为了从赤子爱胜蚓中获得主要表现为纤溶酶原激活活性的蛋白酶,采用盐析、离子交换层析、凝胶过滤层析和疏水吸附层析从蚯蚓组织匀浆中纯化出6种具有纤溶活性的酶组分(F1、F2、F3、F4、F5和F6).它们均为单一多肽链;表观分子量分别为28500、29500、26100、26300、14850和32800;经非还原型SDSPAGE和扫描仪扫描,纯度分别为100%、95.2%、96.5%、93.6%、98%和97.8%;等电点(pI)均不高于3;SDSPAGE后,用Schiff试剂染色显示F5为糖蛋白;纯化的6种酶于20℃～50℃保温1h,酶活力基本不变;F1和F2、F3和F4、F5和F6分别在pH4～10、4～11、7～12范围内稳定;水解BAEE试验及纤溶活性抑制试验表明,F6既是丝氨酸蛋白酶,又是含金属离子的蛋白酶,其它5种酶为胰蛋白酶样的丝氨酸蛋白酶;免疫双扩散试验结果初步表明,F1和F5以及它们和其它4种组分之间无共同的抗原决定簇;用纤维蛋白平板法及以ChromozymU和ChromozymPL为底物测定,除F1外,其余5种酶的纤溶酶原激活活性明显强于其直接纤溶活性.
Abstract
A variety of fibrinoloyic enzymes has been extensively used as thrombolytic agents.However,these agents suffer from number of significant limitations.Therefore,novel thrombolytic agents with better fibrinoloytic efficiency and less adverse side effects are eagerly sought.Six enzymes with fibrinolytic activity were isolated and purified from the tissue homogenate of earthworm, Eisenia foelida. The purification procedure involved ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Sepharose Fast Flow, gel filtration on Sephacryl S-200 HR and hydrophobic interaction chromatography on Phenyl-Sepharose 6 Fast Flow (low substitute). According to analysis with Densitometer System after SDS-PAGE(non-reducing), the purity of each enzyme (F1, F2, F3, F4, F5, and F6) was 100%,95.2%,96.5%,93.6%,98%, and 97.8%, respectively. The apparent molecular weight of each enzyme estimated by SDS-PAGE was 28 500, 29 500, 26 100, 26 300, 14 850, and 32 800, respectively. The isoelectric point (pI) of each enzyme was not higher than 3. The enzymes are single polypeptide chains. F5 was glycoprotein proved by staining with Shiff reagent. The enzymes were stable between 20～50℃ for 1h. F1 and F2, F3 and F4, and F5 and F6 were stable from pH 4～10, 4～11, and 7～12, respectively. On the basis of enzymatic activities against BAEE and various inhibition, F6 was a serine protease and metalloprotease, and the other five enzymes were trypsin-like serine proteases. Double immunodiffusion test showed that anti-F1 serum had no cross-activities with the other five enzymes and nor did anti-F5 serum. All of the enzymes mentioned above but F1 displayed significantly stronger indirect-acting fibrinolytic activity than their direct-acting fibrinolytic activity as determined with fibrin plate method and /or using Chromozym U and Chromozym PL as specific substrates. However,the results from agar immune-double-diffusion assay revealed that common epitopes in these enzymes still wait further elucidation.
关键词
赤子爱胜蚓 /
纤溶酶原激活剂 /
纯化 /
纤溶活性 /
丝氨酸蛋白酶
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Key words
Eisenia foelida /
plasminogen activator /
purification /
fibrinolytic activity /
serine protease
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