentage Relative Activity of cyt-c reductase under the application of various
molarities of GdnHcl in the presence of GdnHcl and Glycerol at different molarities
GdnHcl(M)
0
0 Gly
100
1M Gly
100
2M Gly
100
3M Gly
100
4Mgly
100
0.125
80.23
59.76
59.76
81.62
83.72
0.25
80.23
59.76
59.76
83.72
85.69
0.5
80.23
39.76
39.76
83.72
85.69
1
59.76
39.76
39.76
59.76
63.72
2
0
0
19.76
20.93
20.93
3
0
0
0
0
19.76
4
0
0
0
0
2
5
0
0
0
0
0
6
0
0
0
0
0
30
Table 14: Percentage Relative Activity of cyt-c reductase under the application of various
molarities of GdnHcl in the presence of GdnHcl and Mgcl2 at different molarities
GdnHcl(M)
0mM Mgcl2
5mM Mgcl2
10mM Mgcl2
15mM Mgcl2
0
100
100
100
100
0.125
80.23
80.23
80.23
80.23
0.25
80.23
80.23
59.76
59.76
0.5
80.23
39.76
51.16
55.81
1
59.76
59.76
69.76
75.58
2
0
0
0
0
3
0
0
0
0
4
0
0
0
0
5
0
0
0
0
6
0
0
0
0
31
Table 15: Percentage Relative Activity of cyt-c reductase under the application of various
molarities of GdnHcl in the presence of GdnHcl and sucrose at different molarities
GdnHcl(M)
0M sucrose
0.5M sucrose
0.75M sucrose
1M sucrose
0
100
100
100
100
0.125
80.23
47.2
59.76
39.76
0.25
80.23
59.76
49.76
39.76
0.5
80.23
47.2
63.72
59.76
1
59.76
39.76
29.88
39.76
2
0
0
0
39.76
3
0
0
0
0
4
0
0
0
0
5
0
0
0
0
6
0
0
0
0
32
Table 16: Percentage Relative Activity of cyt-c reductase under the application of various
molarities of GdnHcl in the presence of GdnHcl and Trehalose at different molarities
0M
Trehalose
100
0.25M
Trehalose
100
0.5M
Trehalose
100
0.75M
Trehalose
100
1M
Trehalose
100
0.125
59.76
80.23
80.23
89.53
93.72
0.25
59.76
59.76
59.76
63.72
69.76
0.5
59.76
59.76
59.76
67.44
72.09
1
39.76
59.76
63.7
67.44
72.09
2
0
19.76
39.76
39.76
39.76
3
0
0
0
0
0
4
0
0
0
0
0
5
0
0
0
0
0
6
0
0
0
0
0
GdnHcl(M)
0
33
Table 17: Percentage Relative Activity of cyt-c reductase under the application of various
molarities of GdnHcl in the presence of GdnHcl and DMSO at different molarities.
GdnHcl(M)
0M DMSO
0.25M
DMSO
0.5M DMSO
0.75M
DMSO
1M DMSO
0
100
100
100
89.53
85.69
0.125
59.76
59.76
63.72
67.44
85.69
0.25
59.76
59.76
63.72
63.72
72.09
0.5
59.76
80.23
80.23
80.23
63.72
1
39.76
80.23
80.23
89.53
80.23
2
0
0
0
0
89.53
3
0
0
0
0
0
4
0
0
0
0
0
5
0
0
0
0
0
6
0
0
0
0
0
34