关键词: 苯二胺, 氧化, 过氧化氢, 辣根过氧化物酶, 红外分光光度法, 高速液体色谱, 核磁共振谱法

The oxidation of m-phenylene Diamine (MPD) by H~2O~2 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^-^3 mol/L MPD and 9.0×10^-^4 mol/L H~2O~2 when reacting in the presence of HRP in 0.02 mol/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 reoxidized reversibly back to 2,7-diaminophenazine. This enzymatic reaction can be used in the voltammetric enzyme-linked immunoassay.

Key words: BENZENEDIAMINE, OXIDATION, HYDROGEN PEROXIDE, INFRARED SPECTROPHOTOMETRY, HIGH SPEED LIQUID CHROMATOGRAPHY, NMR SPECTROMETRY