10.analytical biochemistry

Analytical Biochemistry: Isolation of
Protein and Protein Sequencing
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CELL
DISRUPTION
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explanation 
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Extraction buffer
explanation 

1. Ionic strength of 0.1 – 0.2 M and pH 7.0 - 8.0
2. Isotonic solution e.g 0.25 mM sucrose solution
3. Tris buffer and phosphate buffer
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Other additives
1. Antioxidant: Cellular proteins are in highly reducing environment,
but when released into the buffer it is exposed to a more oxidising
environment. Since most proteins contain a number of free
sulphydryl groups (from the amino acid cysteine) these can
undergo oxidation to give inter- and intramolecular disulphide
bridges. To prevent this, reducing agents such as dithiothreitol, β-
mercaptoethanol, cysteine or reduced glutathione are often
included in the buffer.
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Other additives
2. Enzyme inhibitors: The disruption of cell’s integrity, proteolytic enzymes are
released for example from lysosomes. To slow down unwanted proteolysis, all
extraction and purification steps are carried out at 4ºC, and in addition a range of
protease inhibitors is included in the buffer. Each inhibitor is specific for a
particular type of protease, for example serine proteases, thiol proteases, aspartic
proteases and metalloproteases. Common examples of inhibitors include: di-
isopropylphosphofluoridate (DFP), phenylmethyl sulphonylfluoride (PMSF) and
tosylphenylalanyl-chloromethylketone (TPCK) (all serine protease inhibitors);
iodoacetate and cystatin (thiol protease inhibitors); pepstatin (aspartic protease
inhibitor); EDTA and 1,10-phenanthroline (metalloprotease inhibitors); and
amastatin and bestatin (exopeptidase inhibitors).

Other additives
3. Enzyme substrates and cofactors: Low levels of substrate are often
included in extraction buffers when an enzyme is purified, since binding of
substrate to the enzyme active site can stabilise the enzyme during
purification processes. Where relevant, cofactors that otherwise might be
lost during purification are also included to maintain enzyme activity so
that activity can be detected when column fractions, etc. are screened.
4. EDTA: This can be present to remove divalent metal ions that can react
with thiol groups in proteins giving mercaptids.

Other additives
3. Polyvinylpyrrolidone (PVP): Plant tissues contain considerable amounts of
phenolic compounds (both monomeric, such as p -hydroxybenzoic acid, and
polymeric, such as tannins) that can bind to enzymes and other proteins by non-
covalent forces, including hydrophobic, ionic and hydrogen bonds, causing
protein precipitation. Insoluble PVP (which mimics the polypeptide backbone) is
therefore added to adsorb the phenolic compounds which can then be removed by
centrifugation. Thiol compounds (reducing agents) are also added to minimise the
activity of phenol oxidases, and thus prevent the formation of quinones.
4. Sodium azide: For buffers that are going to be stored for long periods of time,
antibacterial and/or antifungal agents are sometimes added at low concentrations.
Sodium azide is frequently used as a bacteriostatic agent.

CELLS
explanation 

Cell types
1. Mammalian cells: 10 mm in diameter, enclosed by a plasma membrane, weakly
supported by a cytoskeleton. Lack any great rigidity and are easy to disrupt by
shear forces.
2. Plant cells: 100 mm in diameter, fairly rigid cell wall, comprising carbohydrate
complexes and lignin or wax that surround the plasma membrane. Although the
plasma membrane is protected by this outer layer, the large size of the cell still
makes it susceptible to shear forces.

Cell types
3. Bacteria: 1 to 4 mm, rigid cell walls. Bacteria can be classified as either Gram
positive or Gram negative depending on whether or not they are stained by the
Gram stain (crystal violet and iodine). In Gram-positive bacteria, the plasma
membrane is surrounded by a thick shell of peptidoglycan (20 – 50 nm), which
stains with the Gram stain. In Gram- negative bacteria (e.g. Escherichia coli) the
plasma membrane is surrounded by a thin (2–3 nm) layer of peptidoglycan but
this is compensated for by having a second outer membrane of
lipopolysaccharide.
4. Fungi and yeast: Filamentous fungi and yeasts have a rigid cell wall that is
composed mainly of polysaccharide (80 –90%). In lower fungi and yeast the
polysaccharides are mannan and glucan. In filamentous fungi it is chitin cross-
linked with glucans. Yeasts also have a small percentage of glycoprotein in the cell
wall, and there is a periplasmic space between the cell wall and cell membrane. If
the cell wall is removed the cell content, surrounded by a membrane, is referred to

METHODS OF CELL DISRUPTION
explanation 

Methods of Cell Disruption
1. Blenders: These are commercially available, although a typical domestic
kitchen blender will suffice. This method is ideal for disrupting mammalian
or plant tissue by shear force. Tissue is cut into small pieces and blended, in
the presence of buffer, for about 1 min to disrupt the tissue, and then
centrifuged to remove debris. This method is inappropriate for bacteria and
yeast, but a blender can be used for these microorganisms if small glass
beads are introduced to produce a bead mill. Cells are trapped between
colliding beads and physically disrupted by shear forces.

