利用等温滴定量热仪测定胰蛋白酶活性  

Measuring enzyme activity by isothermal titration calorimeter

作　　者：李辉[1] 张潇慧 李桂澜[1] Li Hui;Zhang Xiaohui;Li Guilan(Peking Public Instrument Center,School of Life Sciences,Peking University,Beijing 100871,China)

机构地区：[1]蛋白质科学研究国家重大科技基础设施北京大学基地,北京100871

出　　处：《分析仪器》2023年第2期62-67,共6页Analytical Instrumentation

基　　金：国家自然科学基金(3100820)。

摘　　要：等温滴定量热技术可以直接测量化学反应吸收或者放出的热量。酶促反应伴随着吸放热过程,因此利用等温滴定量热仪可以测量酶活性。本文详细描述了等温滴定量热仪测定酶活性的原理、方法并对胰蛋白酶的活性进行了测试,研究了温度、转速、缓冲液等条件对胰蛋白酶活性的影响,并对实验过程中常见的滴定曲线形状进行了分析,结果表明温度变化导致k_(cat)、k_(m)有明显的变化,ΔH没有明显的变化。不同的缓冲液中k_(cat)、k_(m)没有明显的变化,但ΔH有明显的变化。对酶促反应的两种方法单次注入法和多次注入法进行了对比,结果表明单次注入法操作简单,多次注入法对酶浓度的变化更为敏感。Enzyme catalysis is accompanied by endothermic exothermic process,and enzyme activity can be measured by isothermal titration calorimeter.In this paper,the testing principle and method were described in detail.The effects of temperature,rotation speed,buffer solution,and other conditions on trypsin activity were studied.The common titration curve shape in the experiment process was analyzed.The results showed that the change of temperature led to obvious changes of k_(cat) and k_(m),but no obvious change ofΔH.Kcat and Km did not change significantly in different buffer solutions,butΔh changed significantly.In addition,the results showed that the single injection method was simple and the multiple injection method was more sensitive to the change of enzyme concentration.

关 键 词：等温滴定量热仪 胰蛋白酶 酶活性 

分 类 号：O652[理学—分析化学] TQ033[理学—化学]