Interaction of clostripain with natural trypsin inhibitors and its affinity labeling by N α ‐ p ‐nitrobenzyloxycarbonyl arginine chlormethyl ketone

Clostripain (EC 3.4.4.20) is a protease from the culture filtrate of Clostridium histolyticum, with a highly limited specificity directed at the carboxyl bond of arginyl residues in proteins and in synthetic substrates [l] . According to the specificity clostripain is close to trypsin-like enzymes, on the other hand its catalytic site is that of a SH-protease. Recently we have developed new types of affinity chromatography which allow us to obtain highly active clostripain on a preparative scale [2]. The availability of the pure enzyme prompted us to study its inhibition by natural trypsin inhibitors and by a synthetic inhibitor suitable for the affinity labeling of its active site. The experimental results show that the basic pancreatic trypsin inhibitor (Kunitz) is without effect on clostripain whereas the soy bean inhibitor shows a competitive type of inhibition. The synthetic affinity labeling reagent p-NOz-ZACK was prepared for the first time in a pure and stable form by a substantial modification of a synthesis described previously [3], It is highly active as an irreversible inhibitor of both clostripain and trypsin. The active site of clostripain is protected against this substitution by the competitive inhibitor benzamidine.