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

本文试图建立一种简便、通用、精确的以反应热为探针测定酶动力学参数如米氏常数和分子活性的方法。为此，我们利用高灵敏度等温滴定量热法。采用“滴定法”和“单次注射法”两种方法。对于前一种情况，通过将底物溶液以短间隔多次注入酶溶液中，底物浓度逐步增加，并且每次观察到反应热。对于后者，酶溶液和底物溶液仅混合一次，观察到随后的总反应。作为模型情况，检测了抗坏血酸氧化酶、葡糖淀粉酶和脂肪酶催化的反应。用滴定法检测抗坏血酸氧化酶和葡糖淀粉酶的情况，获得了满意的结果，与以前报道的数据一致。表明该方法是一种较有前途的非熟练测定酶动力学参数的方法。单次激发法测得的k值偏小<cat>，Km值偏大，但原因不明;脂肪酶在底物溶液和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仪的反应池的内表面上，并且不容易将酶从细胞中去除。结果，催化反应仅仅通过用底物溶液填充量热计单元而开始。这表明酶作为蛋白质的性质是该方法的重要因素。我们计划运行由此获得的参数的数据库，包括反应热。

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.