蝮蛇毒蛋白C激活物的纯化与活性分析  

Purification and activity analysis of protein C activator from Agkistrodon halys Palls

作　　者：章俊[1] 肖小璞[1] 李长清[1] 王宗奎[1] 林方昭[1] 

机构地区：[1]中国医学科学院北京协和医学院输血研究所,四川成都610052

出　　处：《中国输血杂志》2011年第4期299-303,共5页Chinese Journal of Blood Transfusion

基　　金：四川省科技支撑项目(2008SZ0013)

摘　　要：目的探索从国产蝮蛇毒中分离纯化人血浆蛋白C激活物(PCA)的方法及其用于蛋白C的鉴定。方法以国产蝮蛇毒为原料,通过溶解、离心、透析,利用Q Sepharose Fast Flow离子交换层析和Superdex G75凝胶过滤法分离纯化PCA组份。通过SDS-PAGE观察PCA组份的分子量,用发色底物法和凝固法分别测定活化PC(APC)的酰胺酶活性和抗凝活性。结果经两步层析,从蝮蛇粗蛇毒中纯化出PCA组份,SDS-PAGE图谱显示在分子量51 kD左右呈现1条蛋白带;经生物学活性鉴定:该组份在体外能迅速将PC激活为APC,APC的酰胺酶活性(A405)最高约为Protac(唯一商品化的PC激活剂产品)作用的2.8倍,抗凝活性(秒值)最高约为Protac作用的1.4倍,比活性2.20 IU/mg;不同浓度组份激活PC的酰胺酶活性和抗凝活性均随组份浓度的增大而升高,有明显的量效关系。结论采用离子交换层析和凝胶过滤法,可以从国产蝮蛇粗蛇毒中分离纯化出PCA组份,该组份具有较强的激活PC的能力,其生物活性与浓度呈正相关。Objective To study the purification and biological activity of protein C activator（PCA）separated from Agkistrodon halys Palls for identification of protein C（PC）.Methods Agkistrodon halys Palls was purified by ion exchange chromatography and gel filtration.The molecular weight wasere detected by SDS-PAGE.PC activity was determined with chromogenic substrate assay and one-stage clotting assay.Results Using chromatography purification,PCA was separated from Agkistrodon halys Palls,which showed a 51 kD single wide band in SDS-PAGE.Its specific activity was about 2.20 IU/mg.By the activation analysis,PCA rapidly converted PC into activated protein C（APC）,showing strong amidase activity and anticoagulant activity.The amidase activity was 2.8 times as high as Protac（the only commercial products of PC activator）,and the anticoagulant activity was 1.4 times stronger than Protac.The amidase activity and the anticoagulant activity analysis increased with the PCA concentration,which were positively correlated,as well as the results of anticoagulant activity analysis.Conclusion PCA is prepared by ion exchange chromatography and gel filtration.The PCA activates PC,and the activation activity is positively related to thewhich concentration.

关 键 词：蝮蛇毒 纯化 活性 蛋白C特异性激活剂 PCA组份 

分 类 号：R392.33[医药卫生—免疫学] R456[医药卫生—基础医学]