蚓激酶的提取纯化及性质研究

Purification and characterization of lumbrokinas

目的建立高效的蚓激酶纯化工艺,并研究其酶学性质。方法以赤子爱胜蚓(Eisenia foelide)为原料,经过匀浆抽提、硫酸铵分段盐析、色谱分离、电泳等技术对蚓激酶进行分离纯化及鉴定,并研究pH、温度、金属离子与抑制剂对蚓激酶活性的影响以及蚓激酶的作用机理。结果纯化蚓激酶比活达5 100 U/mg,经SDS-PAGE电泳分析得2条带,相对分子质量为32 000和33 500;体外溶栓实验表明,蚓激酶具有直接溶解纤维蛋白和激活纤溶酶原的双重作用。在中性和略偏碱性环境中稳定且活性高;温度适应范围广;不同种类及浓度的金属离子对蚓激酶活性的影响不尽相同;抑制因子及底物特异性研究发现,蚓激酶属于丝氨酸蛋白酶,不属于金属蛋白酶,同时含有二硫键。结论该分离方法可以得到较纯的蚓激酶。

Purpose To establish an effective method of purifying lumbrokinas（LK） and to study the characteristics.Methods LK was purified through extraction,（NH4） 2SO4 fractional salting out,Sephadex G-75,DEAE-Sepharose 6 Fast Flow and Phenyl Sepharose High Performance from earthworm Eisenia foelide and identified by SDS-PAGE.Effects of pH value,temperature and inhibitions on the activity of LK and the mechanism of LK were studied.Results The specific activity of LK amounts to 5 100 U/ mg.Two fractions of LK,with Mr of 32 000 and 33 500,was obtained through SDS-PAGE.The fibrinolytic experiment in vitro indicated that LK not only hydrolyzed fibrin directly,but also activated the plasminogen to plasmin fibrinolytic enzyme.LK kept high activity in neutral and slightly alkaline environments.LK activity was stable at wide temperature.The effects of different types and concentrations of metal ions on the activity of LK were different.Studies on substrate specificity and inhibition indicated that LK belonged to serine protease but not belonged to metalloroteinase,and it had disulfide bonds.Conclusion Purified LK can be obtained by the techniques of the separation and purification.