柿子单宁对几种蛇毒中主要酶的抑制作用及其机理初探

Study on Inhibitory Effects of Persimmon Tannins on Activities of Major Enzymes from Several Snake Venoms and Its Mechanism

【目的】分析柿子单宁(persimmon tannin,PT)对江浙蝮蛇、尖吻蝮蛇、眼镜蛇毒中主要酶活力的抑制作用,初步研究两者相互作用的机理。【方法】将蛇毒与PT以不同比例混合后在37℃下温育1h,离心分离,测定上清液中主要酶活力变化,并采用SDS-聚丙烯酰胺凝胶电泳对粗毒和作用后所得沉淀及上清液进行分析。【结果】当蛇毒与PT质量比达到1﹕1时,粗毒中蛋白质水解酶、磷脂酶A2、L-氨基酸氧化酶、精氨酸酯酶、乙酰胆碱酯酶、类凝血酶活力被完全抑制,且酶活力的下降与PT含量呈剂量增加效应;SDS-聚丙烯酰胺电泳表明,PT与蛇毒蛋白具有很强的非特异性结合能力。但对不同结构蛋白质结合能力和选择性也明显不同。【结论】柿子单宁在体外条件下对蛇毒中几种酶的活力具有明显的抑制作用,PT与蛋白结合是导致酶失活的重要原因。

【Objective】 The inhibitory effects of persimmon tannins （PT） on the activities of proteolytic enzyme, phospholipase A2, L-amino acid oxidase, arginine esterase, acetylcholinesterase and thrombin-like enzyme from Agkistrodon halys Pallas, Agkistrodon acutus, Naja naja atra venom , and the action mechanism between venom and PT were studied. 【Method】 Different amounts of PT were mixed with snake venoms, after incubation at 37℃ for 1h and centrifugation, the enzyme activities of proteolytic enzyme, phospholipase A2, L-amino acid oxidase, arginine esterase, acetylcholinesterase and thrombin-like enzyme from the supernatants were mensurated, and the precipitates , supernatants and venom were analyzed by SDS-PAGE.【Result】The enzymatic activities of proteolytic enzyme, phospholipase A2, L-amino acid oxidase, arginine esterase, acetylcholinesterase, and thrombin like enzyme were inhibited completely as the rate of snake venom and PT reached 1﹕1 （w/w）, and in a dose-dependent manner. It was found that PT had a very high combining ability with venom proteins in a non-specific manner, but with different selectivity for different proteins.【Conclusion】 It was suggested that the enzyme activities from snake venoms could be inhibited remarkably by persimmon tannin in vitro by non-specific combination.