Purification And Characterization Of Fibrinolytic Enzyme From Snake Venom (Trimeresurus Albolabris)

As the development of people's lives, sufferers of thromboembolic disease increased year by year. Thromboembolic disease seriously hazard health and lives of people. Consequently, to exploit an efficient, hypotoxic, inexpensive thrombolysis medicine is an emergent and impotant work. Snake Venom Fibrinolysin (SVFLE) have a good specificity, longer half-life in vivo, and stabile thermal stability. Up to now, a number of studies aimed at characterizing SVFLE of Crotalidae snakes, numerous studies on SVFLE from the venoms of snake such as Agkistrodon acutus, Agkistrodon halys Pallas, Trimeresurus Stejnegeri, Trimeresurus mucrosquamatus have been reported, but the studies on SVFLE from the venom of snake (T. albolabris ) have been not yet reported. In this work, from the venom of snake (T.albolabris) in China we have isolated and purified a none hemorrhagic activity fibrinolytic enzyme, which was identified as aα~metalloproteinas, and its physical-chemical properties and biological functions have been studied. The main research works are as follows:1. The physic-chemical properties and the biological activities of the venom of snake(T. albolabris) were determined, and the results included that LD50 of the crude venom to mice was 7.01 mg/kg; it contained 81% proteins by the Lowry method; it presented many bands of proteins on SDS-PAGE; it possessed the activity of hydrolyzing casein, but the proteolytic activity was inhibited by EDTA. These results indicated it was possible to contain some metalloproteinase in the venom of snake (T. albolabris).Meanwhile, the fibrinolytic activity of the cude venom have been detected by fibrin plate method, either arginine ester hydrolysing enzyme activity or thrombin like enzyme activity have been detected, and the activities of other components in the crude venom such as PLA, 5'-nucleotidase, alkaline phosphomonoesterase and phosphodiesterase in the venom of the venom were also measured.2. The fibrinolytic metalloproteinases component with 66.3 KDa was isolated and purified from the venom of snake (T. albolabris) by column chromatography of DEAE-SephadexA-25, SephadexG-100 and CM-SephadexC-50. On SDS-PAGE , thefibrinolytic enzyme component exhibited only one band under the reduced and unreduced condition, hence this results indicated that it was a single chain enzyme. When the fibrinolytic enzyme component hydrolyzed casein, its hydrolytic activity was 0.68 U/mg, its maximum temperature was 40℃, and its maximum pH was 7. The inhibitors, such as EDTA, EGTA, could inhibit its fibrinolytic activity, but PMSF orβ-mercaptoethanol have little inhibitory effect on its activity. Cu2+ significantly inhibited its activity, but and Zn2+ weakly affect its activity. In the fibrinolytic enzyme component there were no activities of arginine ester hydrolysing enzyme; and it cleaved Aαchain immediately and Bβchain slowly, but notγchains of fibrinogen when incubated with fibrinogen in 5 minutes at 37℃. These results implied the fibrinolytic enzyme component isolated and purified from snake venom (T. albolabris) isα~ Metalloproteinases, and animal experiments indicated there was no hemorrhagic activity in the enzyme.Conclusion: A novel fibrinolytic enzyme, with the strong fibrinolysis activity( cleaved Aαand Bβof fibrinogen) and without no hemorrhagic activity, was obtained from the venom of snake (T. albolabris) in China. These reuslts may lay a basis to conduct such works as gene clone, clinical development, and the molecular structure, active center and the molecular details of its fibrinolysis mechanism in the future.