Purification and characterisation of trypsins from the spleen of skipjack tuna (Katsuwonus pelamis)

Three trypsin isoforms, trypsins A, B and C, from the spleen of skipjack tuna (Katsuwonus pelamis) were purified by a series of chromatographies including Sephacryl S-200, Sephadex G-50 and diethylaminoethyl-cellulose to obtain a single band on native-PAGE and SDS–PAGE. The molecular mass of all the trypsin isoforms was estimated to be 24 kDa by size exclusion chromatography and SDS–PAGE. The optimum pH and temperature of the three isoforms for the hydrolysis of Nα-p-tosyl-l-arginine methyl ester hydrochloride were 8.5 and 60 °C, respectively. Trypsins were stable to heat treatment up to 50 °C, and over a pH range of 6.0–11.0. All isoforms were stabilised by calcium ions. The trypsin activities were effectively inhibited by soybean trypsin inhibitor, TLCK and partially inhibited by ethylenediaminetetraacetic acid, while E-64, N-ethylmaleimide, iodoacetic acid, TPCK and pepstatin A showed no inhibitory effect. Activities decreased continuously as NaCl concentration (0–30%) increased. Trypsins A, B and C showed Km of 0.11–0.29 mM and Kcat of 57.1–114 s−1. The N-terminal amino acid sequence of 20 residues of three trypsin isoforms was IVGGYECQAHSQPHQVSLNS and had high homology to those of other fish trypsins.