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Purification and Characterization of Thrombin-like Enzyme from Snake Venom (Trimeresurus albolabris)

Author: LiHeng
Tutor: YuXiaoDong
School: Chongqing Normal University
Course: Biochemistry and Molecular Biology
Keywords: Thrombin-like enzyme White-lipped Trimeresurus Venom Serine protease
CLC: R285
Type: Master's thesis
Year: 2010
Downloads: 34
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Abstract

In recent decades, thrombotic diseases serious harm to human health, and the incidence of these diseases, there is an increasing trend. Thus the development of a class of efficient low-cost, non-toxic side effects of new thrombolytic agents has become an important and urgent task. Snake venom thrombin-like enzyme is a serine protease, fibrinolytic activity, the most widely distributed and most abundant in the venom of Viperidae snakes. From Klobusitzki and Konig in 1936 for the first time from the American spearhead viper (Bothrops jararaca) venom has been partially purified thrombin-like enzyme, so far biological science and technology workers have been found in more than 30 species of viper venom containing the component, and isolated and purified more than 20 types of thrombin. At present, China has snake venom thrombin made of antithrombotic drugs into clinical applications, including: defibrase Acutobin DF-521 enzymes. Human thrombin structure, venom thrombin are single-stranded structure; addition, almost all of the venom thrombin-like glycoprotein, its sugar content is quite different, approximately 5% to 20%. At present, domestic and dialogue lips Trimeresurus snake venom thrombin Purification and Characterization studies have not been reported. In this study, the white-lipped Trimeresurus venom as the research object, a hemorrhagic activity of thrombin-like enzyme purified from the main work is as follows: by Sephadex G-100 gel chromatography, DEAE-Sephadex A-25 ion-exchange chromatography and Sepharose-heparin affinity chromatography three-step chromatography, isolated and purified from the white-lipped Trimeresurus venom a molecular weight of 63.1KDa thrombin-like enzyme (TA-2). SDS-PAGE electrophoresis showed that the enzyme in reducing and non-reducing conditions are single band, indicating that the TA-2 only constituted by a single polypeptide chain, and measured obtaining the sugar content of 6.0%. This enzyme and fibrinogen were incubated for 2 hours in 37 ° C, the hydrolysis of fibrinogen Aα chain, no degradation of the Bβ chain and gamma chains; inhibitor EDTA, no effect on the enzyme activity, and PMSF (serine protease inhibitor) significant inhibitory effect on the enzyme activity, the group is divided into a serine protease. Fe2 Zn2 Cu2 significantly inhibited the enzyme activity; enzyme in pH 4.5-9.5, within the range of 25 ° C to -45 ° C have a strong activity. Animal experiments have shown that TA-2 of bleeding activity and edema activity, can prolong the tail bleeding time, the performance of the components in the body for anticoagulant.

Purification and Characterization of Thrombin-like Enzyme from Snake Venom (Trimeresurus albolabris)

Abstract

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