Valero E, Varón R, García-Carmona F
Departamento de Química-Física, E.U. Politécnica, Universidad de Castilla-La Mancha, Albacete, Spain.
Biochem J. 1995 Jul 1;309 ( Pt 1)(Pt 1):181-5. doi: 10.1042/bj3090181.
A kinetic study is made of a system consisting of a specific enzymic cycling assay coupled to an enzymic reaction. A kinetic analysis of this system is presented, and the accumulation of chromophore involved in the cycle is seen to be parabolic, i.e. the rate of the reaction increases continuously with constant acceleration. The system is illustrated by the measurement of alkaline phosphatase activity using beta-NADP+ as substrate. The enzymes alcohol dehydrogenase and diaphorase are used to cycle beta-NAD+ in the presence of ethanol and p-Iodonitrotetrazolium Violet. During each turn of the cycle, one molecule of the tetrazolium salt is reduced to an intensely coloured formazan. A simple procedure for evaluating the kinetic parameters involved in the system and for optimizing this cycling assay is described. The method is applicable to the measurement of any enzyme, and its amplification capacity as well as the simplicity of determining kinetic parameters enable it to be employed in enzyme immunoassays to increase the magnitude of the measured response.
对一个由与酶促反应偶联的特定酶循环分析组成的系统进行了动力学研究。给出了该系统的动力学分析，并且观察到循环中涉及的发色团积累呈抛物线形，即反应速率以恒定加速度持续增加。以使用β-NADP⁺作为底物测量碱性磷酸酶活性为例对该系统进行说明。在乙醇和对碘硝基四氮唑紫存在的情况下，使用醇脱氢酶和黄递酶来循环β-NAD⁺。在循环的每一轮中，一分子四氮唑盐被还原为颜色很深的甲臜。描述了一种评估系统中涉及的动力学参数以及优化这种循环分析的简单程序。该方法适用于任何酶的测量，其放大能力以及确定动力学参数的简便性使其能够用于酶免疫测定中以增加测量响应的幅度。
