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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 To isolate a novel phospholipase A2 (PLA2) from the Naja naja atra in Guangxi and study its properties. Methods Phospholipase A2 (PLA2) was isolated and purified using CM5 2, CM-Sepharose CL-6 B ion exchange column and Sepharose CL-6 B gel column. The properties of phospholipase A2 (PLA2) were performed by classical methods. Results The isolated phospholipase A2 was subjected to polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis, and HPLC detection as a single component. Its molecular weight was determined to be 1 500 ± 1 0 0 0 by SDS-PAGE. Isoelectric focusing measured its isoelectric point 6.3. Its optimum temperature is 5 5 °C, and its optimal p H value is 8.5. Amino acid composition analysis showed that the enzyme is composed of 126 amino acids, mostly Asp, Ala, Gly, and Cys. Fe3 +, Zn2 +, and EDTA inhibited their enzyme activity; K+, Ca2 +, and detergents promoted their activity. Fluorescence analysis showed that the tryptophan residues and histidine residues of the enzyme may be located on the surface of the molecule. Pharmacological experiments showed that the enzyme has antitrypsin effect, anticoagulant effect, indirect hemolysis and cardiotoxicity to frogs. Conclusion The phospholipase A2 gene of Guangxi cobra venom was highly homologous to PLA2 from other sources, but its properties were not identical.