Quantitative affinity chromatography of alpha-chymotrypsin

Affinity chromatography on a column of 4-phenylbutylamine, immobilized on succinylated polyacrylic hydrazide agarose, has been employed to study binding of ligands to α-chymotrypsin. In contrast to earlier studies of competitive elution phenomena, where an added soluble ligand interferes with enzyme binding to an affinity matrix, benzyloxycarbonyl derivatives of aromatic acids have been shown to facilitate binding of chymotrypsin to this matrix. This behavior has been analyzed in terms of an expanded binding scheme for affinity chromatography including the formation of a ternary complex (α-chymotrypsin · benzyloxycarbonyl-amino acid · 4-phenylbutylamine · matrix) where the soluble ligand and immobilized ligand bind to different sites. Equations to describe the phenonema have been derived and utilized to quantitate equilibrium constants for dissociation of the binary and ternary complexes. Benzyloxycarbonyl-Ala-Ala was found to promote earlier elution of the enzyme from its affinity matrix. Other ligands known to bind to the active site do not alter the binding to the 4-phenylbutylamine affinity matrix. These results illustrate the conclusion that binding of a small molecule can alter affinity retention in positive, negative, or neutral modes. This suggests that affinity chromatography could be “fine-tuned” by appropriate selection of cosolutes and illustrates the value of relatively weakly binding affinity matrices in enzyme studies.