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  Separation of Peptides-II, Biology tutorial

Further technique to Separation of Peptides:

Proteins can be purified and separated by just taking benefit of differences in their solubility and in their sizes. These are not the only parameters utilized in purifying polypeptides. As proteins and peptides are charged molecules and have different functional groups which could act as potential anchors for other molecules, these dual properties have been utilized to create separation methods for protein/peptides and even other macromolecules with such properties like carbohydrates and nucleic acids.

Separation Based on Charges:

Polypeptides have different charged groups and, in solution, do have characteristic pH at which they suppose electrical neutrality (net charge of zero). This pH is known as isoelectric point (pI). Both electrophoresis and isoelectric focus on separate proteins/peptides on the basis of rates of their movement in the electric field.

i) Method of Electrophoresis:

Term electrophoresis refers to migration of charged molecules in the electric field. Electrophoretic method is extensively utilized analytical separation process for proteins and peptides (among several other charged biomolecules). Under the effect of electric field of few milliamperes of current, proteins migrate and separate. Those with the net negative charge migrate towards anode while those with net positive charge move towards cathode of electrophoresis appliance. This migration is stated as electrophoretic mobility (μ) and is mathematically given as: μ = v/E=q/f . Where v is velocity of migration, E is electric field strength; q is charge on ion while f is frictional coefficient that is a function of size, shape, state of salvation of ion and viscosity of solvent. Every protein has its characteristic electrophoretic mobility. The above equation exhibits that μ (rate of migration) depends on many factors, like:

a) Potential gradient of voltage applied;

b) Net charge on protein/peptide molecule- molecule with larger net charge migrate faster; and

c) Molecular size and shape (from f in equation)-larger proteins migrate at the slower rate than smaller ones.

There are numerous kinds of electrophoresis. In gel electrophoresis, one of the most common electrophoretic methods, electrophoresis is performed in polyacrylamide gel that acts as molecular sieve. Molecular separation is based on gel filtration and electrophoretic mobility.

ii) Isoelectric Focusing:

In isoelectric focusing, the mixture of low molecular weight polyaminopolycarboxyl acid ampholytes is utilized to establish pH gradient by distributing them across a gel. When the solution having proteins is applied on gel, every protein moves until it gets to pH which equals its isoelectric point. Proteins with different isoelectric points, thus, separate on gel even if they have the same size on gel filtration. Isoelectric focusing, thus, has very high resolution power. To get even higher resolution, isoelectric focusing has been combined with SDS-PAGE to make the technique called as two dimensional electrophoresis.

Separation Based on Affinity to other Biological Molecule:

i) Ion Exchange Chromatography:

This method depends on electrostatic interactions between charged groups of proteins/peptides and oppositely charged groups on ion exchanger (also known as ion exchange resin). These ion exchangers comprises of insoluble matrix to which charged groups have been covalently bound. There are two kinds of ion exchangers. Ionic groups in cation exchangers are negatively charged while those of anion exchangers are positively charged.

Carboxymethyl-sephadex, phosphocellulose and DEAE-cellulose are ion exchangers of choice for protein /peptide separation. Separation process engages passing sample solution through the column packed with the kind of ion exchanger using the buffer solution. Separation is performed by reversible adsorption in two stages:

Stage 1: Protein mixture is permitted to interact with resin so that protein of interest gets absorbed unto exchanger. Again, proteins with comparatively low affinities for ion exchanger move through column faster than bound protein with higher affinities.

Stage2: This engages separation and elution of bound polypeptide from exchanger and is performed by using fresh eluting buffer of different pH so that affinity of bound proteins to matrix is really reduced.

In ion exchange chromatography, one may select whether to bind protein/peptide of interest or absorb out contaminants and permit substance of interest to elute out of column.

ii) Affinity Chromatography:

Proteins are known to have high affinity for different substances like substrates, receptors, inhibitor, prosthetic groups, and antibodies elevated against them. When any of these affinity compounds (known as ligands) is covalently attached (immobilised) to insoluble resin (matrix), it can be utilized to purify its conjugate protein by permitting the mixture having protein of interest to pass through the column of immobilized ligand.

During their passage, only protein with complimentary site to that of immobilized ligand is retarded and therefore separated from others. Desired protein can then be recovered from column by changing elution conditions.

The major needs of affinity chromatography are:

(i) Biospecific ligand that can be covalently coupled to the chromatographic matrix;

(ii) That such ligands retains their biological activities.

Ligand ideal for affinity chromatography must should the following characteristics:

i) Specific and reversible binding;

ii) Presence of chemically changeable groups which can be utilized for attachment to matrix while it still retains its binding properties;

iii) Its affinity for binding site of biomolecule must be within 10-4 and 10-8 M in free solution; and

iv) It must interact with biomolecule noncovalently

Affinity chromatography can be utilized to purify proteins and peptide like enzymes, antibodies, lectins, hormones and even whole cells.

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