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本文采用亲和层析分离纯化了结晶慈菇蛋白酶抑制剂A和B,抑制剂A和B均为双头多功能蛋白酶抑制剂,抑制剂A能同时等当量抑制胰蛋白酶和胰凝乳蛋白酶,对激肽释放酶的抑制作用较弱,抑制剂B能当量抑制2克分子的胰蛋白酶,对激肽释放酶的抑制活力高于抑制剂A,但对胰凝乳蛋白酶的抑制作用远比抑制剂A弱。化学修饰以及胰蛋白酶与胰凝乳蛋白酶对抑制剂A的竞争性结合表明:抑制剂A和B的两个活性中心均为Lys和Arg残基,其中Lys活性中心专一抑制胰蛋白酶,而由Arg活性中心构成的活性区域则表现为多功能,能抑制多种蛋白酶,从抑制剂A和B的结构特征推测,两活性中心应分别为Lys-Ser(44—45)及Arg-Tyr-Lys(76—78),在抑制剂A中还存在一疏水性残基参与对胰凝乳蛋白酶的抑制,此残基位于由Arg活性中心所构成的活性区域中。
In this paper, affinity chromatography was used to separate and purify the crystal saponins A and B. The inhibitors A and B were double-headed multifunctional protease inhibitors. Inhibitor A could simultaneously inhibit trypsin and chymotrypsin at the same time, Inhibitory effect on kallikrein weaker, inhibitor B can equivalently inhibit 2 mol of trypsin, kallikrein inhibitory activity was higher than inhibitor A, but the inhibition of chymotrypsin than the inhibition Agent A is weak. Chemical modification and competitive binding of trypsin and chymotrypsin to inhibitor A show that both of the active sites of inhibitors A and B are Lys and Arg residues, of which Lys activity centers exclusively inhibit trypsin and The active region formed by Arg active site is multifunctional and can inhibit a variety of proteases. Based on the structural characteristics of inhibitors A and B, the two active sites should be Lys-Ser (44-45) and Arg-Tyr-Lys (76-78), there is also a hydrophobic residue in inhibitor A that is involved in the inhibition of chymotrypsin, which residue is located in the active region consisting of the Arg active site.