生物酶HRP催化H_2O_2氧化间苯二胺反应的研究

Studies on the Oxidation of m - Phenylene Diamine by H_2O_2 Catalyzed by Horseradish Peroxidase

应用电化学分析，高效液相色谱（ＨＰＬＣ），紫外－可见光谱（ＵＶ－ｖｉｓ），红外光谱（ＩＲ）和核磁共振（ＮＭＲ）等技术，对辣根过氧化物酶（ＨＲＰ）催化Ｈ２Ｏ２氧化间苯二胺（ＭＰＤ）的反应进行了研究。伏安法和高效液相色谱实验说明，在所选择的酶催化反应条件下，酶催化反应生成一种产物，用化学方法制得了ＨＲＰ酶催化Ｈ２Ｏ２氧化ＭＰＤ的产物纯品。以ＵＶ－ｖｉｓ，ＩＲｔ ＾１ＨＮＭＲ谱鉴定，产物为２。

The oxidation of m - phenylene Diamine (MPD) by H2O2 catalyzed by horseradish peroxidase (HRP) has been investigated using electrochemical analysis, high performance liquid chromatography (HPLC), UV - vis, IR and NMR spectroscopy. The experiments of voltammetry and HPLC indicate that only one enzymatic product is present under the selected reaction conditions. The best results for the enzymatic reaction were obtained with 2.0 × 10-3mol/L MPD and 9.0 × 10-4mol/L H2O2 when reacting in the presence of HRP in 0.02mol/L BR buffer solution for 60 min at room temperature. The authentic sample of 2,7 -diaminophenazine, the product of the enzymatic reaction, was prepared by chemical means and characterized by UV - vis, IR and 1H NMR spectroscopy. The processes of the enzymatic reaction are described. The enzymatic product shows a pair of reversible redox peaks in the cyclic voltammogram. The reduction peak and the oxidation peak are completely symmetric, which demonstrates that the enzymatic product behaves as a complete adsorption reversible redox process on the mercury electrode. The product ,2,7 ?diaminophenazine, undergoes a reversible two - electron reduction to give N, N' - dihydro - 2,7 - diaminophenazine, which can be re-oxidized reversibly back to 2,7 - diaminophenazine. This enzymatic reaction can be used in the voltammetric enzyme - linked immunoassay.