GENERAL BIOCHEMISTRY
(BCMB 205)
Prof. Osbourne Quaye
Introduction to Biochemistry
• Biochemistry is the language of biology (life).
The study of biochemistry is essential to understand the basic functions of the body.
Give information regarding the functioning of cells at the molecular level.
Why Biochemistry?
• How the food that we eat is digested, absorbed, and used to make ingredients for the
body?
• How does the body derive energy for normal day-to-day work?
• How are the various metabolic processes interrelated?
•
• What is the function of genes?
•
• What is the molecular basis for immunological resistance against invading organisms?
Definition of Biochemistry
• Therefore we can say:
• “It is a branch of science which deals with the study of biomolecules and their role in
living systems”
• “Chemistry of Life or Living Being is Biochemistry”
• Biochemistry is a branch of science that seeks to describe the structure, organization,
and functions of living matter in molecular terms.
Biomolecules
• A biomolecule or biological molecule is any molecule that is present in living
organisms.
• Large Macromolecules Such as proteins, carbohydrates, lipids, and nucleic acids.
• Small Molecules such as primary metabolites, secondary metabolites, and natural
products.
• Metabolites (Small Molecules) are small molecules that are the intermediates and
products of metabolism e.g: enzymes.
What do biochemists do?
• Provide new ideas and experiments to understand how life works
• Support our understanding of health and disease
• Contribute innovative information to the technological revolution
• Work alongside chemists, physicists, healthcare professionals, policymakers,
engineers, and many more professionals
Branches of Biochemistry
Vast and diverse
Structural biochemistry
Neurochemistry
Bio-organic chemistry
Chemotaxonomy
Enzymology
Chemical ecology
Metabolic biochemistry
Virology
Xenobiotics
Molecular biology
Immunology
Cell biology
Endocrinology
Molecular genetics and Genetic engineering
Where do biochemists work?
• Hospitals
• Universities
• Agriculture
• Food institutes
• Education
• Cosmetics
• Forensic crime research
• Drug discovery and development
Cell structure and function
• Cell theory:
• Cell theory is a collection of ideas and conclusions from many different
scientists over time that describes cells and how cells operate
• The cell theory states that:
1. All known living things are made of one or more cells.
2. Cells are the basic unit of structure and function in living things.
3. All living cells arise from pre-existing cells by division.
Cell structure and function
• Cells vary considerably in size and shape but they share certain common
features:
1. Every living cell is surrounded by a membrane, which separates the cell contents
from everything else outside.
2. Cells contain genetic material which stores all of the instructions needed for the
cell’s activities.
3. Many of these activities are chemical reactions, catalysed by enzymes produced
inside the cell.
4. Cells have their own energy release system that powers all of the cell’s activities.
• So, cells can be thought of as the smallest living structures – nothing smaller
can survive.
Size of Cells
• Cells vary in size.
• Most cells are very small (microscopic), some may be very large (macroscopic).
• The unit used to measure size of a cell is micrometer. (1 μm = 1/1000
millimeter)
Shape of Cells
• Cells vary in shape
• Variation depends mainly upon the
function of cells
• Some cells like Euglena and
Amoeba can change their shape,
but most cells have a fixed shape
Structure of Cell
• The detailed structure of a cell can be
studied under a compound
microscope and electron microscope.
• Certain structures can be seen only
under an electron microscope.
• The structure of a cell as seen under
an electron microscope is called
Ultrastructure.
Categories of Cells
1. Prokaryotic cells: Prokaryotes are unicellular organisms that lack
membrane-bound structures, the most noteworthy of which is the nucleus.
Prokaryotic cells tend to be small, simple cells, measuring around 0.1-5 μm
in diameter.
2. Eukaryotic Cells: Eukaryotes are organisms whose cells have a nucleus and
other organelles enclosed by a plasma membrane.
• Organelles: are internal structures responsible for a variety of functions, such
as energy production and protein synthesis.
Prokaryotic cells
• The primarily single-celled organisms found in the Bacteria domains are
known as prokaryotes.
• These organisms are made of prokaryotic cells — the smallest, simplest,
and most ancient cells.
Prokaryotic cells
Eukaryotic cells
• Organisms in the Eukarya domain are made of the more complex eukaryotic
cells. These organisms, called eukaryotes, can be unicellular or multicellular
and include animals, plants, fungi and protists
Eukaryotic cells
Eukaryotic cells
What do prokaryotes and eukaryotes have in common?
