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

Blood Coagulation & Fibrinolysis : an International Journal in Haemostasis and Thrombosis
Yanfang LiuJianhua Zhang

Abstract

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...Continue Reading

References

Aug 1, 1993·Computer Applications in the Biosciences : CABIOS·A Trubuil
Nov 26, 2009·Analytical Biochemistry·Stefanie MädlerRenato Zenobi
Oct 12, 2010·Journal of Biotechnology·Joris BeldDonald Hilvert
Nov 19, 2013·Biosensors & Bioelectronics·Lijun LiShouzhuo Yao

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Methods Mentioned

BETA
electrophoresis

Software Mentioned

Visilog4
Noésis E1D
INRA

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