Purification And Characterization Of A Kind Of Snake Metalloproteinase From Gloydius Saxatilis

Snake originated 135million years ago.It is one of the oldest animals.Statistics indicates that there are 2700 species snake in the world.Among them,poisonous snake are more than 600 species.In China,there are 165 species snake and 47 species of them are poisonous.Particularly,there are more than 10 species have deadly poison.The poisonous snake in China are commanded of 4 families: Hydrophidae, Elapedae, Colubridae and Viperidae.The crude snake venom is composed of more than 10~20 toxin proteins molecular and enzymes.Among the enzymes of snake venom,one of the most important is snake venom metalloproteinases (SVMPs). Now,there are more than 100 SVMPs from about 40 species snake are reported all over the world,then, about 70 of them have hemorrhagic activity and nearly 20 of them have not hemorrhagic activity.Some SVMPs are called as hemorrhagin, hemorrhagic toxin,hemorrhagic principle, hemorrhagic factor, fibrogenase, hemorrhagic protease and protease.A lot of SVMPs are wildly applied in clinic.According to the component of the domain,SVMPs are classified to 4 classes.Class P-Ⅰhas only one part:proteinase domain. The molecular weight is 20~30kDa.Class P-Ⅱhas two parts: proteinase domain and disintegrin domain. The molecular weight is 30~50kDa.Class P-Ⅲhas three parts: proteinase domain, disintegrin- like domain and Cysteine-rich domain. The molecular weight is 50~80kDa.Class P-Ⅳhas four parts: proteinase domain,disintegrin- like domain, Cysteine-rich domain and c-lectin-like domain. The molecular weight is 80~100 kDa.Now there are few reported about Gloydius saxatilis. Pro. Sun Dejun of our lab has secreened the cDNA of the Ussurin and selecte the positive clones by using the ploy(A+)isolation system.By analyze the sequence,it contain a metalloproteinase domain,a disintegrin and a characteristic RGD.So, Ussurin belongs to P-ⅡSVMPs.By means of one time DEAE Sepharose Fast Flow column chromatography, two times Sephadex G-100 column chromatography and two times Sephadex G-50 column chromatography,a new SVMPs(T7) from Gloydius saxatilis was purified to homogeneity.This purified principle is singal -chain polypeptide with a molecular weight of 24kDa as estimated by SDS-PAGE.According to the molecular weight, T7 belongs P-ⅠSVMPs.Mice were injected subcutan- eously with various density of T7.After 24h,hemorrhagic spot was observed.The size of hemorrhagic spot dependents the density of T7.So T7 belongs P-Ⅰ-A SVMPs. T7 had fibrinogenolytic activity. T7 acted theα-chain of fibrinogen first,thenβ-chain,and leftγ-chains intact.The fibrinogenolytic activity of T7 was inhibited by EDTA. T7 belongsα-fibrogenase. T7 could not inhibit human platelet aggregation stimulated by ADP.It indicates that the hemorrhagic activity of T7 had nothing to do with the platelet aggregation. T7 had no procoagulant and anti-coagulant activitys.Above all, T7 can be appled in the treatment of thrombus.