蚓激酶中激酶活性蛋白质组成分析及效价测定

Active Protein Components and Potency Detection of Kinases in Lumbrokinase

将来自不同生产企业的蚓激酶样品采用胰蛋白酶酶解,采用超高效液相色谱-四极杆-飞行时间质谱(UPLC-QTOF-MS)技术对蚓激酶的蛋白质及多肽进行来源检定,采用PLGS 3.0软件及蛋白质谱库分析处理数据,鉴定了5家企业的蚓激酶样品中激酶样活性蛋白组分,结果均鉴定出8~9种与纤溶活性相关的蛋白,其中各有4~5种属于激酶蛋白组分。以尿激酶为对照,使用琼脂糖-纤维蛋白平板法对蚓激酶中的激酶活性进行确证,并初步建立了蚓激酶中激酶活性效价的测定方法。结果每1 000 IU蚓激酶相当于尿激酶1.509 1~2.819 1 IU。本试验通过UPLC-Q-TOF-MS技术分析,鉴定了蚓激酶中的具有激酶活性的蛋白成分,并对其激酶活性进行了初步定量。

The lumbrokinase samples from different manufactures were digested by trypsin, then were separated and analyzed by UPLC-Q-TOF-MS technology. The MS data was processed by PLGS 3.0 software to identify the protein components that related to fibrinolytic activity of lumbrokinase. In five enterprises' products, 8-9 proteins related to fibrinolytic activity were identified, 4-5 proteins among them belonged to the kinase activity components. The kinase activity of lumbrokinase was investigated by agarose-fibrin plate method with urokinase as the reference. The potency determination results showed that every 1 000 IU of lumbrokinase was equivalent to 1.509 1-2.819 1 IU urokinase. The proteins of kinase activity in lumbrokinase were identified and the kinase activity were quantitatively detected.