一种筛选明胶酶抑制剂的微量分析方法

A Versatile Microassay for Screening of Gelatinases Inhibitors

目的:建立一种通用的微量分析法筛选明胶酶抑制剂。方法:以琥珀酰明胶为底物,在 pH=8.5,含 Ca^(2+)10mmol·L^(-1)的50mmol·L^(-1)硼酸缓冲液中与明胶酶在37℃反应30min,然后加入0.03%的TNBSA,室温放置20min,在450nm 处测定吸收度。结果:本方法对明胶酶的检测限为7.5ng,灵敏度随反应时间的延长而增加,吸收度在0-360min 内与时间成线性,在7.5-480ng 内与明胶酶的量成线性;以1,10-二氮菲为对照进行抑制率测定的日内及日间精密度 RSD 分别为1.5%和2.5%。结论:本方法不仅在检测明胶酶活性上可实现灵敏、快速、简便和微量,并可用于筛选明胶酶抑制剂,有望成为明胶酶抑制剂的高通量筛选法。

Objective:To develop a versatile microassay method to screen gelatinases inhibitors.Methods:Using succinylated gelain as substrate,reacted with gelatinases(or gelatinases with added drugs)at 37℃ for 30 min in pH 8.5 borate buffer(50 mmol·L^(-1))with CaCl_2(10mmol·L^(-1)),then added 0.03% solution of TNBSA and allowed to incubate at room temperature for 20 min.The absorbance was determined at 450 nm.Results:The detection limit of the method for gelatinases is 7.5 ng;The sensitivity of the assay could be increased significantly by increas- ing the incubation time; The rate of hydrolysis of substrate by gelatinases,measured as the increase in absorbance at 450 nm,was linear up to 6 hours and was linear in 7.5-480 ng of gelatinases.When using 1,10-phenanthroline as inhibitor reference,the precision of interday and intraday for the method is 1.5% and 2.5% respectively. Conclusions:The assay method is specific,sensitive,rapid and easy,suitable for measuring gelatinolytic activity of enzymes and high throughout screening of gelatinases inhibitors.