Development and evaluation of kinetic spectrophotometric assays for horseradish peroxidase by catalytic coupling of paraphenylenediamine and mequinol.
This paper presents a novel spectrophotometric method to measure peroxidase activity using paraphenylenediamine dihydrochloride (PPDD) and Mequinol (MQ). The PPDD traps the free radical, and gets oxidized to electrophilic 1,4-diimine; this couples with MQ to an give intense violet-colored chromogenic species with the maximum absorbance at 560 nm. This assay was adopted for the quantification of hydrogen peroxide between 10 x 10(-6) to 80 x 10(-6) M. From the kinetic data, a two-substrate ping-pong mechanism of peroxidase was established. Catalytic efficiency and catalytic power of commercial peroxidase were 0.204 x 10(6) M(-1) min(-1) and 2.86 x 10(-4) min(-1), respectively. The catalytic constant (k(cat)) of the proposed method was 0.2080 x 10(3) min(-1). As a simple, rapid, precise and sensitive technique, PPDD-MQ stands as a potential replacement for the traditional guaiacol method. Applications to the plant extracts increase its relevance in the field of biochemical analysis.
Nagaraja
Padmarajaiah
P
Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, India. drpn58@yahoo.co.in
Shivakumar
Anantharaman
A
Kumar Shrestha
Ashwinee
A
D000042:Absorption; D000840:Anisoles / Q000378:metabolism*; D055162:Biocatalysis; D003116:Color; D006735:Horseradish Peroxidase / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000032:analysis / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010655:Phenylenediamines / Q000378:metabolism*; D010944:Plants / Q000201:enzymology; D013053:Spectrophotometry / Q000379:methods*
D000840:Anisoles; D010655:Phenylenediamines; C009760:mequinol; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; C029728:4-phenylenediamine