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Test of hirudin activity by tracking the binding of hirudin to thrombin in the presence of BS3 cross-linking

文献类型： 外文期刊

作者： Liu, Yanfang 1 ; Yang, Jian 2 ; Wang, Jiangmin 1 ; Huang, Qingmei 1 ; Yang, Xiaohong 1 ; Zhang, Jianhua 1 ;

作者机构： 1.Yunnan Acad Agr Sci, Qual Stand & Testing Technol Res Inst, Kunming 650205, Peoples R China

2.Guangxi Teachers Educ Univ, Sch Chem & Life Sci, Nanning, Peoples R China

关键词： binding activity;cross-linking;hirudin;thrombin

期刊名称：BLOOD COAGULATION & FIBRINOLYSIS （ 影响因子：1.276； 五年影响因子：1.328 ）

ISSN： 0957-5235

年卷期： 2015 年 26 卷 7 期

页码：

收录情况： SCI

摘要： Hirudin has a great potential in inhibiting thrombin, and its antithrombin activity has direct bearing on its clinical application. Using bovine alpha-thrombin and recombinant hirudin of Poecilobdella javanica purified from Phichia pastoris as materials, this study introduced a novel method to testing antithrombin activity of hirudin visually and dynamically by tracking the binding of hirudin to thrombin. After incubating the mixture of thrombin and hirudin at 37 degrees C for 5 min, the binding of hirudin to thrombin was crosslinked by bis[sulfosuccinimidyl] suberate for 30 min and visualized by SDS-polyacrylamide gel electrophoresis. With the aid of image analysis on the basis of INRA-Noesis E1D analysis software, antithrombin activity of hirudin was calculated through intensity variations of protein bands of either thrombin-hirudin compound, unbound thrombin, or unbound hirudin. In this regard, activity of the given hirudin was tested to be 5625ATU/mg based on a single reaction, and 5675.3 ATU/mg based on a series of reactions in a stepwise manner, close to the result of 6000ATU/mg concluded by titration method. The superiorities of the method include good accuracy (the minimum testable concentration of hirudin is 1.5 mu g/ml) and little sample consumption (sample consumption of hirudin is generally 1-11.5 mu l using the apparatus of Mini Protean 3 Cell). Easy operation, low input, and equipment requirement also grant it as an effective way. Copyright (c) 2015 Wolters Kluwer Health, Inc. All rights reserved.

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