Hydroxylation of 4-Amino-5-hydroxynaphthalene-2,7-disulfonic Acid Monosodium Salt Catalysed by Horseradish Peroxidase and Hydrogen Peroxide: Computation of Kinetic Parameters Including Its Application to Crude Plant Extracts

摘要

The new spectrophotometric assay method for the quantification of peroxidase activity uses 4-amino-5-hydroxynaphthalene-2,7-disulfonicacid monosodium salt (AHNDSA) as chromogenic co-substrate. The method is based on hydroxylation of AHNDSA in presence of H_2O_2/peroxidase forming quinone, having λ_(max) = 460 nm in the acetate buffer (pH = 4.0) at 30 ℃. The linearity of H_2O_2 by kinetic method was 10-332 μM and for peroxidase by kinetic and fixed time methods were 1.18-18.92 and 1.18-9.46 nM, respectively. Catalytic efficiency and catalytic power for peroxidase assay were 7.965 × 10~4M~(-1)min~(-1) and 3.76 × 10~(-4)min~(-1), respectively. From the plot of d(1/A_0) vs d(1/V_0) and d(1/H_0) vs d(1/V_0), the apparent Michaelis-Menten constants for H_2O_2 and AHNDSA were K_m~(H_2O_2) = 68 K_m~A = 275 μM, respectively. The method was tested with some plant extracts and also compared with guaiacol/peroxidase system. Except Boerhavia diffusa, all other tested plant samples showed highest peroxidase activity. The proposed method is a rapid and convenient method to determine peroxidase activity by spectrophotometer. This method for the first time reports peroxidase activity in Lantana camara and Oplismenus compositus plants. Kinetic results showed that AHNDSA/peroxidase system can be better hydrogen donor for peroxidase assay than guaiacol system.