Practical Protein Chemistry - A. Darbre 1989

Affinity Chromatography of Proteins
General Procedures in Affinity Chromatography
Immobilized Substrates, Inhibitors, and Enzyme Cofactors

Although appropriate substrates are optimal ligands for enzyme Affinity Chromatography, their use is generally hindered by their rapid conversion into enzymatic reaction products, leading to reduced efficiency and sorbent degradation. Nevertheless, substrates can be employed as ligands if catalytic reaction rates are slowed under specific conditions while maintaining sufficiently strong enzyme binding. Such conditions can be achieved by lowering the Temperature (from —2 to —50°C), where the strength of the enzyme-substrate complex is sufficient to retain the enzyme on the Column, whereas desorption is readily accomplished by raising the temperature [5].

More generally, ligands include inhibitors as well as effectors that do not bind to the Active Site of the enzyme. An immobilized substrate can occasionally serve as a highly specific inhibitor. The choice of a particular inhibitor depends entirely on the relative rates of its association and dissociation with the enzyme, which dictate the conditions required for adsorption and desorption.

To avoid the need to synthesize new sorbents every time a specific individual enzyme must be isolated, researchers have sought "universal" affinity ligands. Adenine nucleotide Coenzymes, such as 5'-АМР, 2',5'-ADP, NAD⁺, and NADP⁺, are best suited for this purpose, since approximately one-third of the 2,000 known Enzymes exhibit activity in the presence of such Cofactors. Blue dextran has been used as a structural analogue of NAD⁺-containing Supports for synthesizing group-specific sorbents.

Characteristically, The Use of group-specific ligands has contributed to a better understanding of cofactor Mechanisms of action, as well as the effects of salts and accompanying Proteins that determine the strength of biospecific interactions and the conditions for enzyme Adsorption and Elution. Thus, despite the relatively low Specificity of group-specific ligands, a high degree of purification of the target enzyme can be achieved by optimizing these conditions.

An elegant variation of this "cascade" technique was recently demonstrated for the isolation of citrate synthase (EC 4.1.3.7) and fumarase (EC 4.2.1.2) using a single affinity column containing pyromellitic acid (PMA) coupled to Sepharose 4B via a diaminopropanol spacer. The tissue extract is applied to the PMA-Sepharose 4B column in 0.01 M phosphate buffer (pH 7.3) containing 0.01 M mercaptoethanol. Citrate synthase is completely adsorbed, whereas fumarase binding is inhibited in the presence of phosphate (which acts as a competitive inhibitor for this enzyme). Citrate synthase is eluted with the same buffer containing 0.01 M citric acid. The resulting eluate is applied to a Blue Sepharose 4B gel column, where the enzyme binds quantitatively, and is subsequently eluted with 0.01 M phosphate buffer (pH 7.3) containing mercaptoethanol (but lacking citric acid). The fumarase fraction, which is not adsorbed on the PMA-Sepharose 4B under phosphate elution conditions and retains 100% of its enzymatic activity, is dialyzed against 0.01 M Tris-acetate buffer (pH 7.3) containing 0.014 M mercaptoethanol and applied to a PMA-Sepharose 4B column in the same buffer. This results in quantitative binding of the enzyme, which is then eluted in the presence of L-malic acid; following the removal of the latter, fumarase is adsorbed onto a Blue Sepharose column and eluted by re-adding malic acid to the working Tris-acetate buffer.