General properties of a protease inhibitor from the seeds of kidney bean.

Abstract

A protein isolated from kidney bean was found to react and form a 1:1 enzyme-inhibitor complex with trypsin, pancreatic elastase and, at low enzyme concentrations, with human plasmin. A similar reaction of the inhibitor with chymotrypsin occurred in two stages: at low enzyme concentrations a 1:1 complex formed while at high enzyme concentrations more chymotrypsin reacted in a non-stoichiometric manner. The inhibitor had separate binding sites for trypsin and chymotrypsin. Plasmin in low concentrations was probably also bound at the trypsinreactive site by the inhibitor. The inhibitor had no effect on the activity of pepsin, papain, a protease from Bacillus subtilis and carboxypeptidase-A but depressed the activity of a carboxypeptidase-B and a thrombin preparation slightly. Complexes of the inhibitor with trypsin and chymotrypsin were prepared and isolated on a Sephadex G-75 column and their properties were investigated. These complexes could be dissociated at or below pH 3. Free enzyme and inhibitor respectively were recovered by chromatography on a Sephadex G-75 column from the dissociated chymotrypsin-inhibitor complex. Under similar conditions, the extent of dissociation of a trypsininhibitor complex and the recovery of its components was much less. The inhibitor was found to be a small protein with a most probable molecular weight value of about 10,000. It was found to be unaffected by exposure to heating (up to 90°) or to pepsin and to low pH values (pH 2). Oxidation with performic acid or reduction with 2-mercaptoethanol reduced the inhibitory activity to very low values. Re-oxidation in air of the reduced and nearly inactive inhibitor failed to restore activity to any significant degree.