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

[Display omitted] ► 2,5-Dimethoxyaniline is used as a chromogenic co-substrate for peroxidase assay. ► A mathematical model has been proposed for the evaluation of catalytic parameters. ► Peroxidase activity in some of crude medicinal plant species has been assessed. ► Peroxidase activity has been r...

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Published in:	Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy Vol. 102; pp. 75 - 81
Main Authors:	Krishna, Honnur, Nagaraja, Padmarajaiah, Shivakumar, Anantharaman, Chamaraja, Nelligere A., Aradhana, Narayan
Format:	Journal Article
Language:	English
Published:	England Elsevier B.V 01-02-2013
Subjects:	2,5-Dimethoxyaniline Aniline Compounds - metabolism Armoracia - enzymology Bio-catalyst Crude plant extracts Electrons Enzyme Assays - methods Horseradish Peroxidase - metabolism Horseradish peroxidase assay Hydrogen Peroxide - analysis Hydrogen Peroxide - metabolism Kinetics Mathematical model Mediated electron transfer system Models, Biological Peroxidase - metabolism Plant Extracts - metabolism Plants, Medicinal - enzymology Plants, Medicinal - metabolism Spectrophotometry - methods 2,5-Dimethoxyaniline [H2O2]o HRP [D]o KeffD and KeffG KmH2O2 VmaxH2O2 Bio-catalyst KpowD and KpowG DMA Vo 2,4-DMA Horseradish peroxidase assay Mathematical model Crude plant extracts Mediated electron transfer system
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Summary:	[Display omitted] ► 2,5-Dimethoxyaniline is used as a chromogenic co-substrate for peroxidase assay. ► A mathematical model has been proposed for the evaluation of catalytic parameters. ► Peroxidase activity in some of crude medicinal plant species has been assessed. ► Peroxidase activity has been reported first time in T. fruticosum and S. paniculatum. 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.
ISSN:	1386-1425 1873-3557
DOI:	10.1016/j.saa.2012.10.024