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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 establish a chromogenic substrate enzymatic hydrolysis method for quantitative analysis of the total activity and its single-stranded zymogen proportion in recombinant urokinase-type plasminogen activator (u_PA). Methods: U_PA was transformed by thermolysin to catalytically active double-stranded urokinase, and the total activity of u_PA was measured by urokinase chromogenic substrate S 2 44 4. Under non-activating conditions, the single-chain proenzyme in the u_PA product does not catalyze the hydrolysis of the S 2 44 4 substrate and thus the single-stranded zymogen fraction can be determined. Results: The thermophilic protease activation u_PA reaction conditions and urokinase catalytic hydrolysis S_2 44 4 reaction conditions were optimized. Using this method, the gene recombinant u_PA product expressed in CHO cells was determined to have a single chain ratio of more than 98% and a specific activity of about 11 × 10 4 U / mg. CONCLUSIONS: The determination of total activity and single-stranded ratio in recombinant human u_PA products by the chromogenic substrate method has good reproducibility and accuracy.