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

Citation
Mj. Cao et al., Purification and characterization of two anionic trypsins from the hepatopancreas of carp, FISHERIES S, 66(6), 2000, pp. 1172-1179
Citations number
32
Categorie Soggetti
Aquatic Sciences
Journal title
FISHERIES SCIENCE
ISSN journal
09199268 → ACNP
Volume
66
Issue
6
Year of publication
2000
Pages
1172 - 1179
Database
ISI
SICI code
0919-9268(200012)66:6<1172:PACOTA>2.0.ZU;2-F
Abstract
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.