一种新的纤溶酶原激活反应动力学研究方法

 在溶栓研究中 ,纤溶酶原激活剂 (plasminogen activator,PA)激活纤溶酶原 (plasminogen,Plg)反应的动力学常数的测定占有重要地位 .在前人的研究中 ,虽然进行了这项实验 ,但是并未给出一个方便、快捷的测定方法 .所以建立一种更准确 ,更适合一般的实验室条件的 PA分子激活 Plg反应动力学常数的测定方法是必要的 .在对该反应进行数学分析的基础上 ,得到由可测量表示的纤溶酶 (plasmin,Plm)生成速度 (v(Plm) )的计算公式 ,由 v(Plm)及相应已知量可进一步推导出 Km、kcat的表达式 ,最终测得相应动力学常数 .用这种方法测定的由甲醇酵母 (pichia pastoris)表达的人单链尿激酶型纤溶酶原激活剂 (human single chain urokinase- PA,scu- PA)激活 Plg的反应的 Km=0 .648μmol·L-1,kcat=0 .0 62 6s-1,与文献报导相符 (Km=0 .4～ 1 .1 μmol· L-1,kcat=0 .0 2～ 0 .0 93) s-1,说明此方法是可靠的 .又因该法只需相应底物及酶标仪且为连续测定 ,所以十分简便 .

A New Method of Kinetic Study on the Reaction of Plasminogen Activation

In the study of the activity of all kinds of plasminogen activator,it is important to determine the kinetic constants of the plasminogen activation catalyzed by plasminogen activator.But there is no suitable method for this experiment.To find an expedite,shortcut plasminogen activation study method is very important.Expression of plasmin generating rate( v (Plm))expressed in detectable variable was derived from mathematics reasoning of this reaction,the kinetic constants K m and k cat can be easily calculated by the value of v( Plm)and other known quantities.This method was used to determine the kinetic constant of the reaction of plasminogen activation which was catalyzed by Pichia pastoris expressed human single chain urokinase type plasminogen activator.The result is: K m=0.648 μmol·L -1 , k cat =0.0626 s -1 coincidental with the former result( K m=0.4-1.1 μmol·L -1 , k cat =0.02-0.093 s -1 ),which means this method is reliable.This method is also expedite for it only needs some kinds of substrate and a microreader.