Time-Resolved Ligand Exchange Reactions: Kinetic Models for Competitive Inhibitors with Recombinant Human Renin
作者:Maurice M. Morelock、Christopher A. Pargellis、Edward T. Graham、Daniel Lamarre、Grace Jung
DOI:10.1021/jm00010a019
日期:1995.5
peptidomimetic, competitive inhibitors of human renin using a novel binding assay. The method entails analyzing a pair of ligand exchange reactions in which a dansylated inhibitor serves as the fluorescent probe. The first in the pair of reactions involves preincubating renin with the probe and initiating the reaction by addition of a sample inhibitor; the second reaction involves preincubating renin with the
使用新型结合测定法,确定了一系列人肾素的拟肽竞争性抑制剂的开和关速率常数(kon和koff)。该方法需要分析一对配体交换反应,其中以丹磺酰化的抑制剂作为荧光探针。该对反应中的第一个涉及将肾素与探针预孵育,并通过添加样品抑制剂来引发反应;第二个反应包括将肾素与样品抑制剂预孵育,然后通过添加探针来引发反应。这两个反应均产生了进度曲线,其中包含有关每个配体的kon和koff的补充信息。使用从描述配体交换过程的微分速率方程式衍生的模型同时拟合两条曲线。探针的kon和koff速率常数分别为6.85 x 10(6)M-1 s-1和2.96 x 10(-4)s-1,得出的Kd为43.2 pM。抑制剂系列的Kd值变化超过2个数量级(27-2320 pM),而单个kon(10(6)-10(7)M-1 s-1)和koff(10(-4)- 10(-3)s-1)常数仅在1个数量级上变化。