单甲氧基聚乙二醇修饰蚓激酶的制备及性质研究

Preparation and Properties of Earthworm Fibrinolytic Enzyme Modified with mPEG

采用以三聚氯氰活化的单甲氧基聚乙二醇(mPEG-5000),对从赤子爱胜蚓体内提取的抗血栓酶——蚓激酶进行了化学修饰,以期降低其抗原性及增加它的稳定性。实验表明,在37℃下,15.0mL,pH9.2,0.1mol?L?1的硼酸缓冲液中,按每1.0mg的蚓激酶加入25.5mg活化的mPEG,水浴保温1h,经透析、超滤浓缩、冻干得mPEG修饰酶,测得蚓激酶的修饰率为63.2%,酶活回收率为56.6%。在此基础上,对修饰蚓激酶的性质进行了研究。结果显示,修饰后的蚓激酶对底物的亲和力有所增加(天然酶表观米氏常数Km值为0.267mg.mL?1,修饰酶Km'值为0.115mg.mL?1);修饰蚓激酶的抗胰蛋白酶水解能力、热稳定性均优于天然酶;同时修饰酶的抗原性有了较大程度的降低,与抗血清的结合能力大约是天然酶的25.0%左右。

In order to reduce the antigenicity and enhance the stability of the Earthworm Fibrinolytic Enzyme (EFE) which is extracted from the Eisenia faetida earthworm, the EFE was modified with the activated monomethoxypolyethylene glycol (mPEG). The suitable modification procedures were found as follows: for every 1.0 mg EFE, desolved it in 37°C, 15.0 mL sodium tetraborate buffer solution of concentration of 0.1 mol &middot L-1 and pH=9.2, then 25.5 mg activated mPEG is added in the solution and keep the solution under 37°C for 1 hr. After that the solution is dialysised in the buffer solution of phosphoric acid (0.1 mol &middot L-1 and pH=7.0) for 24 hr and is filtrated by the ultrafilter membrane. The intercepted concentrated liquid is freeze dried for 24 hr to get the modified EFE (mPEG-EFE). The experiments show the activity of prepared mPEG-EFE can remain about 56.6% of the activity of the native EFE and its modification rate of amino residue is 63.2%. Other properties of the prepared mPEG-EFE were studied and the results show that the effinity of mPEG-EFE to casein is higher than that of the native EFE (the native EFE km=0.267 mg &middot L-1 and the modified EFE km'=0.155 mg &middot L-1), the ability of counteracting hydrolyzation of trypsin and the thermal stability of the prepared mPEG-EFE are both improved, the antigenicity of the mPEG-EFE is remarkly reduced and its binding ability with antiserum retains only 25.0% of that of the native EFE.