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目的从广东舟山眼镜蛇毒中分离纯化磷脂酶A(2Phospholipase A2,PLA2)组分,并分析其理化性质、酶活性质以及影响其酶活力的因素。方法采用化学沉淀、Sephadex G-50凝胶过滤及UND Sphere S阳离子交换层析法对PLA2进行分离纯化;采用BCA法测定其蛋白浓度,HPLC法测定其纯度,SDS-PAGE测定其相对分子质量,PLA2测定方法测定其活力;并考察反应温度、反应pH值及金属离子对酶活性的影响及其热稳定性。结果从广东舟山眼镜蛇毒中分离的PLA2蛋白浓度为0.6 mg/ml,回收率为9.5%,HPLC纯度为99%,相对分子质量为14 300,比活力为133μmol/(mg.min);最适反应温度及pH值分别约为60℃和8.5;Ca2+和Mg2+可增强酶活力,K+、Ni2+、Fe2+、Cu2+、Zn2+、Co2+和Cr2+对酶活力均有抑制作用;该酶具有较高的热稳定性。结论从广东舟山眼镜蛇毒中分离纯化出一种高纯度的PLA2,该酶具有较高的最适反应温度和热稳定性。
OBJECTIVE To separate and purify the components of phospholipase A2 (PLA2) from the cobra venom in Zhoushan, Guangdong Province, and analyze its physico-chemical properties, enzyme activity and factors affecting its enzyme activity. Methods PLA2 was separated and purified by chemical precipitation, Sephadex G-50 gel filtration and UND Sphere S cation exchange chromatography. The protein concentration was determined by BCA method. The purity of PLA2 was determined by HPLC. The relative molecular mass, PLA2 determination of its activity; and investigated the reaction temperature, the reaction pH and metal ions on the enzyme activity and its thermal stability. Results PLA2 protein was isolated from Zhoushus cobra venom in Guangdong Province. The concentration of PLA2 was 0.6 mg / ml, the recovery was 9.5%, the HPLC purity was 99%, the relative molecular mass was 14 300 and the specific activity was 133μmol / (mg.min) The reaction temperature and pH value were about 60 ℃ and 8.5 respectively. Ca2 + and Mg2 + could enhance the enzyme activity, while K +, Ni2 +, Fe2 +, Cu2 +, Zn2 +, Co2 + and Cr2 + all inhibited the enzyme activity. The enzyme had high thermal stability Sex. Conclusion A highly purified PLA2 was isolated and purified from the venom of Zhoushan, Guangdong Province. The enzyme has high optimum reaction temperature and thermal stability.