广西产眼镜蛇毒酸性磷脂酶A_2的分离纯化及性质研究

Study of Purification and Properties Phospholipase A_2 from Chinese Cobra Venom

目的 从广西产中华眼镜蛇毒 ( N aja naja atra)中分离了一种新的磷脂酶 A2 ( PLA2 ) ,并研究其性质。 方法 磷脂酶 A2 ( PLA2 )的分离纯化采用 CM5 2、CM-Sepharose CL-6 B离子交换柱和 SepharoseCL-6 B凝胶柱 ,磷脂酶 A2 ( PLA2 )的性质采用经典方法进行。 结果 分离后的磷脂酶 A2 经过聚丙烯酰胺凝胶电泳、SDS-聚丙烯酰胺凝胶电泳和 HPLC检测 ,其为单一组分。SDS-PAGE测定它的分子量为1 5 0 0 0± 1 0 0 0。等电聚焦测得它的等电点为 6 .3。它的最适温度为 5 5℃ ,最适 p H值为 8.5。氨基酸成分分析表明该酶由 1 2 6个氨基酸组成 ,以 Asp、Ala、Gly、Cys居多。Fe3 + 、Zn2 + 、EDTA对其酶活力起抑制作用 ;K+、Ca2 +、去垢剂对其活力起促进作用。荧光光谱分析表明该酶的色氨酸残基、组氨酸残基可能位于分子表面。药理实验表明 :该酶具有抗胰蛋白酶作用、抗凝血作用、间接溶血作用以及对青蛙具有心脏毒性。 结论 广西产中华眼镜蛇毒磷脂酶 A2 与其它来源的 PLA2 同源性高 ,但性质不尽相同。

Objective In order to isolate and Purify an acidic phospholipase A 2 from Chinese cobra (Naja naja atra) in Guangxi province of China, and study its properties Methods CM 52 ,CM Sepharose CL 6B exchange chromatography and Sepharose CL 6B gel filtration were used to isolate PLA 2 from Naja naja atra(Chinese cobra) the study of its Properties employed the classic methods Result The purified PLA 2 was homogeneous, as confirmed by PAGE、SDS PAGE and HPLC the molecular weight was estimated to be 15 KD by SDS PAGE, isoelectric point determined by isoelectric focusing was 6 3 ,the PLA 2 is composed of 126 amino acid group, the pH and temperature optima were 8 5 and 55℃ It had a fluorescence emission maxima at 350nm The PLA 2 did not exhibit direct haemolytic activity on washed intact human erythrocytes, but showed indirect haemolytic activity in addition, it induced anticoagulant activities,antitrypsinase activity and cardiotoxicity on the heart of frog Conclusion The PLA 2 of Chinese cobra was high homologous with other PLA 2, but the Properties of it not all same as others