Diazo reaction

Diazo reaction

Methods of affinity chromatography

Affinity chromatography techniques are not generally appropriate for the fractionation of mixtures because of the very positive and specific affinity between the ligand and solute molecules. Separation may be achieved in a column where the solute molecules bind to the matrix, and, after washing to remove unwanted substances, the test substance can be eluted. Alternatively, a batch procedure may be used in which the affinity medium is mixed with the sample solution and allowed to react. The solid-phase complex is then removed from the bulk solution by filtration or centrifugation and after washing, the purified solute is separated from the complex.

In order to elute molecules which are strongly bound to the ligand group it is necessary either to reduce their affinity for each other or to introduce molecules that are more strongly bound or in greater concentration and will therefore be able to displace the test molecules. Generally, non-specific methods are preferred and involve altering either the ionic strength or the pH of the buffer. These will result in conformational changes in the proteins and hence their binding characteristics. Changes in the dielectric constant of the solvent caused by the introduction of organic solvents will result in altered hydrophobic bonding and again aid the dissociation of the complex. It is possible to use specific agents which compete for the binding sites, such as alternative substrates and inhibitors for enzymes.

> A phytohaemagglu-tinin is a plant extract which is capable of agglutinating blood cells.


The nature of the binding between the ligand and its complementary molecule restricts affinity chromatography to a particular type of biological compound and some examples are given below.

1. Enzymes. The specificity of an enzyme for its substrate, coenzyme or competitive inhibitor provides the basis for many affinity chromatographic separations. Enzymes may be extracted and purified using insolubilized substrates, coenzyme or inhibitors. Less frequently, enzymes are used as the ligands.

2. Antibodies. The reaction between an antibody and its antigen does not result in the chemical modification of the antigen compared with the action of an enzyme and provides the basis for producing chromatographic media capable of selecting the complementary molecules. Either the antigen is insolubilized and used to isolate and purify the appropriate antibodies or with the increased availability of monoclonal antibodies, the reverse procedure is used.

3. Lectins. Some proteins extracted from certain seeds are capable of binding compounds containing carbohydrate groups. These proteins are known as phytohaemagglutinins or lectins. Affinity chromatographic media using such lectins have been used to investigate cell membrane structures and aid in the study of cell interactions. They are also used in conjunction with quantitative column chromatographic methods and in some electrophoretic separations of carbohydrate-rich proteins.

4. Receptor proteins. Hormones act on cells via specific membrane receptors and can be used to purify these receptors from cell homogenates. Receptors for compounds such as insulin, oestrogens and acetylcholine among others have been purified in this way.

5. Nucleic acids. Immobilized polynucleotides can be used to extract nucleic acid binding proteins as well as complementary strands of nucleic acids.

Self test questions

Sections 3.4/5

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