发色底物法定量测定重组u-PA产品的总活性和单链比例  

Quantitative measurement of total activity and scu-PA ratio of recombinant u-PA product with a chromogenic substrate

作　　者：胡显文[1] 高丽华[1] 李世崇[1] 胥照平[1] 张正光[1] 肖成祖[1] 饶春明 张翊 姜燕[3] 胡铁强[3] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071 [2]中国药品及生物制品检定所,北京100050 [3]天津天士力集团,天津300402

出　　处：《军事医学科学院院刊》2003年第5期349-352,376,共5页Bulletin of the Academy of Military Medical Sciences

基　　金：国家高技术研究发展计划项目资助 (Z18_0 3_12 )

摘　　要：目的 :建立一种发色底物酶水解方法定量分析重组尿激酶型纤溶酶原激活剂 (u_PA)中的总活性及其单链酶原比例。方法 :用嗜热菌蛋白酶激活u_PA转化成具有催化活性的双链尿激酶 ,再用尿激酶的发色底物S_2 44 4测定u_PA的总活性。在非激活条件下 ,u_PA产品中的单链酶原不会催化水解S_2 44 4底物 ,因而可以测定其单链酶原比例。结果 :优化了嗜热菌蛋白酶激活u_PA的反应条件和尿激酶催化水解S_2 44 4的反应条件。用这种方法测定了用CHO细胞表达的基因重组u_PA产品 ,单链比例大于 98%,比活性约为 11× 10 4 U/mg。结论 :用发色底物法测定重组人u_PA产品中的总活性和单链比例具有很好的重复性和准确性。Objective:To develop a chromogenic enzymatic assay to quantify the total activity and the inactive single-chain zymogen form (scu-PA or pro-urokinase) ratio of recombinant urokinase-type plasminogen activator (u-PA) product.Methods:The u-PA product was converted into its enzymatically active two-chain urokinase (tcu-PA or urokinase) with thermolysin, and then the total activity of u-PA product was measured with a specific chromogenic substrate S-2444. Scu-PA is the inactive zymogen form of u-PA and incapable of enzymatic hydrolysis of S-2444, so the scu-PA ratio in u-PA product could be determined when u-PA product was not activated by thermolysin.Results:The reaction conditions of activation of u-PA by thermolysin, as well as the reaction conditions of catalytic hydrolysis of S-2444 by tcu-PA, were optimized. When the recombinant u-PA product expressed by CHO cells was analyzed with this method, the scu-PA ratio was greater than 98%, and the specific activity was about 11×10 4 U/mg.Conclusion:The method developed for quantitative assay of total activity and scu-PA ratio of rhu-PA was accurate with high reproducibility.

关 键 词：发色底物法 定量测定 重组u-PA产品 总活性 单链比例 尿激酶型纤溶酶原激活剂 

分 类 号：R346[医药卫生—基础医学]