Purification And Characterization Of Thrombin-like Enzyme From Snake Venom (trimeresurus Albolabris)

Recent decades, thrombotic disease has become a serious harm to human health, one of the diseases and the incidence of these diseases have increased year by year. Developed a class of efficient low-cost, but without the side effects of new thrombolytic agents become very important and urgent work. Snake Venom thrombin-like enzyme (TLE) is a class of fibrinolytic activity of serine proteinases, In the Snake Venom of the most widely distributed Viperidae, the most abundant. Since 1936, the first time from the Americas Klobusitzki and Konig directed viper (Bothrops jararaca) venom was partially purified thrombin-like enzyme has so far been found containing more than 30 kinds of snake venom thrombin-like enzyme component, and has so far received more than 20 kinds of separation and purification. China's current clinical application of snake venom preparations is mainly concentrated in the thrombin-like enzyme, such as batroxobin, Agkistrodon enzymes defibrase enzymes. Different structures and human thrombin, snake venom thrombin-like enzyme are single-chain structure; apart from the yellow, and green tip (T. flavovirdis) and from the angle of flavoxobin viper (Cerastes Vipera) of the V iperabin not sugar, is an almost All the snake venom thrombin-like enzyme are glycoproteins, and the more acidic glycoprotein, whose sugar content quite different, about 5%～20%.At present, the right domestic and foreign-made white-lipped Trimeresurus snake venom thrombin-like separation and purification of the study have not been reported. In this study, made in China, white-lipped Trimeresurus venom as the research object is not extracted out of a bleeding effect of thrombin, the main research work are as follows:With Sephadex G-100 gel chromatography, DEAE-Sephadex A-25 ion-exchange chromatography and Sepharose-heparin affinity chromatography to separate three-step chromatography purification, from the white-lipped Trimeresurus venom purified 63.1KDa out the molecular weight of the serine protease. SDS-PAGE electrophoresis results showed that reducing and non-reducing conditions, are single band, the table understand the lip venom of Trimeresurus stejnegeri fibrinolytic components by a single peptide chains of protein molecules. Inhibitors EDTA, has no effect on the enzyme activity, while PMSF (serine protease inhibitor) significantly inhibited the enzyme activity; and fibrinogen at 37℃for 2 hours, can significantly hydrolyzed fibrinogen A a chain, Bβ-chain and Y chain, there is no degradation, suggesting that the group was divided into a serine protease; Fe2+Zn2+Cu2+ on the enzyme activity significantly inhibited; enzyme at pH 4.5～9.5,25℃-45℃, within the scope of activity of a strong; not detected hemorrhagic activity and edema activity; enzyme can promote platelet aggregation; animal experiments show that The component can be extended tail bleeding time in mice.