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

第一作者： Liu, Yanfang

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

作者机构：

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.

分类号：

  • 相关文献

[1]Preparation of a Xanthine Sensor Based on the Immobilization of Xanthine Oxidase on a Chitosan Modified Electrode by Cross-linking. Liu Yuge,Li Weiming,Wei Changbin,Lu Lingling. 2012

[2]Preparation of Cross-Linked Enzyme Aggregates of Trehalose Synthase via Co-aggregation with Polyethyleneimine. Zheng, Jianfeng,Chen, Ying,Yang, Liwei,Zhang, Jun,Zheng, Jianfeng,Li, Mingchun.

[3]Immunolocation and enzyme activity analysis of Cryptosporidium parvum enolase. Mi, Rongsheng,Chen, Zhaoguo,Mi, Rongsheng,Yang, Xiaojiao,Huang, Yan,Cheng, Long,Lu, Ke,Han, Xiangan,Chen, Zhaoguo,Mi, Rongsheng,Yang, Xiaojiao,Huang, Yan,Cheng, Long,Lu, Ke,Han, Xiangan,Chen, Zhaoguo. 2017

[4]Fusion expression of bovine lactoferricin in Escherichia coli. Feng, Xing-jun,Wang, Jian-hua,Shan, An-shan,Teng, Da,Yang, Ya-lin,Yao, Yi,Yang, Guan-pin,Shao, Yan-chun,Liu, Shuo,Zhang, Fan.

[5]SpALF4: A newly identified anti-lipopolysaccharide factor from the mud crab Scylla paramamosain with broad spectrum antimicrobial activity. Zhu, Lei,Huang, Yan-Qing,Zhou, Jun-Fang,Fang, Wen-Hong,Yao, Xiao-Juan,Wang, Hao,Li, Xin-Cang,Zhu, Lei,Huang, Yan-Qing,Zhou, Jun-Fang,Fang, Wen-Hong,Yao, Xiao-Juan,Wang, Hao,Li, Xin-Cang,Lan, Jiang-Feng,Zhang, Chao. 2014

[6]Improving sensitivity of gold nanoparticle based fluorescence quenching and colorimetric aptasensor by using water resuspended gold nanoparticle. Liu, Jinchuan,Guan, Zheng,Lv, Zhenzhen,Jiang, Xiaoling,Yang, Shuming,Chen, Ailiang,Liu, Jinchuan,Guan, Zheng,Lv, Zhenzhen,Jiang, Xiaoling,Yang, Shuming,Chen, Ailiang.

[7]CkDREB gene in Caragana korshinskii is involved in the regulation of stress response to multiple abiotic stresses as an AP2/EREBP transcription factor. Wang, Xuemin,Chen, Xiaofang,Gao, Hongwen,Wang, Zan,Sun, Guizhi,Liu, Yun.

[8]Newly identified invertebrate-type lysozyme (Splys-i) in mud crab (Scylla paramamosain) exhibiting muramidase-deficient antimicrobial activity. Zhou, Jian,Zhao, Shu,Fang, Wen-Hong,Zhou, Jun-Fang,Zhang, Jing-Xiao,Li, Xin-Cang,Zhou, Jian,Zhao, Shu,Fang, Wen-Hong,Zhou, Jun-Fang,Zhang, Jing-Xiao,Li, Xin-Cang,Zhou, Jian,Ma, Hongyu,Lan, Jiang-Feng. 2017

[9]A convenient sandwich assay of thrombin in biological media using nanoparticle-enhanced fluorescence polarization. Yue, Qiaoli,Shen, Tongfei,Wang, Lei,Xu, Shuling,Li, Haibo,Xue, Qingwang,Zhang, Yuanfu,Liu, Jifeng,Gu, Xiaohong,Zhang, Shuqiu.

[10]A label-free and high sensitive aptamer biosensor based on hyperbranched polyester microspheres for thrombin detection. Sun, Chong,Han, Qiaorong,Wang, Weijuan,Zhao, Wenbo,Sun, Chong,Wang, Daoying,Xu, Weimin,Zhou, Min.

[11]A simple and sensitive label-free fluorescent approach for protein detection based on a Perylene probe and aptamer. Lv, Zhenzhen,Liu, Jinchuan,Bai, Wenhui,Yang, Shuming,Chen, Ailiang,Lv, Zhenzhen,Liu, Jinchuan,Bai, Wenhui,Yang, Shuming,Chen, Ailiang,Lv, Zhenzhen.

[12]Chiral beta-arylalkyl-1H-1,2,4-triazoles as demethylase inhibitors: Biological evaluation and its stereoselective interaction with sterol 14 alpha-demethylase from Penicillium digitatum. Cao, Xiufang,Chen, Changshui,Ke, Shaoyong.

[13]A novel myeloid differentiation factor 88 homolog, SpMyD88, exhibiting SpToll-binding activity in the mud crab Scylla paramamosain. Li, Xin-Cang,Zhu, Lei,Li, Lin-Gui,Huang, Yan-Qing,Lu, Jian-Xue,Fang, Wen-Hong,Kang, Wei,Ren, Qian,Ren, Qian.

作者其他论文 更多>>

微信小程序