Test of hirudin activity by tracking the binding of hirudin to thrombin in the presence of BS3 cross-linking.
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 °C for 5 min, the binding of hirudin to thrombin was cross-linked 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-Noésis 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 5625 ATU/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 6000 ATU/mg concluded by titration method. The superiorities of the method include good accuracy (the minimum testable concentration of hirudin is 1.5 μg/ml) and little sample consumption (sample consumption of hirudin is generally 1-11.5 μl using the apparatus of Mini Protean 3 Cell). Easy operation, low input, and equipment requirement also grant it as an effective way.
aQuality Standard and Testing Technology Research Institute, Yunnan Academy of Agricultural Sciences, Kunming bSchool of Chemistry and Life Sciences, Guangxi Teachers Education University, Nanning, China *Yanfang Liu and Jian Yang contributed equally to writing of this article.
Yanfang
Y
Liu
Jian
J
Yang
Jiangmin
J
Wang
Qingmei
Q
Huang
Xiaohong
X
Yang
Jianhua
J
Zhang
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D006629:Hirudins / Q000378:metabolism*; D006801:Humans; D013917:Thrombin / Q000378:metabolism*
D006629:Hirudins; D013917:Thrombin