Purification And Properties Research Of Fibrinolytic Enzyme From Phascolosoma Esculenta

Objective To purify Fibrinolytic Enzyme from Phascolosoma esculenta, research its enzymatic and partial pharmacological effects.Methods The fibrinolytic enzyme from Phascolosoma esculenta was purified by extracting,homogenizing,centrifuging,QAE-sephadex A-25 ion exchange and gel filtration column sephacryl S-300. Its purity and molecular weight was identified through SDS-PAGE. Making fibrin plate was to know the mechanism and study the effect of pH,temperature,metallic ion,enzyme inhibitor on fibrinolytic activity of the fibrinolytic enzyme from Phascolosoma esculenta. The purified enzyme was subcutaneously injected in the mice to judge hemorrhagic activity. The influences of the purified enzyme on hemolysis test,the blood clotting dissolution test,euglobulin lysis time(ELT),prothrombin time(PT) and activated partial prothrombin time(APTT) were observed in vitro.Results A purified fibrinolytic enzyme was obtained from Phascolosoma esculenta, with molecular weight 29.8×10³, purify multiple 87, recovery rate 5.4% . The activity of fibrinolytic enzyme was stable within 50℃and at pH2.0～11.0, reached highest at pH 8.0 . Metallic ion(Cu²⁺,Mg²⁺,Ba²⁺,Al³⁺,Na⁺,K⁺ ) had no effect on the activity of fibrinolytic enzyme from Phascolosoma esculenta. PMSF,EDTA and Ag⁺,Zn²⁺ could partly inhibit the activity, whereas Hg²⁺completely inhibited the enzyme. The purified fibrinolytic enzyme from Phascolosoma esculenta could not only directly degrade fibrin but also take effect by activating plasminogen. This enzyme, without any hemorrhagic activity would not cause hemolytic reaction. The vitro thrombolytic experiments showed that the purified fibrinolytic enzyme could obviously dissolve blood clotting, shorten euglobulin lysis time(ELT),prolong significantly prothrombin time(PT) and activated partial thromboplastin time(APTT).Conclusion A single fibrinolytic enzyme was purified from Phascolosoma esculenta,which had stable enzymatic properties and some thrombolytic effect in vitro.