Purification Bufrudin From Guangxi Hirudinaria Manillensis And Its Effects On Human And Rabbit Plasma Coagulation

Objective :To purify bufrudin from the GuangxiHirudinaria manillensis and study the physical and biochemicalcharacterization and anticoagulation effect in vitro on human andrabbit plasma coagulateon. Methods :The anticoagulation proteinbufrudin was purified from the Saliva of Guangxi Hirudinariamanillensis by 30 % ammonium sulfate preparation and columnchromatography on DEAE cellulose ,Sephadex G-50 and Re-HPLC .UsingSDS-PAGE calculated their molecular weights and then estimatedisoelectric point by the isoelectric focusing electrophoresis , thenassayed anticoagulant activities in vitro by Activated partialthromboplastin time(APTT) , Prothrombin time(PT),Thrombin time (TT)on human and rabbit plasma and their affection on Blood clotting factor.Result :The purified Bufrudin A (BDA) showed a single band by SDS-PAGEwith a molecular weight of 15.2KD,and Bufrudin B(BDB) show the samecharacter but the molecular weight is 14.6 KD.The isoelectric pointof BDA was PH 3.97 and BDB was PH 4.61 by the isoelectric focusingelectrophoresis .This protein BDA or BDB all prolonged acitivatedpartial thromboplsatin time(APTT) ,thrombin time (TT)and prothrombintime(PT),but there were some different charater between them .Comparatively,BDA effected stronger on thrombin time (TT) but BDBeffected more on prothrombin time(PT). BDA didn't affect other bloodclotting factor but inhibited the blood clotting factor â…¡,â…§,â…«.BDBonly inhibited the blood clotting factorâ…¡,â…§,â…©,â…«. Conclusion:Both of the anticoagulantion protein BDA and BDB have stronganticoagulantion activity ,BDA and BDB inhibited the coagulationprocess by effecting on the thrombin .