抗凝蛋白EH体外活性检测条件的建立

EH蛋白是一种水蛭素衍生物,它是在水蛭素的N端引入了一小段寡肽,该寡肽可被凝血因子XIa(factor XIa,FXIa)和Xa(factor Xa,FXa)裂解释放水蛭素的抗凝血酶活性.比较在不同条件下FXa裂解EH蛋白的效果,包括裂解时间(2h,4h,6h,8h,10h,20h)、裂解温度(25℃,37℃,40℃)...

The Antithrombin Activity Detection of EH in Vitro

Recombinant protein EH originates from hirudin by linking three amino acids to its N-terminal. The three amino acids are recognized and cut by coagulate factor Xa (FXa) and XIa (FXIa), that will release the antithrombin activity of hirudin.The cleavage efficiency of FXa to EH was compared under different conditions, incuding emzymolytic time and temperature, the molar ratio of FXa and EH, and reaction solution. Results showed that the antithrombin activity of EH increased proportionally, under certain conditions, as the reaction time and temperature increased, Moreover, the FXa cleavage activity is enhanced when the molar ratio of FXa and EH was increased. It seems that saline is more suitable reaction solutions for FXa to cut EH. So the optimal cleavage conditions for EH with FXa in vitro is that EH was reacted with FXa at 37℃ for 6 hours in saline solution with the molar ratio (1 ∶180) of FXa and EH. A practical and reproducible method for the activity determination and quality control of EH in vitro was developed.