目的 通过探讨影响胃蛋白酶和胰蛋白酶纤溶活性的因素及其纤溶活性失活方法,排除胃蛋白酶和胰蛋白酶对中药鼠妇Armadillidium vulgare活性粗蛋白纤溶活性检测的干扰,以在鼠妇活性粗蛋白酶解过程,获取相对分子质量更小、效价更高的蛋白质或多肽。方法 采用纤溶蛋白平板法分别研究pH值、温度、金属离子、酶抑制剂和表面活性剂对胃蛋白酶和胰蛋白酶纤溶活性的影响,以获取较佳的酶失活工艺,并研究较佳酶失活工艺对尿激酶、蚓激酶、鼠妇纤溶酶纤溶活性的影响。结果 pH值、温度、金属离子、酶抑制剂和表面活性剂均可对胃蛋白酶和胰蛋白酶纤溶活性产生影响,其中胃蛋白酶的最优失活方案为pH 6.0~8.0;胰蛋白酶的最优失活方案为质量浓度25 mg/mL TLCK和浓度1 mmol/L EDTA的混合制剂。结论 研究所获取的较佳酶失活工艺,可用于以纤维蛋白平板法检测胃蛋白酶、胰蛋白酶降解产物的纤溶活性,操作简单,重复性好,稳定性好。

Objective To investigate the method for study on the effect of factors on pepsin and trypsin fibrinolytic activity and deactivation of fibrinolytic activity and to eliminate the interference of pepsin and trypsin on the detection of crude protein fibrinolytic activity of Armadillidium vulgare(porcellio plasmin) in order to obtain the proteins or peptides which have the smaller molecular weight but higher titer during the pepsin and trypsin degradation. Methods To study the effect of pepsin and trypsin deactivation on pH value, temperature, metal ions, enzyme inhibitor, surfactant, and responsing fibrinolytic by fiber fibrin plate assay. The better enzyme deactivation process was obtained and used for studying the effect on the fibrinolytic activity of urokinase, lumbrokinase, and porcellio plasmin. Results All the pH value, temperature, metal ions, enzyme inhibitor, and surfactant have had an impact on pepsin and trypsin fibrinolytic activity. Among them the optimum deactivation of pepsin was pH 6.0-8.0, while the optimum deactivation of trypsin was mixed preparation with TLCK at the concentration of 25 mg/mL and EDTA at the concentration of 1 mmol/L. Conclusion This study has obtained the better enzyme deactivation process which could be used for the detection of fibrinolytic activity of pepsin and trypsin degradation product by fiber fibrin plate assay, the operation is simple, and the repeatability and stability are good.

国家自然科学基金资助项目(81274096,81303230);江苏省科技厅产学研联合创新资金-前瞻性研究项目(BY2012036);江苏省科技支撑计划-社会发展项目(BE-2012763);中国博士后科学基金(2013M541704);江苏省博士后科学基金(1301133C)