Purification and characterization of two anionic trypsins from the hepatopancreas of carp

Two trypsins, designated as trypsin A and trypsin B, have been purified fro m the hepatopancreas of carp, The purification procedures consisted of ammo nium sulfate fractionation, and chromatographies on DEAE-Sephacel, Ultrogel AcA54 and Q-Sepharose. Trypsin A was purified to homogeneity with the mole cular mass of approximately 28 kDa, while trypsin B gave two close bands of 28.5 kDa and 28 kDa on sodium dodecylsulfate polyacrylamide gel electropho resis both under reducing and non-reducing conditions. On native-PAGE, both trypsin A and trypsin B showed a single band. Trypsin A and trypsin B reve aled optimum temperature of 40 degreesC and 45 degreesC, respectively, and shared the same optimum pH 9.0 using Boc-Phe-Ser-Arg-MCA as substrate. Both enzymes were effectively inhibited by trypsin inhibitors and their suscept ibilities were similar. The NH2-terminal amino acid sequences of trypsin A and trypsin B were determined to 37th and 40th amino acid residue, respecti vely. Their sequences were very homologous, but not identical to that of a trypsin-type serine proteinase from carp muscle and these of other trypsins . Immunoblotting test using the antibody raised against trypsin A cross-rea cted with trypsin B positively.