Purification and Some Properties of Trypsins from the Pyloric Caeca of Chum Salmon

Abstract

Trypsins were purified from the pyloric caeca of chum salmon Oncorhyncus keta in order to study the properties of these enzymes and to know the mechanism of protein digestion in the pyloric caeca.
Six anionic trypsins (designated as DI-1, DI-2, DI-3, DII-1, DII-2 and PI-1) having isoelectric point (i.p) of 4.1-4.3 and one cationic trypsin (designated as PII-1) having i.p of 11.1 were isolated by ammonium sulfate precipitation, batch methods of P- and DEAE-cellulose, gel filtration chromatography on Sephadex G-100, DEAE- and P-cellulose column chromatography from the water extract of the pyloric caeca which was autoactivated at 3-5°, pH 8.5 for 20 hours.
Results of polyacrylamide gel electrophoresis showed that isolated trypsins except DII-2 were homogenous or highly purified enzymes and that DII-2 was mixture of two trypsins.
The activitiess of all purified trypsins were affected by metal-chelating agents, alkylating agents, sulfhydryl compounds and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) in similar fashion to bovine cationic trypsin. These were completely inhibited by diisopropylfluorophosphate(DFP), N-tosyl-L-lysine chrolomethyl ketone (TLCK), soybean trypsin inhibiter and chicken ovomucoid.
These results suggested that their functional properties were similar to those of the other vertebrate trypsins.