Affinity chromatography of chymotrypsin on a sepharose derivative coupled with a chymostatin analogue

Journal of Biochemistry
M Nishikata


A Sepharose derivative coupled with a chymostatin analogue, Gly-Gly-L-Leu-L-phenylalaninal (Pheal), was prepared. A number of native and chemically modified proteases were applied on a column of the adsorbent. Bovine chymotrypsins [EC] and Streptomyces griseus protease B were adsorbed strongly at pH 8.2. The affinities of these enzymes under various conditions were measured quantitatively by frontal chromatography in terms of the dissociation constant (Kd) of the enzyme-immobilized ligand complex. The pH dependence of the Kd value of alpha-chymotrypsin was consistent with that of the inhibition constant (Ki) of the enzyme for a corresponding soluble peptide aldehyde. Anhydro-chymotrypsin, in which the active site Ser-195 is converted to dehydroalanine, was not adsorbed. Ser-195 proved to be essential for the binding. The frontal chromatography method also gave the amount of the immobilized ligand that can interact with the enzyme. It was extremely small compared with the amount of the immobilized ligand determined by amino acid analysis. This was explained on the basis of the structural features of the agarose gel.

Related Concepts

Peptide Hydrolases
Chromatography, Agarose
Binding (Molecular Function)
Analogs & derivatives

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