1. DNA: Genetic coding that determines all the characteristics of living things.
2. Cell (or plasma) membrane: Outer layer that separates the cell from the
surrounding environment and acts as a selective barrier for incoming and
outgoing materials.
3. Cytoplasm: Jelly-like fluid within a cell that is composed primarily of
water,salts and proteins.
4. Ribosomes: Organelles that make proteins.
How do prokaryotes and eukaryotes differ?
Prokaryotic cell
Eukaryote cell
1. Nucleus is undeveloped
2. Only one chromosome is present
3. Membrane bound organelles are
absent
4. Size ranges from 0.5-5 μm
5. Examples: Bacteria and blue
green algae
1. Nucleus is well developed
2. More than one chromosomes
are present
3. Membrane bound organelles are
present
4. Size ranges from 5-100 μm
5. Examples: All other organisms
How do plant and animal cells differ?
Plant cell
Animal cell
1.
2.
3.
4.
1.
2.
3.
4.
Generally large in size
Cell wall is present
Plastids are present
Vacuoles are larger in size and
more in number
5. Centrioles are absent
Generally small in size
Cell wall is absent
Plastids are absent
Vacuoles are smaller in size and
less in number
5. Centrioles are present
Cell structure
1. Plasma Membrane
2. Nucleus
3. Cytoplasm
A.Cytosol
B. Cell Organelles
a) Endoplasmic reticulum
b) Golgi body
c) Lysosomes
d) Vacuoles
e) Mitochondria
f) Plastids
g) Centrosome
h) Cytoskeleton
Plasma Membrane
• Extremely delicate, thin and elastic semipermeable membrane
• Made up of phospholipid bilayer
• Thickness varies from 75-110 A˚
• Can be observed under an electron
microscope only
• Functions:
• Maintains shape & size of the cell
• Protects internal contents of the cell
• Regulates entry and exit of substances in
and out of the cell
• Maintains homeostasis
Cell wall
• Non-living and outermost covering of a cell
(plants & bacteria)
• Can be tough, rigid and sometimes flexible
• Made up of cellulose, hemicellulose and
pectin
• Functions:
• Provides definite shape, strength & rigidity
• Prevents drying up(desiccation) of cells
• Helps in controlling cell expansion
• Protects cell from external pathogens
Nucleus
• Dense spherical body located near the centre
of the cell
• Present in all the cells except red blood cells
and sieve tube cells
• Double layered covering called nuclear
membrane
• Functions:
• Control all the cell activities like metabolism,
protein synthesis, growth and cell division
• Nucleolus synthesizes ribonucleic acid (RNA)
to constitute ribosomes
• Store hereditary information in genes
Cytoplasm
• Jelly-like material formed by 80% of water
• Found between the plasma membrane and
the nucleus
• Contains a clear liquid portion called cytosol
and various organelles with distinct structure
and function
• Some of these organelles are visible only
under an electron microscope
• Granular and dense in animal cells and thin
in plant cells
Endoplasmic Reticulum
• Network of tubular and vesicular
structures which are interconnected with
one another
• Some parts are connected to the nuclear
membrane, while others are connected to
the cell membrane
• Two types: SER (lacks ribosomes) and RER
(studded with ribosomes)
• Functions:
• Gives internal support to the cytoplasm
• RER synthesize secretory proteins and
membrane proteins
• SER synthesize lipids for cell membrane
• In liver cells SER detoxify drugs & poisons
• In muscle cells SER store calcium ions
Golgi body
• Discovered by Camillo Golgi
• Formed by stacks of 5-8 membranous sacs
• Sacs are usually flattened and are called
the cisternae
• Functions:
• Modifies, sorts and packs materials
synthesized in the cell
• Delivers synthesized materials to various
targets inside the cell and outside the cell
• Produces vacuoles and secretory vesicles
• Forms plasma membrane and lysosomes
Lysosomes
• Small, spherical, single membrane sac
• Found throughout the cytoplasm
• Filled with hydrolytic enzymes
• Occur in most animal cells and in few type
of plant cells
• Functions:
• Help in digesting of large molecules
• Protect cell by destroying foreign invaders
like bacteria and viruses
• Degradation of worn out organelles
• In dead cells perform autolysis
Vacuoles
• Single membrane sac filled with liquid or
sap (water, sugar and ions)
• In animal cells, vacuoles are temporary,
small in size and few in number
• In plant cells, vacuoles are large and more
in number
• May be contractile or non-contractile
• Functions:
• Store various substances including waste
products
• Maintain osmotic pressure of the cell
• Store food particles in amoeba cells
• Provide turgidity and rigidity to plant cells
Mitochondria
• Small, rod shaped organelles bounded by
two membranes - inner and outer
• Outer membrane is smooth and encloses
the contents of mitochondria
• Inner membrane is folded in the form of
shelf like inward projections called cristae
• Inner cavity is filled with matrix which
contains many enzymes
• Contain their own DNA which are
responsible for many enzymatic actions
• Functions:
• Synthesize energy rich compound ATP
• ATP molecules provide energy for the vital
activities of living cells
Plastids
• Plastids are double membrane-bound
organelles found inside plants and some
algae.