2. Grinding with abrasives: Grinding in a pestle and mortar, in the presence
of sand or alumina and a small amount of buffer, is a useful method for
disrupting bacterial or plant cells; cell walls are physically ripped off by the
abrasive. However, the method is appropriate for handling only relatively
small samples. The Dynomill is a large-scale mechanical version of this
approach. The Dynomill comprises a chamber containing glass beads and a
number of rotating impeller discs. Cells are ruptured when caught between
colliding beads. A 600 cm3 laboratory scale model can process 5 kg of
bacteria per hour.

3. Presses: The use of a press such as a French Press, or the Manton–Gaulin Press,
which is a larger-scale version, is an excellent means for disrupting microbial cells.
A cell suspension (approximately 50 mL) is forced by a piston-type pump, under
high pressure (10 000 PSI = lbfin.-2 approximately 1450 kPa) through a small
orifice. Breakage occurs due to shear forces as the cells are forced through the
small orifice, and also by the rapid drop in pressure as the cells emerge from the
orifice, which allows the previously compressed cells to expand rapidly and
effectively burst.
4. Enzymatic methods: The enzyme lysozyme, isolated from hen egg whites, cleaves
peptidoglycan. The peptidoglycan cell wall can therefore be removed from Gram-
positive bacteria by treatment with lysozyme, and if carried out in a suitable buffer,
once the cell wall has been digested the cell membrane will rupture owing to the
osmotic effect of the suspending buffer. Gram-negative bacteria can similarly be
disrupted by lysozyme but treatment with EDTA (to remove Ca2+, thus destabilising
the outer lipopolysaccharide layer) and the inclusion of a non-ionic detergent to
solubilise the cell membrane are also needed. This effectively permeabilises the
outer membrane, allowing access of the lysozyme to the peptidoglycan layer.

5. Sonication: This method is ideal for a suspension of cultured cells or microbial
cells. A sonicator probe is lowered into the suspension of cells and high frequency
sound waves (<20 kHz) generated for 30 – 60 s. These sound waves cause
disruption of cells by shear force and cavitation. Cavitation refers to areas where
there is alternate compression and rarefaction, which rapidly interchange. The gas
bubbles in the buffer are initially under pressure but, as they decompress, shock
waves are released and disrupt the cells. This method is suitable for relatively small
volumes (50–100 cm3). Since considerable heat is generated by this method,
samples must be kept on ice during treatment.

CENTRIFUGATION
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more explanation 
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Centrifugation technique
1. Employs sedimentation
2. It is a powerful tool use in cutting edge biochemical research such
as genomic and proteomic research
3. Centrifugation is important to purify cells, subcellular organelles,
viruses, proteins and nucleic acids as an integral part of their work.

Principles
The rate of separation in a suspension of particles by way of gravitational force mainly
depends on the particle size and density.
This can be explained by stoke law:
v = sedimentation rate or velocity of the sphere
d = diameter of the sphere
p = particle density
L = medium density
n = viscosity of the medium
g = gravitational force

Thus from the Stoke’s equation the following affect the separation of particles under
centrifugal force i.e the important behaviours of particles under centrifugal force
1. The rate of particle sedimentation is proportional to the particle size i.e the massive
the biological particle moves faster in a centrifugal field
2. The sedimentation rate is proportional to the difference between the density of the
particle and medium
3. The sedimentation rate is zero when the particle density is the same as the medium
density.
4. The sedimentation rate decreases as the medium viscosity increases.
5. The sedimentation rate increases as the gravitational force increases.
Principles

TYPES OF CENTRIFUGATION TECHNIQUES
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1. Differential centrifugation and
2. Density gradient centrifugation, which can be further
classified as
a. Rate-zonal and
b. Isopycnic centrifugation
Types

