摘要
蚓激酶(Earthworm fibrinolytic enzymes，EFE)是一组具有激酶和纤溶酶活性的丝氨酸蛋白酶，可以作为治疗血栓类疾病的药物。本实验以赤子爱胜蚓(Eisenia faetida)为原料，经分离纯化得到较高纯度的蚓激酶(EFE)，并用大分子结合修饰法及肽链有限水解修饰法对蚓激酶进行化学修饰，并对修饰反应的部分条件进行了优化及对修饰前后蚓激酶的部分酶学性质进行了比较。
经过自溶、(NH_4)_2SO_4分步盐析、DEAE-纤维素离子交换层析、超滤等纯化过程，蚓激酶纯化倍数达6.67倍，比活力为2.29×10~4u／mg，酶活力回收率为17.0％。
通过实验优化了部分修饰条件。实验表明，聚乙二醇修饰蚓激酶的较佳条件：在37℃下，pH9.2，0.1M的硼酸缓冲液中，每1.0mg的蚓激酶加25.5mgPEG_1，水浴保温1h，修饰率为63.2％，酶比活为1.30×10~4u／mg而残留酶活为56.6％。右旋糖苷修饰蚓激酶的较佳条件：在室温下，pH8.4、0.1M的硼酸缓冲液中，将酶液与活化的右旋糖苷以1.0：152.7(W／W)的比例反应18h，则酶的修饰率为58.5％时，修饰后的酶比活为6.28×10~3u／mg，酶活回收率为27.4％。固定化胰蛋白酶水解修饰蚓激酶的较佳条件：在45℃下，固定化胰蛋白酶与
被修饰叫激酶以350mg:1.Omg的比例加入一定体积的磷酸缓冲液
(0.lmol/L，pH7.4)中，水浴保温lh，修饰率为72.1%，酶比活为l.46X
1了川mg，酶活回收率为63.6%。
另外，对修饰前后的部分酶学性质进行比较。结果表明，修饰后
的PEG一EFE和DEX一EFE对底物(酪蛋白)的亲合力都增加了、抗
胰蛋白酶的水解能力、热稳定性也提高了，抗原性也大大的降低了，
其中PEG一EFE与抗血清的结合能力大约是天然酶的25 .0%左右，修
饰酶DEX一EFE的抗原抗体的结合能力大约是天然酶的犯.5%。而被
固定化胰蛋白水解后的叫激酶其对酪蛋白的亲和力降低了，热稳定性
也略有下降，与抗血清的结合能力大约是天然酶的42.0%左右。x
Earthworm fibrinolytic enzymes (EFE)is a kind of serine type protease, which can hydrolyze fibrin.Because of its strong ability to hydrolyze fibrin,EFE can be used as a medicine to treat thrombus desease. In the study,EFE was separated and purified from Eisenia faetida.Then EFE was chemically modified with big molecule,orEFEwas hydrolyzed restrictedly. Furthermore,serverl conditions of modifying reaction arid the character of the modified EFE were also studied .
Through autolyzation,ethanol fraction precipitation,DEAE-cellulose ion exchange chromstography and ultrafiltration,EFE was purified by 6.67 folds with a specific of 2.29 104U/mg and a yield of 17.0%.
The reactive conditions for EFE modified were optimized.The optimum conditions for EFE modified with PEG1 are as follows: EFE (1.0mg) was dissolve in 2.0ml of 0.1M sodium tetraborate, pH9.2. the solution was brought to 37 and activated PEG(25.5mg) was added .after
lh,the activity of modified EFEis 56.6% of free EFE,and the modification rate of amino residues is63.2%.Theoptimum conditions for EFE modified with Dextran are as follows: EFE(l.Omg) was dissolved in 2.0ml of 0.1M sodium tetraborate,pH8.4 . the solution was brought to 25 and activated Dextran (152.7mg) was added .after 18h,the activity of modified EFE is 27.4% of free EFE,and the modification rate of amino residues is 58.5%. The optimum conditions for EFE hydrolyzed by immobiled trypsin are as follows: EFE (l.Omg)was dissolved in 2.0ml of 0.1M'phosphate,pH7.4 . the solution was brought to 45 and immobiled trypsin 0.3 5g was added .after lh,the activity of modified EFE is 63.6% of free EFE, and the modification rate of amino residues is 72.1%.
In addition ,the characters are compared between the modified EFE and the native EFE. And the results indicate that the affinity of PEG-EFE to casein is the higher than that of native EFE .Further more ,the ability is improved in counteracting hydrolyzation of trypsin to EFE and the thermal stability of PEG-EFE is enhanced.the Dextran-EFE has the similar changes as the PEG-EFE. The binding ability of PEG-EFE with antiserum retains 25.0% of that of native EFE and DEX-EFE is 32.5%. whereas the affinity of EFE to casein reduces when EFE was hydrolyzed by immobiled trypsin. and the thermal stability of hydrolyzed EFE is lowered. The binding ability of hydrolyzed EFE with antiserum retains 42.0% of that of native EFE.
引文
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