• They are responsible for activities related
to making and storing food.
• They often contain different types of
pigments that can change the colour of
the cell.
Chromoplasts
• Chromoplasts are plastids that produce
and store pigments
• They are responsible for different colours
found in leaves, fruits, flowers and
vegetables.
Leucoplasts
• Leucoplasts are colourless plastids that
store foods.
• They are found in storage organs such as
fruits, tubers and seeds.
Chloroplasts
• Double membrane-bound organelles found
mainly in plant cells
• Usually spherical or discoidal in shape
• Shows two distinct regions-grana and
stroma
• Grana are stacks of thylakoids
(membranebound, flattened discs)
• Thylakoids contain chlorophyll molecules
which are responsible for photosynthesis
• Stroma is a colourless dense fluid
• Functions:
• Convert light energy into chemical energy in
the form of food
• Provide green colour to leaves, stems and
vegetables
Centrosome
• Centrosome is the membrane bound
organelle present near the nucleus
• Consists of two structures called
centrioles
• Centrioles are hollow, cylindrical
structures made of microtubules
• Centrioles are arranged at right angles
to each other
• Functions:
• Form spindle fibres which help in the
movement of chromosomes during cell
division
• Help in the formation of cilia and flagella
Cytoskeleton
• Formed by microtubules and
microfilaments
• Microtubules are hollow tubules made
up of protein called tubulin
• Microfilaments are rod shaped thin
filaments made up of protein called
actin
• Functions:
• Determine the shape of the cell
• Give structural strength to the cell
• Responsible for cellular movements
Methods for studying cells
Cell fractionation
pH
Buffers
Cell fractionation
• Cell fractionation is a procedure for rupturing cells, separating and suspension
of cell constituents in an isotonic medium in order to study their structure,
chemical composition, and function.
Cell fractionation involves 3 steps:
• Extraction: It is the first step toward isolating any sub-cellular structures. In
order to maintain the biological activity of organelles and bio-molecules, they
must be extracted in mild conditions. For these, the cells or tissues are
suspended in a solution of appropriate pH and salt content, (usually isotonic
solution).
Cell fractionation
• Homogenization: The suspended cells are then disrupted by the process of
homogenization.It is usually done by:
• Grinding (with abrasive materials, e.g sand)
• Shearing (with pestle, beads, blades)
• Chemical, ultrasonic or pressure disintegration, to obtain cell/tissue
homogenate
• Centrifugation: Centrifugation is used to separate cellular components.
Samples are spun at high speeds, resulting in a centrifugal force thousands to
hundreds of thousands times “normal” gravity after spinning:
• Pellet – what gets packed down to the bottom (densest material)
• Supernatant – solution above the pellet
Cell fractionation
• Density gradients can be used to subdivide pellet components based on their
density
• Can be used to better separate similar organelles from each other, for example
Golgi complex from ER
Intracellular pH
• pH is commonly expressed as –log₁₀[H⁺]
• I.e the negative log (base 10) of the molar concentrations of hydrogen ions H⁺
in solution
• E.g HCl solutions with pH values of 2.0 and 5.0
• Pure water has [H⁺]=10¯⁷ and thus pH=7.
• Acids have a high [H⁺] and thus a low pH.
• Bases have a low [H⁺] and thus a high pH
Ways to measure pH
• pH meter
• Electrode measures H⁺ concentration
• Must standardize (calibrate) before using
• Indicator dyes and test strips
• Less precise
• Each indicator is only good for a small pH
range (1-2 pH units)
• But may be good for field usage or
measuring small volumes, or dealing with
noxious samples.
