Studies on the oxidation of m-phenylene diamine by H2O2 catalyzed by horseradish peroxidase

The oxidation of m - phenylene Diamine (MPD) by H2O2 catalyzed by horseradi sh peroxidase (HRP) has been investigated using electrochemical analysis, h igh performance liquid chromatography (HPLC), UV - vis, IR and NMR spectros copy. The experiments of voltammetry and HPLC indicate that only one enzyma tic product is present under the selected reaction conditions. The best res ults for the enzymatic reaction were obtained with 2.0 x 10(-3)mol/L MPD an d 9.0 x 10(-4)mol/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 H-1 NMR spectroscop y. The processes of the enzymatic reaction are described. The enzymatic pro duct shows a pair of reversible redox peaks in the cyclic voltammogram. The reduction peak and the oxidation peak are completely symmetric, which demo nstrates that the enzymatic product behaves as a complete adsorption revers ible redox process on the mercury electrode. The product, 2,7 - diaminophen azine, undergoes a reversible two - electron reduction to give N, N' - dihy dro - 2,7 - diaminophenazine, which can be reoxidized reversibly back to 2, 7 - diaminophenazine. This enzymatic reaction can be used in the voltammetr ic enzyme - linked immunoassay.