Purification and characterization of three trypsin isoforms from viscera of sardinelle (Sardinella aurita)

摘要

Three trypsin isoforms A, B and C were purified to homogeneity from the viscera of sardinelle (Sardinella aurita). Purification was achieved by ammonium sulfate precipitation (20-70% (w/v)), Sephadex G-100 gel filtration and Mono Q-Sepharose anion-exchange chromatography. The molecular weights of these purified enzymes were estimated to be 28.8 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Based on the native PAGE and casein-zymography, each purified trypsin appeared as a single band. Trypsins A and C exhibited the maximal activity at 55pC, while trypsin B at 50pC. All isoforms showed the same optimal pH (pH 9.0) using Nl-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as a substrate. The three trypsins were stable at temperatures below 40pC and over a broad pH range (7.0-11.0). The activities of the three isoforms were strongly inhibited by soybean trypsin inhibitor and phenylmethylsulfonyl fluoride, a serine protease inhibitor, and partially inhibited by ethylenediaminetetraacetic acid, a metalloenzyme inhibitor. Kinetic constants of trypsins A, B and C for BAPNA were evaluated at 25pC and pH 9.0. The values of K m and k cat were 0.125, 0.083 and 0.10 mM, and 2.24, 1.21 and 5.76 sp#, respectively. The N-terminal sequences of the first 10 amino acids were I V G G Y E C Q K Y for trypsin A and I V G G Y E A Q S Y for trypsins B and C. These sequences showed highly homology to other fish trypsins.