Why is pH important in biology?
• Intracellular pH is an important aspect of the intracellular environment.
• Changes in intracellular pH can potentially affect virtually all cellular processes.
including metabolism, membrane potential, cell growth, movement of
substances across the surface membrane, state of polymerization of the
cytoskeleton and ability to contract in muscle cells.
• pH affects the solubility of many substances.
• pH affects the structure and function of most proteins - including enzymes.
• Many cells and organisms (esp. plants and aquatic animals) can only survive in
a specific pH environment.
Why is pH important in biology?
Importance of pH
• pH is an important quantity that reflects the chemical conditions of a solution.
• It controls the availability of nutrients, biological functions, microbial activity,
and the behavior of chemicals.
• Because of this, monitoring or controlling the pH of soil, water, and food or
beverage products is important for a wide variety of applications:
• Agriculture and gardening, Aquaculture and aquatic ecosystems, Water
treatment, Swimming pool maintenance, Food Industry, Brewing and
winemaking
Buffer solution
• Buffer Solution is a solution that resists a change in pH upon dilution or upon
the addition of small amounts of acid/alkali to them.
• It consists of a mixture containing a weak acid and the conjugate base of the
weak acid, or a weak base and the conjugate acid of the weak base.
• Blood: pH = 7.35-7.45
• Too acidic? Increase respiration rate expelling CO₂, driving the reaction to the
left and reducing H⁺ concentration.
• Excretory system – excrete more or less bicarbonate
Buffers in biological systems
Blood plasma
H2CO3(aq)
H+(aq) + HCO3-(aq)
In this buffer, carbonic acid (H2CO3) is the hydrogen-ion
donor (acid) and hydrogen carbonate ion (HCO3-) is the
hydrogen-ion acceptor (base)
 The pH of arterial blood plasma is 7.40
 Acidosis: pH below normal
 Alkalosis: pH above normal
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Buffers in biological systems
 Internal fluid of cells
H2PO4-(aq)
H+(aq) + HPO42-(aq)
This buffer system consists of dihydrogen phosphate ions
(H2PO4-) as hydrogen ion donor (acid) and hydrogen
phosphate ions (HPO42-) as hydrogen-ion acceptor (base)
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How buffer works
• Equilibrium between acid and base must be established.
• Example: Acetic acid/Acetate buffer:
CH₃COOH ↔ CH₃COO¯ + H⁺
• If more H⁺ is added to this solution, it simply shifts the equilibrium
to the left, absorbing H⁺, so the [H⁺] remains unchanged.
• If H⁺ is removed (e.g. by adding OH¯) then the equilibrium shifts to
the right, releasing H⁺ to keep the pH constant
Chemistry of buffer
• Kₐ = equilibrium constant for H⁺ transfer…
• Also described as the dissociation constant…the tendency of acid to dissociate.
AH
A¯ + H⁺
• Kₐ = [A¯] [H⁺]/ [AH]
= [base] [H⁺] / [acid]
• Weak acids have low value and contribute few H⁺ ions…
• Because we are usually dealing with very small concentrations, log values are
used…
• The log constant =
Relationship between pH and pKₐ
• First rearrange the first equation and solve for [H⁺]
• [H+] = Kₐ x [acid]/[base]
• Then take the -log of both sides
• -log₁₀[H⁺] = -log₁₀Kₐ -log₁₀ [acid]/[base]
pH
pKₐ
Henderson-Hasselbach equation
pH = pKₐ - log₁₀ [acid]/[base]
Relationship between pH and pKₐ
• What happens when the concentration of the acid and base are equal?
• Example: Prepare a buffer with 0.10M acetic acid and 0.10M acetate
pH = pKₐ - log₁₀ [acid]/[base]
pH = pKₐ - log₁₀ [0.10]/[0.10]
pH=pKₐ
• Thus, the pH where equal concentrations of acid and base are present is
defined as the pKₐ
• A buffer works most effectively at pH values that are + or - 1 pH unit from the
pKa (the buffer range)
Introduction to Chromatography
• Chromatography is a physical technique of separation, identification, and
purification of components of a mixture.
• It is used in many areas of study, particularly in chemistry, biology, and
medicine.
• Pigments, dyes, amino acids, vitamins, polymers, etc can be separated by
using the chromatography technique.
