Broad-specificity protease-isoinhibitors for trypsin, chymotrypsin, plasmin and kallikrein from snails (Helix pomatia). Isolation, inhibition characteristics and amino-acid composition.

Abstract

A low‐molecular‐weight fraction of 88% pure inhibitors, inhibitor preparation I, could be obtained from aqueous extracts of snails (Helix pomatia) using a series of steps involving ammonium sulfate precipitation at 63% saturation, affinity chromatography on trypsin resin and gel filtration on Sephadthx G‐50 (fine). A mixture of heat‐ and acid‐stable inhibitors of broad specificity inhibiting the proteolytic activity of bovine α‐ and β‐trypsin, bovine α‐ and β‐chymotrypsin, porcine plasmin and porcine pancreatic and serum kallikrein could thus be separated from a heat‐ and acid‐instable trypsin‐plasmin inhibitor, inhibitor preparation 11, which is of higher molecular weight and is derivcd from the albumin gland of the snail.Inhibitor preparation I is a mixture of at least 17 active components, which could be separated by equilibrium chromatography using a pH gradient from pH 4.9 to 8.0. Three of the predominant, most active components were obtained as pure homogenous proteins and identified as isoinhibitors composed of 58 amino acid residues. The three purified isoinhibitors closely resemble the basic trypsin‐kallikrein inhibitor of bovine organs with respect to amino acid composition and inhibitory specificity. They inhibit the proteolytic activity of trypsin, chymotrypsin, plasmin and organ kallikrein, but not serum kallikrein. They have been characterized according to their interaction with the enzymes, time dependence of inhibition and reactive‐site residues. Their locus of biosynthesis are the external organs, probably the subepithelial glands of the skin.