Biochemical properties of two isoforms of trypsin purified from the Intestine of skipjack tuna (Katsuwonus pelamis)

Two trypsins (A and B) from the intestine of skipjack tuna (Katsuwonus pelamis) were purified by Sephacryl S-200, Sephadex G-50 and DEAE-cellulose with a 177- and 257-fold increase in specific activity and 23% and 21% recovery for trypsin A and B, respectively. Purified trypsins revealed a single band on native-PAGE. The Molecular weights of both trypsins were 24 kDa as estimated by size exclusion chromatography and SDS-PAGE. Trypsin A and B exhibited the maximal activity at 55 degrees C and 60 degrees C, respectively, and had the same opt inial pH at 9.0. Both trypsins were stable up to 50 degrees C and in the pH range from 6.0 to 11.0. Both trypsin A and B were stabilised by calcium ion. Activity of both trypsins continuously decreased with increasing NaCl concentration (0-30%) and were inhibited by the specific trypsin inhibitors - soybean trypsin inhibitor and N-p-tosyl-L-lysine chloromethyl ketone. Apparent K., and K,,, of trypsin A and B were 0.22-0.31 mM and 69.5-82.5 S-1, respectively. The N-terminal amino acid sequences of the first 20 amino acids of trypsin A and B were IVGGYECQAHSQPPQVSLNA and IVGGYECQAHSQPPQVSLNS. respectively. (C) 2008 Elsevier Ltd. All rights reserved.