DIFFERENTIAL CENTRIFUGATION
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1. It is also called differential pelleting
2. The simplest form of separation involving successive centrifugation (single or
repeated steps) with increasing centrifugal force (g).
3. Particles of different densities or sizes in a suspension will sediment at different
rates, with the larger and denser particles sedimenting faster.
4. During subcellular fractionation, various markers can be used as a quality control
measure, giving an assessment of the quality of separation of individual fractions
e.g.
a. DNA can be used as a marker for the step sedimenting nuclei,
b. while the enzyme succinate dehydrogenase can be used as a marker for the step
sedimenting mitochondria.
Differential centrifugation

Minced liver
Liver homogenate
Homogenization
in
sucrose
solution
Homogenate in
centrifuge tube
Nuclei & cell
debris
Supernatan
t
Mitochondria
Supernata
nt
Lysosome
&
Perixosome
Supernatan
t
Endoplasmic reticulum
microsome & golgi
apparatus
Soluble
proteins
Mince into pieces
Liver
600 g x 5
min

DENSITY GRADIENT CENTRIFUGATION
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1. This was developed to address the main limitation of differential centrifugation by
allowing preparation of homogeneous organelle fractions
2. It also facilitates detailed analysis of cell organelles and function
3. As the name implies, this technique utilizes a specific medium that gradually
increases in density from top to bottom of a centrifuge tube in the separation of
particles on the basis of their size, shape, and density.
4. Implying that under centrifugal force, particles will move through the medium and
density gradient and stop (are suspended) at a point in which the density of the
particle equals the density of the surrounding medium.
5. At this point, the particles appear as bands or zones in the gradient with the more
dense and larger particles migrating furthest
Density gradient centrifugation

The medium used can be classified into four and depends on the
desired outcome
1. Alkali metal salts (e.g. caesium chloride)
2. Neutral water-soluble molecules (e.g. sucrose)
3. Hydrophilic macromolecules (e.g. dextran); and
4. Synthetic molecules (e.g. methyl glucamine salt of triiodobenzoic
acid).
Types of density gradient media

1. The problem of cross-contamination of particles of different sedimentation rates
may be avoided by layering the sample as a narrow zone on top of a density
gradient.
2. In this way, the faster sedimenting particles are not contaminated by the slower
particles as occurs in differential centrifugation.
3. The narrow load zone limits the volume of sample (typically 10%) that can be
accommodated on the density gradient.
4. The gradient stabilizes the bands and provides a medium of increasing density and
viscosity.
5. The speed at which particles sediment depends primarily on their size and mass
instead of density.
Rate zonal

Time of
centrifugation
Centrifugal
field
Small
Medium
Large

1. Also called buoyant or equilibrium separation, particles are separated solely on the
basis of their density.
2. Particle size only affects the rate at which particles move until their density is the
same as the surrounding gradient medium.
3. The density of the gradient medium must be greater than the density of the particles
to be separated.
4. By this method, the particles will never sediment to the bottom of the tube, no
matter how long the centrifugation time
5. Upon centrifugation, particles of a specific density sediment till they reach the point
where their density is the same as the gradient media (i.e., the equilibrium
position).
6. The gradient is then said to be isopycnic and the particles are separated according
to their buoyancy.
Isopycnic

SPECTROPHOTOMETRY
explanation 

Spectrophotometry is a scientific method based on the absorption of light
by a substance, and takes advantage of the two laws of light absorption.
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Theories of absorption
When a material absorbs light, valence (outer) electrons are promoted from their
normal (ground) states to higher energy (excited) states

Origin of absorption
explanation 

1. Valence electrons can generally be found in one of three types of electron orbital:
2. single, or σ , bonding orbitals;
3. double or triple bonds (π bonding orbitals); and
4. non-bonding orbitals (lone pair electrons).
5. Sigma bonding orbitals tend to be lower in energy than π bonding orbitals, which
in turn are lower in energy than non-bonding orbitals. When electromagnetic
radiation of the correct frequency is absorbed, a transition occurs from one of these
orbitals to an empty orbital, usually an antibonding orbital, σ* or π*.

Laws of absorption
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There are two laws of absorption, Lambert’s and Beer’s laws
1. Lambert’s Law
The proportion (fraction) of light absorbed by a medium is independent of the intensity
of incident light. A sample that absorbs 75% (25% transmittance) of the light will
always absorb 75% of the light, no matter the strength of the light source.
Lambert’s law is expressed as
Where I = Intensity of transmitted light
Io = Intensity of the incident light
T = Transmittance

1. Thus transmittance of a sample held in a cell (or cuvette) is the fraction of incident
light that is transmitted. The transmittance is usually defined for a single
wavelength i.e. for monochromatic light.
2. This allows different spectrophotometers with different light sources to produce
comparable absorption readings independent of the power of the light source.

