广西眼镜蛇毒L-氨基酸氧化酶的分离纯化及体外抗血小板聚集作用  

Purification and Characterization of L - amino Acid Oxidase from Guangxi Cobra Venom and Its Antiplatelet Effect on Human in Vitro

作　　者：王辉[1] 张文宇[1] 李玉媚[1] 欧和生[1] 

机构地区：[1]广西医科大学药学院,广西南宁530021

出　　处：《时珍国医国药》2013年第6期1294-1297,共4页Lishizhen Medicine and Materia Medica Research

基　　金：国家自然科学基金(No.30871186)

摘　　要：目的从广西眼镜蛇粗毒中分离纯化L-氨基酸氧化酶(L-amino acid oxidase,LAAO),并对其物理、生化性质及体外抗血小板聚集作用进行研究。方法用Sephacryls-200凝胶过滤,DEAE-Sepharose CL-6B离子交换,CM-SepharoseCL-6B离子交换层析法分离纯化广西眼镜蛇毒中的LAAO,用SDS-聚丙烯酰胺凝胶电泳在还原与非还原条件下测定其蛋白分子量,等电聚焦法测定等电点,比浊法测定广西眼镜蛇LAAO对二磷酸腺苷、胶原、凝血酶、花生四烯酸引起的血小板聚集率的影响。结果从广西眼镜蛇毒中分离出的LAAO分子量约为55.2 KD,等电点为7.6;生物活性检测发现,该蛋白不具有磷脂酶A2和精氨酸酯酶活性,无纤溶及出血活性,能明显抑制二磷酸腺苷、胶原、凝血酶、花生四烯酸引起的血小板聚集,并呈明显的正相关。结论分离纯化的广西眼镜蛇毒LAAO为电泳纯,并具有较强的抑制体外血小板聚集作用。Objective To investigate the purification method of a L-amino acid oxidase（LAAO） from the venom of Guangxi cobra and research its biochemical characterizations and antiplatelet effect on human in vitro.Methods The LAAO was purified from the venom of Guangxi cobra by Sephacryls-200 gel filtration,DEAE-Sepharose CL-6B ion-exchange chromatography and CM-Sepharose CL-6B ion-exchange chromatography.The molecular weight was estimated with SDS-PAGE.The pI was determined through isoelectric focusing.Turbidimetric method was used for determination of the LAAO to inhibit the aggregation of human platelet induced by ADP,collagen,thrombin,arachidonic acid in vitro.Results The purified LAAO was shown to be homogeneous in SDS-PAGE,which acted as an antiplatelet effect in vitro.Its molecular weight was estimated to be 55,200 D and isoelectric point was 7.6.It was devoid of phospholipase A2,arginine,esterase,fibrinolytic,hemorrhagic activities.The purified L-amino acid oxidase dose-dependently inhibited the aggregation of Human platelet induced by ADP,thrombin,collagen and AA in vitro.Conclusion The purified LAAO is a Single component in SDS-PAGE,which could inhibit the aggregation of human platelet in vitro.

关 键 词：蛇毒 L-氨基酸氧化酶 分离纯化 血小板聚集 

分 类 号：R284.2[医药卫生—中药学] R962[医药卫生—中医学]