中文摘要：

采用硫酸铵盐析、DEAE-Sephacel,DEAE-Sepharose阴离子交换柱层析、SP-Sepharose阳离子交换柱层析和Sephacryl S-200凝胶过滤柱层析,从青石斑鱼的胃中分离纯化到3种胃蛋白酶原（PG-1、PG-2和PG-3）,SDS-PAGE结果显示,它们的分子量分别为35、36和37 ku。在pH 2.0下,3种胃蛋白酶原转化为有活性的胃蛋白酶P-1、P-2和P-3,分子量分别约为33、33和34 ku。3种酶的最适pH分别为3.0、2.5和2.5,最适温度均为40℃,且均能被典型的天冬氨酸蛋白酶抑制剂pepstatin A有效抑制。免疫交叉反应结果表明,3种胃蛋白酶原能与大鼠抗黄鳍鲷PG-Ⅰ,PG-Ⅱ,PG-3b,PG-3a,PG-4a及PG-4b多克隆抗体发生不同程度的免疫交叉反应。动力学参数测定结果表明,P-1、P-2和P-3对酸变性牛血红蛋白的Km、kcat及kcat/Km值分别为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）和5.2×10-5 mol/L,18.7 S-1,3.6×105 mol/（L.S）。

英文摘要：

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,