2. Beer’s Law
The absorbance of light is by an absorbing medium is directly proportional to both the
concentration of the absorbing medium and the thickness of the medium. In
Spectrophotometry the thickness of the medium is called the path length. In normal
cuvette-based instruments the path length is 10 mm (1 cm). Beer’s law allows us to
measure samples of differing path length, and compare the results directly with each
other.
A= εCl
Where A = absorbance
ε = molar absorption (extinction) coefficient of the absorbing medium unit mol-1 dm3
cm-1 . However, the unit is not always quoted by convention
C = concentration of the absorbing species in mol dm-3
L = path length = 1 cm

Beer-Lambert’s Law
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The conditions under which Beer-Lambert’s law will only be valid are:
1. The absorbing solution must be of low concentration (i.e dilute), as higher
concentration might lead to association of molecules and therefore cause deviation
from ideal behavior
2. The light ray passing through the absorbing medium must be a monochromatic
light
3. The path length, l, (cm) of the light path through the sample must equal 1
4. There is no complex formation

Spectrophotometer
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For spectrophotometer to perform this function, it is designed with the following as its
component:
1. Light source, which emits light along a broad spectrum,
2. Focusing device/culminator, which transmits an intense beam of light,
3. Monochromator, which selects the desired wavelength
4. A device for selecting the desired wavelength
5. A compartment in which the cuvette or test tube containing the sample is placed
6. Photoelectric detector, which measure the transmitted light
7. Electric meter, which record the output of the detector

Applications of spectrophotometer
explanation 

The applications of spectrophotometer includes:
1. Estimation of nucleic acids (DNA or RNA). This is done at 260 nm or 280 nm.
Purines and pyrimidines absorbed light at 260 nm
2. Application in protein
➢ Lowry protein assay
➢ Bicinchoninic Acid (BCA) Assay
➢ Biuret method
➢ Bradford assay
➢ Kjedhal analysis

CHROMATOGRAPHY
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Chromatography is usually introduced as a technique for separating
and/or identifying the components in a mixture. The basic principle is
that components in a mixture have different tendencies to adsorb onto a
surface or dissolve in a solvent. It is a powerful method in industry,
where it is used on a large scale to separate and purify the
intermediates and products in various syntheses.

Basic principles
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The principle of all chromatographic techniques is the partition or
distribution of solute between two immiscible phases. This phenomenon
is often refer to as distribution or partition coefficient (Kd). For such
two immiscible phases, the value for this coefficient is a constant at a
given temperature and is given by the expression:

All chromatographic systems consist of:
1. Stationary phase (solid, gel, liquid or solid/liquid mixture
immobilized)
2. Mobile phase (liquid or gaseous). The mobile phase is passed over
or through the stationary phase after the mixture of analytes to be
separated has been applied to the stationary phase.

These phase makes these techniques to rely on any of the following
phenomena:
1. Adsorption;
2. Partition;
3. Ion exchange; or
4. Molecular exclusion.
When considering chromatography, two major principles require
attention, namely retention and plate theory.

Retention theory
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Retention measures, retention time Rt or retention factor Rf, the speed
at which a biomolecule moves in a given chromatographic system.
1. Retention time (Rt) is used in high-performance liquid
chromatography (HPLC) and gas chromatography,
2. While Rf is used in paper and thin-layer chromatography. Rf is
calculated using the following formula:

The following factors affect the Rf values of biomolecules:
1. Temperature
2. Humidity
3. Solvent
4. Type of stationary phase (paper, alumina and silica)
Arising from the above, it is important to report along with Rf values,
the exact details of solvent used, temperature, stationary phase and
humidity for reproducibility of data by other scientist who may often be
in other laboratories.

Plate theory
explanation 

Plate theory measures the rate of migration of a biomolecule through a
stationary phase in a given chromatography system. This migration is
determined by the distribution ratio (Kd), otherwise known as
distribution constant (Kc) that is given by the following formula:
Biomolecules with large Kc values will be retained more strongly by the
stationary phase than those with smaller Kc values. In other words, as
Kc increases it takes longer for solutes to separate.

