Pepsin is a member of the large family of aspartic proteinases and plays a critical role in the digestion of food proteins.In the present study,three pepsinogens(PG-1,PG-2 and PG-3)were highly purified from the stomach of marine fish banded grouper(Epinephelus awoara)by ammonium sulfate fractionation and a series of column chromatographies.In the first anion exchange column DEAE-Sephacel,three peaks of hemoglobin-digesting activity were detected.These fractions were pooled respectively and applied to SP-Sepharose,DEAE-Sepharose or Sephacryl S-200 for further purification.As a result,4.0 mg of PG-1,12.2 mg of PG-2 and 4.4 mg of PG-3 were obtained with purification folds of 12.8,13.0 and 18.7,respectively.The molecular mass of the three purified pepsinogens was estimated to be 35,36 and 37 ku,respectively,by SDS-PAGE.Under acid conditions,pepsinogens converted into their active form pepsins(P-1,P-2 and P-3)with molecular masses of approximately 33,33 and 34 ku,respectively.PG-1 converted into its active form by a one-step pathway while PG-2 and PG-3 converted into their active forms by a stepwise pathway.NativePAGE analysis showed that all of the three purified pepsinogens reveal single band with different migration rates.The results indicated these three enzymes had different types of pepsinogens.To investigate the effect of pH or temperature on activity of pepsins,pepsinogens were first converted to pepsins at pH 2.0.They showed maximal activity at pH 3.0,2.5 and 2.5,respectively and the optimal temperatures of all PGs were 40 ℃,using acidic-denatured bovine hemoglobin as substrate.All of the enzymatic activity of the three pepsins decreased when the temperature is higher than 50 ℃,suggesting their susceptibility to higher temperature.The activity of all three pepsins could be completely inhibited by a typical aspartic proteinase inhibitor pepstatin A and these pepsins exhibited different sensitivities to pepstatin A.When the molar ratios of pepstatin A to pepsins(P-1,P-2 and P-3)were 8∶1,6∶1 and 4∶1,respectively,the enzymatic activities could be completely inhibited.These results strongly indicated that the three purified pepsins purified in the present study are aspartic proteinases.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.These results further confirmed that PG-1 and PG-2 were closely related pepsinogens and share high identity with each other,whereas PG-3 was different.The results of the kinetic constants of different pepsins were determined by Lineweaver-Burk plots using acidicdenatured bovine hemoglobin as substrate.The kinetic constants of K_m、k_cat and k_cat/K_m of pepsins(P-1,P-2 and P-3)for acidic-denatured bovine hemoglobin were 7.0?10^-5 mol/L,17.6 S^-1, 2.5?10⁵ mol/(L?S);5.5?10^-5 mol/L,22.8 S^-1,4.1?10⁵ mol/(L?S) and 5.2?10^-5 mol/L,18.7 S^-1, 3.6?10⁵ mol/(L?S),respectively.The specificity constant of P-2 was higher than those of the other two pepsins,suggesting that its catalytic efficiency is higher than those of the other two pepsins under physiological conditions.