Chromatography
• Chromatography is a separation technique based on the different interactions
of compounds with two phases, a mobile phase and a stationary phase, as
the compounds travel through a supporting medium.
Components:
• Mobile phase: a solvent that flows through the supporting medium
• Stationary phase: a layer or coating on the supporting medium that interacts
with the analytes
• Supporting medium: a solid surface on which the stationary phase is bound or
coated
Chromatography
• Those components that are strongly retained by the stationary phase move
slowly with the flow of mobile phase.
• In contrast, components that are weakly held by the stationary phase travel
rapidly.
• As a consequence of these differences in mobility, sample components
separate into discrete bands that can be analyzed qualitatively and/or
quantitatively.
Chromatography
Chromatography
Chromatography
• There are many types of chromatography e.g.,
• liquid chromatography, gas chromatography, ion-exchange chromatography,
affinity chromatography
• All of these employ the same basic principles.
Chromatography
Chromatography
• The analyte is loaded over the silica bed packed in the column (stationary
phase) and allowed to adhere to the silica.
• Solvent (mobile phase) is then made to flow through the silica bed under
gravity.
• The different components of the analyte exhibit varying degrees of adhesion
to the silica and as a result they travel at different speeds through the
stationary phase as the solvent flows through it, indicated by the separation of
the different bands.
• The components that adhere more strongly to the stationary phase travel
more slowly compared to those with a weaker adhesion.
• Analytical chromatography can be used to purify compounds ranging from
milligram to gram scale.
Thin Layer Chromatography
• The separation relies on the relative affinity of compounds towards both
phases.
• The compounds in the mobile phase move over the surface of the stationary
phase.
• The movement occurs in such a way that the compounds which have a higher
affinity to the stationary phase move slowly while the other compounds travel
fast.
• Therefore, the separation of the mixture is attained.
• On completion of the separation process, the individual components from the
mixture appear as spots at respective levels on the plates.
• Their character and nature are identified by suitable detection techniques.
Experiment on Chromatographic separation
.
Experiment on TLC separation
• Take a few leaves and crush them in a mortar.
• Spot a drop of the leaf extract on a strip of chromatographic paper ~ 0.5 cm
above the edge of the paper. (Chromatographic paper is made of cellulose and
is quite polar in nature)
• Place the strip of paper in a jar that contains a small volume of propanone
(acetone). There should be just enough propanone that the edge of the paper
dips in it comfortably.
• Place a lid on the jar to avoid any evaporation of the solvent.
• Let the solvent rise up the paper by capillary action.
• Remove the paper strip from the jar once the solvent has reached the ‘solvent
front’ level.
What do you think you will notice?
Uses of chromatography
• Drug testing: Urine is analyzed for known drugs
• Firefighting: To identify chamical substances found in fires… e.g during fire
disaster
• Forensics: Separate and analyze substances found on the crime scene
• Pharmaceutical: Chemists make and purify compounds
Electrophoresis
• Electrophoresis may be defined as the migration of the charged particle
through a solution under the influence of an external electrical field.
• It is a technique used for the separation and analysis of nucleic acids i.e., DNA,
RNA, and proteins. The separation is based on the size, charge, density, and
purity of molecules.
• Ions that are suspended between two electrodes tend to travel toward the
electrodes that bear opposite charges.
Principle of Electrophoresis
• The rate of migration of an ion in an electrical field depends on 5 factors:
1. Net charge of molecule
2. Size and shape of particle
3. Strength of electrical field
4. Properties of supporting medium
5. Temperature of operation
Migration depends on…
• Strength of electric fields.
• Temperature
• Features of the molecule
• –Net charge of molecule
• –Size of molecule
• –Shape of molecule
• Features of the Gel
• –Gel type
• –Gel concentration
• Buffer Type/pH.
What can be separated?
• Nucleic acids
• Proteins
• Peptides
• Amino acids
• Organic acids/bases
• Drugs
• Pesticides
• Non organic anions/cations.
• Everything that can carry a charge.!
Molecular pathology
• Nucleic acids.
• Determining quality of DNA/RNA
• Analyses of PCR products
• Mutation detection
• Southern and Northern blotting
• Sequencing
• Proteins
• Western blotting
• Protein purification
Types of electrophoresis
• Gel electrophoresis
• Agarose gel
• Polyacrylamide gel
• Capillary Electrophoresis
• Isoelectric focusing
• 2D electrophoresis