Classes of chromatography
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1. Adsorption chromatography
2. Partition chromatography

Adsorption chromatography
explanation 

1. This is the first chromatography to be developed.
2. It has a solid stationary phase and liquid or gaseous mobile phase
3. The different solutes travelled different distances through the solid,
carried along by the solvent.
4. Each solute has its own equilibrium between adsorption onto the surface
of the solid and solubility in the solvent, the least soluble or best adsorbed
ones travel more slowly.
5. The result is a separation into bands containing different solutes.

Partition chromatography
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1. In partition chromatography, the stationary phase is a non-volatile
liquid, which is held as a thin layer (or film) on the surface of an
inert solid.
2. The mixture to be separated is carried by a gas or a liquid as the
mobile phase.
3. The solutes distribute themselves between the moving and the
stationary phases, with the more soluble component in the mobile
phase reaching the end of the chromatography column first.
4. Paper chromatography is an example of partition chromatography.

Common chromatographic techniques
explanation 

1. Paper chromatography
2. Thin layer chromatography
3. Ion-exchange chromatography
4. Molecular size exclusion chromatography
5. Affinity chromatography

Thin layer
chromatography
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TLC
plate
Point of
origin
A B C
Solvent
front

Ion-exchange chromatography
explanation 

Principle
This form of chromatography relies on the attraction between
oppositely charged stationary phase, known as an ion exchanger, and
analyte.
The following are important terms
1. Cation exchanger: possess negatively charged ion, thus attracts
positively charged biomolecules
2. Anion exchanger: are positively charged, as such attracts
negatively charged biomolecules

Molecular sieve chromatography
explanation 

Principle
Molecular size exclusion separates biomolecules on the basis of their molecular size
and shape.
1. Unlike other types of chromatography, no equilibrium state is established between
the solute and the stationary phase. Instead, the mixture passes as a gas or a
liquid through a porous gel.
2. The pore size is designed to allow the large solute particles to pass through
uninhibited (i.e completely excluded from the pores).
3. The small particles, however, permeate the gel and are slowed down so the
smaller the particles, the longer it takes for them to get through the column, hence
appearing last in the eluate. Thus separation is according to particle size.

Principle
1. Unlike other separation techniques (other chromatographic techniques,
electrophoresis and centrifugation), this technique relies on differences in the
physical properties of the analytes.
2. It exploits the unique property of extremely specific biological interactions to
achieve separation and purification.
3. Consequently, affinity chromatography is theoretically capable of giving
absolute purification, even from complex mixtures, in a single process.
4. The technique was originally developed for the purification of enzymes, but it
has since been extended to nucleotides, nucleic acids, immunoglobulins,
membrane receptors and even to whole cells and cell fragments.

Gas chromatography
explanation 

Principle
1. This technique uses a gas as the mobile phase, and the stationary phase can
either be a solid or a non-volatile liquid (in which case small inert particles
such as diatomaceous earth are coated with the liquid so that a large surface
area exists for the solute to equilibrate with).
2. If a solid stationary phase is used the technique is described as gas-solid
adsorption chromatography, and if the stationary phase is liquid it is called
gas-liquid partition chromatography.
3. The latter is more commonly used, but in both cases the stationary phase is held
in a narrow column in an oven and the stationary phase particles are coated
onto the inside of the column.

High pressure liquid chromatography
explanation 

ELECTROPHORESIS
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Electrophoresis describes the migration of a charged particle under the
influence of an electric field.
Biological molecules, such as amino acids, peptides, proteins,
nucleotides and nucleic acids, possess ionisable groups. Thus, at any
given pH, exist in solution as electrically charged species either as
cations (+) or anions (-).
Under the influence of an electric field these charged particles
will migrate either to the cathode or to the anode, depending on the
nature of their net charge.

Support media
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1. Cellulose acetate (formed from cellulose and acetic anhydride)
2. Agarose
3. Polyacrylamide gel

Factors affecting separation of biomolecules
in an electric field

1. Frictional force (V =Eq/f)
2. Heat
a. An increased rate of diffusion of sample and buffer ions leading to broadening
of the separated samples.
b. The formation of convection currents, which leads to mixing of separated
samples.
c. Thermal instability of samples that is rather sensitive to heat. This may
include denaturation of proteins (and thus the loss of enzyme activity).
d. A decrease of buffer viscosity, and hence a reduction in the resistance of the
medium

PROTEIN SEQUENCE DETERMINATION

Amino terminal sequence determination
explanation 

2. Dansyl chloride degradation

Protease digestion
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Specificities of Endopeptidases

Carboxyl terminal sequence determination
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Specificities of Exopeptidases

10.analytical biochemistry

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