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Development of a rapid and general reaction-oriented method for determining enzyme activity by calorimetry

开发一种快速、通用的反应导向方法，用于通过量热法测定酶活性

基本信息

批准号：
15580076
负责人：
TANAKA Akiyoshi
金额：
$ 1.73万
依托单位：
Mie University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15580076/
关键词：
enzyme reaction kinetic parameter calorimetry rapid determination general method

项目摘要

We have tried to develop a convenient, general, and precise method for determining the enzyme kinetic parameters such as Michaelis constant and the molecular activity using reaction heat as a probe. For this purpose, we utilized high-sensitivity isothermal titration calorimetry. Two methods, "titration method" and "single-shot method" were employed. For the former case, a substrate concentration increases stepwise by injecting the substrate solution several times with short intervals into the enzyme solution and reaction heat was observed each time. For the latter, an enzyme solution and its substrate solution were mixed only once and the following total reaction is observed.As model cases, ascorbate oxidase, glucoamylase, and lipase-catalyzed reactions were examined.Satisfactory results were obtained for the cases of ascorbate oxidase and glucoamylase by the titration method, consisting with the previously reported data, indicating that this method is a promising approach for determining the enzyme kinetic parameters without skilled job. On the other hand, the single-shot method tends to give somewhat smaller k_<cat> and larger K_m values with unknown reason.For the case of lipase, huge amount of dilution heat was observed when a substrate solution and the enzyme solution were mixed, which masked the enzyme reaction-heat. What is worse, the lipase molecules were adsorbed on the inner surface of the reaction cell of the titration calorimeter tightly, and it was not easy to remove the enzyme from the cell. As a result, the catalytic reaction started simply by filling the calorimeter cell with a substrate solution. This indicates that property of an enzymes as a protein is an important factor for this method.We are planning to operate the data base of the parameters thus obtained, including reaction heat.

我们试图建立一种方便、通用和精确的方法来测定酶的动力学参数，如米切里斯常数和分子活性，以反应热为探针。为此，我们采用了高灵敏度等温滴定量热法。采用“滴定法”和“单针法”两种方法。在前一种情况下，将底物溶液以短时间间隔注入酶溶液数次，使底物浓度逐步增加，每次观察反应热。对于后者，酶溶液和底物溶液只混合一次，观察到如下总反应。研究了抗坏血酸氧化酶、葡萄糖淀粉酶和脂肪酶催化的反应。该方法对抗坏血酸氧化酶和葡萄糖淀粉酶的测定结果满意，与文献报道的结果一致，表明该方法是一种很有前途的测定酶动力学参数的方法，无需人工操作。另一方面，单次方法往往会给出较小的k_<cat>和较大的K_m值，原因不明。以脂肪酶为例，底物溶液与酶溶液混合时产生大量的稀释热，掩盖了酶的反应热。更糟糕的是，脂肪酶分子被紧密地吸附在滴定量热计反应细胞的内表面，酶不容易从细胞中去除。因此，催化反应只需在量热计电池中填充底物溶液即可开始。这表明酶作为蛋白质的性质是这种方法的一个重要因素。我们正计划操作由此获得的参数数据库，包括反应热。