[31] Affinity chromatography

Publisher Summary The selective isolation and purification of enzymes and other biologically important macromolecules by “affinity chromatography” exploits the unique biological property of the proteins or polypeptides to bind ligands specifically and reversibly. Affinity chromatography exploits the phenomenon of specific biological interaction in a large variety of protein–ligand systems. A solution containing the macromolecule to be purified is passed through a column containing an insoluble polymer or gel to which a specific competitive inhibitor or other ligand has been covalently attached. Proteins not exhibiting appreciable affinity for the ligand pass unretarded through the column, whereas those which recognize the inhibitor are retarded in proportion to the affinity existing under the experimental conditions. The specifically adsorbed protein can be eluted by altering the composition of the solvent so that dissociation occurs. Affinity chromatography may be useful in concentrating dilute solutions of proteins, in removing denatured forms of a purified protein, and in the separation and resolution of protein components resulting from specific chemical modifications of purified proteins. Inherent advantages of this method of purification are the rapidity and ease of a potentially single-step procedure, the rapid separation of the protein to be purified from inhibitors and destructive contaminants, such as proteases, and protection from denaturation during purification by active site ligand-stabilization of protein tertiary structure.