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Three pepsinogens (PG-1、 PG-2 and PG-3) were highly purified from the stomach of Epinephelus awoara by ammonium sulfate fractionation, DEAE-Sephacel and DEAE-Sepharose anionic exchange column chromatoraphy, SP-Sepharose cationic exchange column chromatography, and Sephacryl S-200 gel filtration.Their molecular masses were estimated to be 35, 36 and 37 kDa, respectively, by SDS-PAGE.Pepsinogens converted into their active form pepsins (P-1、 P-2 and P-3) with molecular masses of approximately 33, 33 and 34 kDa, respectively, under pH 2.0.They showed maximal activity at pH 3.0, 2.5 and 2.5.And their optimal temperatures were 40 C.All three pepsins were completely inhibited by pepstatin A, a typical aspartic proteinase inhibitor.Western blot analysis revealed that these pepsinogens had different cross reaction with anti-sea bream PG-Ⅰ, PG-Ⅱ, PG-3b, PG-3a, PG-4a and PG-4b polyclonal antibodies.The kinetic constants of Km, kcat and kcat/Krn of pepsins (P-1、 P-2 and P-3) for acidic-denatured bovine hemoglobin were calculated as 7.0× 10-5 M, 17.6 s-1, 2.5×105 M-1·s-1; 5.5x10-5 M, 22.8 s-1, 4.1×105 M-1·s-1 and 5.2×10-5 M, 18.7 s-1, 3.6×105 M-1·s-1 , respectively.