Affinity Chromatography, Principles and Applications

Abstract

Since the time the term “affinity chromatography” was first coined a few years ago (Cuatrecasas et al., 1968), the basic idea has been widely exploited as a powerful tool for the separation and purification of a wide variety of biological macromolecules. Its effectiveness for purification rests on the selectivity of interaction, and thus of adsorption, of a biological macromolecule on an affinity adsorbent which is prepared by the covalent immobilization of a specific ligand on a solid polymeric matrix. In the case of an enzyme, an appropriate reversible competitive inhibitor is immobilized. Substrates or cofactors of the enzyme may also be used under selected experimental conditions (Cuatrecasas, 1970; Cuatrecasas and Anfinsen, 1971). The desorption (i.e., elution) of the macromolecule from the affinity column is achieved either by perturbing the interaction between the macromolecule and the adsorbent, or by including a competing ligand in the eluting buffer. For obvious reasons, the design of a new affinity-chromatographic system for a given macromolecule requires individual attention in the selection and attachment of the ligand as well as selection of the buffer conditions for the adsorption and desorption processes. In principle, virtually any specifically interacting system composed of two or more species can be approached by this method.