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| Fig. Protein purification steps |
Proteins differ in their molecular size and
charge. They can be separated on the basis of their following properties:
1.
molecular size
2.
solubility
3.
electrical charge
4.
adsorption properties
5.
specific bioaffinity.
Many techniques for protein purification
exist, but the emphasis here is on some of the most popular procedures and the
principles involved in their use.
Protein fractionation is required to separate and characterize a protein
in detail.
Differential Centrifugation
A typical crude broken cell preparation
contains disrupted cell membranes, cellular organelles and a large number of
soluble proteins all dispersed in an aqueous buffered solution. The membranes
and the organelles can be separated from the soluble proteins by differential
centrifugation.
This type of centrifugation involves the
use of different speeds and different durations. For eg. If the protein of
interest is in the mitochondrial fraction, the crude cell lysate is first
centrifuged at 1000g for removing nuclei, debris etc. The supernatant contains
among other elements the mitochondria, which can be pelleted at 3300g for 10
minutes.
Salt Fractionation
Proteins show a variation in solubility
that depends on the concentration of salts in the solution. This method is
frequently used to separate serum proteins into albumins and globulins. Albumin is soluble in water whereas globulins
are not. Globulins are soluble in weak
salt solutions, going into solution at salt concentrations of 0.1 mol/L. This
phenomenon called "salting
in". This is thought to be due to electrostatic attraction between
salt ions and the charged groups on the protein, which decreases the
intermolecular electrostatic attraction of proteins & increases the
interactions of protein molecules with water, a polar solvent, thus making them
soluble. Salts with divalent ions are
more effective than those with monovalent ions.
As the salt concentration is increased,
however, salt ions compete for the water molecules of hydration of the hydrated
groups of proteins, resulting in the decreased solubility and precipitation of
protein out of the solution. The larger
proteins are usually precipitated first. This phenomenon is referred to as “salting out” of protein. Ammonium sulphate is commonly used for
salting out proteins. Globulins are the proteins precipitated by half
saturation of a solution with ammonium sulphate, while Albumin is precipitated
on fully saturating the solution.
Electrophoresis
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| Fig. Electrophoresis apparatus |
Electrophoresis
is the movement of charged particles through an electrolyte in an electric
field. The positively charged particles move towards
the cathode and the negative ions to the anode. The rate of migration of
particles of like charge will depend among other things on the number of
charges it carries. Different rates of migration separate a complex mixture
such as plasma proteins into a number of fractions according to mobility.
Electrophoresis is not used to purify proteins because some alteration in
protein structure and ultimately function. This is used as a analytical method.
It permits to estimate number of proteins in a mixture. This is also useful to
determine isoelectric point and approximate molecular weight.
Chromatography
This technique was originally used to separate chlorophyll from plant extracts on silica, hence the name chromatography, which means separation of colored compounds. It is the name given to any technique in which the members of a group of similar substances are separated by a continuous redistribution between two phases. One is the stationary phase, which may be solid, liquid, gel or solid/liquid mixture which is immobilised. The second mobile phase may be liquid or gaseous and flows over or through the stationary phase. The choice of stationary or mobile phases is made so that the compounds to be separated have different distribution coefficients.
The movement of any substance being
chromatographed is the outcome of
a. Forces resulting from its
interaction with the mobile phase which tend to pull it along with the mobile
phase
b. Forces resulting from its
attraction to the stationary phase, which tend to retard its movement.
Since the forces are different for
different substances chromatography will be able to separate out the components
of a mixture of substances.
Based on the properties of the stationary
phase and the acting forces, several different types of chromatographic
procedures have been described.
1. Adsorption chromatography: Adsorption
equilibrium between a stationary solid and a mobile liquid phase. This is used
to separate the non ionic, water insoluble compounds such as vitamins,
triglycerides drugs etc.
2. Partition chromatography: Partition
equilibrium between a stationary liquid and a mobile liquid phase. This is used
to analyze drugs, insecticides, pestisides,amino acids .
3. Gas-Liquid chromatography: Partition
equilibrium between a stationary liquid and a mobile gaseous phase. The mobile
phase is generally an inert gas - steroid separation.
4. Ion-exchange chromatography: Ion exchange
equilibrium between an ion-exchange stationary resin phase and a mobile
electrolyte phase. So amino acids proteins which have ionized groups can be
separated on the basis of this.
5. Gel permeation chromatography: Equilibrium
between a liquid phase inside and outside a porous molecular sieve depending on
molecular size - preparative procedure.
6. Affinity chromatography: Equilibrium between a
macromolecule and a small molecule (Ligand) for which it has high affinity -
monoclonal antibody purification.
Types of solid support media
a.
Dextran (Sephadex),
b.
Agarose (Sepharose),
c.
Silica Gel,
d.
Cellulose (Sephacel)
Dextran is a polysaccharide built from glucose residues. When the dextran is
cross-linked by epichlorhydrin to form a gel, the polysaccharide chains of the
gel from a 3-dimensional network. The product is insoluble unless it is broken
down chemically. Dextran gels are commercially known under the name of
Sephadex. The different types of sephadex beads differ in their degree of
cross-linking. The various types are characterized by the letter G, followed by
a number. Based on their extent of cross-linkage, proteins of different MW are
separated from each other.
Principle of the method:
In the column, proteins of different
molecular size penetrate into the internal pores of the beads to different
degrees and thus travel down the column at different rates. In other words,
very large protein molecules cannot enter the pores of the beads and they are
excluded, while small proteins can enter the pores of the beads freely and are
retarded. This is called molecular sieving or gel permeation. The different
peaks of separation can be monitored by UV detectors and recorded. The peak
containing required material can be identified depending upon the properties of
the substance for e.g. measurement of enzyme activity, Ag-Ab binding, etc.
Bed volume
Volume of the gel which is available to
those molecules which are smaller than the fractionation range for the gel.
Void volume
Volume of the liquid in the interstitial
spaces between beads of the gel. It is available to those molecules whose size
is bigger than the fractionation range of the gel eg. Blue Dextran (2 million
MW).
Applications of protein purification:
1.
Protein concentration
2.
Monoclonal antibody
purification
3.
Removal of endotoxins from a
protein solution
4. Removal of proteins from a sugar solution.
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| Fig. Paper chromatography |
Amino acid separation by paper chromatography:
In this, the amino acids partition themselves
on the paper between an organic solvent of choice and water.
Procedure: There are two techniques, which may be employed for the development of
paper chromatograms: ascending or descending methods. In ascending technique, the paper is kept in
a glass jar containing the solvent such that the lower edge of the paper is in
contact with the solvent. The sample is applied to the position just above the
surface of the solvent. As the solvent moves vertically up the paper by
capillary action, separation of the amino acids is achieved. In the descending
techniques, the solvent moves downwards under gravity.
Component
Detection: Spraying of the paper with ninhydrin will stain
amino acids. The identification of a
given amino acid may be on the basis of its relative fraction (Rf) value,
which is defined as:
This value is constant for a particular
compound under standard conditions.
Quantification of the amino acid may be carried out after eluting it
with a suitable solvent.
Clinical applications:
1. Screening and diagnosis of
inherited disorders of amino acid metabolism e.g. Phenylketonuria and
Tyrosinaemia.
2. Peptide fingerprinting for the
diagnosis of diseases like sickle cell anemia.
3. One dimensional thin layer chromatography (TLC) can detect
aminoacidopathies and is the most adequate for screening.
Specimens
reference solution (mixture of known amino acids at known concentration) and
controls are applied to thin layer plates, run in an appropriate solvent-vapor
system and then dried and stained with ninhydrin
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