Quantification and evaluation of kinetic bio-catalytic pathway of horseradish peroxidase in an electron mediated reaction system and its applications in plant extracts

Abstract

The intermolecular coupling of 2,5-dimethoxyaniline (DMA) as mediated electron transfer reaction in presence of H2O2 and peroxidase in acetate buffer of pH 4.2 resulting green colored product having maximum absorption at λmax=740nm was investigated by spectrophotometer. Under optimum conditions, linearity range for the quantification of H2O2 was 2.0–288.0μM and for peroxidase were 0.59–9.46 and 0.443–9.46nM by kinetic and fixed-time method, respectively. The catalytic efficiency and catalytic power were KeffD=2.354×105M−1min−1 and KpowD=4.59×10−4min−1, respectively. From the plot of d(1/Do) vs d(1/Vo) and d(1/Ho) vs d(1/Vo), Michaelis–Menten constants for DMA and H2O2were found that KmD=1458μM and KmH2O2=301μM. Applicability of the method was tested for peroxidase activity in some plant extracts and compared with guaiacol/peroxidase system. Regarding superiority of the method, it is suggested that DMA/peroxidase system can be a better hydrogen donor for HRP assay than guaiacol system as evident from kinetic data.