Purification and characteristics of trypsins from cold-zone fish, Pacific cod (Gadus macrocephalus) and saffron cod (Eleginus gracilis)

摘要

Trypsins from the pyloric ceca of Pacific cod (Gadus macrocephalus) (GM-T) and saffron cod (Eleginus gracilis) (EG-T) were purified by gel filtration on Sephacryl S-200 and Sephadex G-50. The final enzyme preparations were nearly homogeneous on SDS-PAGE and the molecular weights of both enzymes were estimated to be approximately 24 kDa by SDS-PAGE. The specific trypsin inhibitors, soybean trypsin inhibitor and TLCK, strongly inhibited the activities of GM-T and EG-T. The optimum pH and optimum temperature of both trypsins were around pH 8.0 and 50 ℃, respectively, using N~α-p-tosyl-L-arginine methyl ester as substrate. The GM-T and EG-T were unstable above 30 ℃ and below pH 5.0, and they were stabilised by calcium ion. The N-terminal amino acid sequences of GM-T (IVGGYECTRHS-QAHQVSLNS) and EG-T (IVGGYECPRHSQAHQVSLNS) were found. The percentage of hydrophobic amino acid in the N-terminal 20 amino acids sequences of these cold-zone fish trypsins was lower (28%) than those of temperate-zone fish trypsins (34%), tropical-zone fish trypsins (37%) and mammalian trypsins (34%). Whereas the content of charged amino acids in the GM-T and EG-T was relatively higher than those of trypsins from temperate-zone fish, tropical-zone fish and mammals. Moreover, the GM-T catalyzed synthesis of N~α-(rerf-butoxycarbonyl)-l-alanyl-l-alanine-p-nitroanilide (N~α-Boc-l-Ala-l-Ala-pNA) has been studied by using N~α-(terr-butoxycarbonyl)-l-alanine-p-guanidinophenyl ester [N~α-Boc-l-Ala-OpGu (inverse substrate)] as acyl donor and l-alanine-p-nitroanilide (l-Ala-pNA) as acyl acceptor, respectively.