【摘要】 目的研究五步蛇毒蛋白C激活剂(PCA)组分的分离纯化及其抗凝活性。方法借助DEAE-Cel-lulose、CM-Sephadex C-50和Sephadex G-75柱层析,以离子交换和凝胶过滤法从皖南产五步蛇粗毒中分离纯化PCA组分;以SDS-PAGE凝胶电泳检测其纯度和分子量,发色底物法(Chromogenic Substrate Assay)测定PCA组分的生色反应能力;测定PCA对正常血浆KPTT和PT的影响。结果从五步蛇粗毒中纯化的PCA组分经SDS-PAGE测定为单一区带,相对分子质量约为18.5 kD,等电点pH4.9;发色底物法显示其具有生色反应能力;该PCA1 mg/L在体外明显延长KPTT,但PT不受影响。结论采用离子交换和凝胶过滤法,可从皖南产五步蛇毒中分离纯化出高纯度的PCA组分,该组分可抑制内凝途径影响血液凝固过程。更多还原

【Abstract】 Objective To investigate the purification and anticoagulation activity of protein C activator(PCA) from Agkistrodon acutus venom(AAV).Methods Isolation and purification of the PCA fraction from AAV were carried out by a combination of DEAE-Cellulose,CM-Sephadex C-50 and SP-Sephadex G-75 column chromatography.Homogenicity and molecular weight of the fraction were determined in SDS-polyacrylamide gel electrophoresis(SDS-PAGE).Anticoagulant activity of the PCA fraction were measured by KPTT,PT of normal rabbit plasma and color producing reaction ability were performed with Chromogenic substrate assay.Results The purified PCA fraction showed a single band in SDS-PAGE and the molecular mass was about 18.5 kD.The isoelectric point of PCA was pH 4.9.The fraction showed a color producing reaction ability,and prolong obviously KPTT on normal rabbit plasma(1 mg/L) in vitro.Conclusion The purified PCA fraction from AAV in Wannan area,using ion exchange and gel filtration method,possessed distinct anticoagulant activity by inhibiting endogenous blood clotting pathway.更多还原