Sequence of Tryptic Cleavages in Porcine Pancreatic Secretory Inhibitor II

Abstract

Abstract The appearance of sequential forms of inhibitor in a mixture of inhibitor and less than equimolar quantities of porcine trypsin at pH 2.75 has been monitored by polyacrylamide gel electrophoresis. Each of the detected forms has been isolated and identified by its Rf in gel electrophoresis, its composition, and its NH2-terminal and COOH-terminal amino acids. The sequence of the bond cleavages by trypsin during temporary inhibition is Lys 14-Ile 15, followed by Arg 40-Gln 41, Lys 48-Ser 49, and Lys 38-Lys 39. The modified inhibitor (with one cleavage at the reactive site) was active while all other forms were inactive. The second cleavage, Arg 40-Gln 41, is the temporary (inactivating) cleavage. The modifying cleavage of the Lys 14-Ile 15 bond was found to be an obligatory step for the further digestion of the inhibitor. The reversible nature of the reactive site bond cleavage was demonstrated in gel electrophoresis by the examination of separate incubations of virgin and modified inhibitors with trypsin. Starting with either form, a mixture of virgin and modified inhibitor was obtained. The modified inhibitor was susceptible to digestion by α-chymotrypsin.