Articles
IDENTIFICATION AND CHARACTERIZATION OF PEROXIDASES ACTIVITY IN LEMON THYME (THYMUS CITRIODORUS) ROOTS
Article number
853_36
Pages
307 – 318
Language
English
Abstract
Enzymes are of critical importance in maintaining a species and protecting it against various forms of environmental stresses.
In particular, the formation of reactive oxygen species and ensuing oxidative stress is a constant menace for any organism.
Plants are equipped with a battery of antioxidative stress enzymes, including peroxidases that fulfill a variety of functions while scavenging H2O2. In this study, peroxidase activities were identified in an extract obtained from Thymus citriodorus roots by monitoring spectrophotometrically the H2O2-mediated oxidation of either o-dianisidine (at 460 nm), guaiacol (at 470 nm) or ferulic acid (at 310 nm) (lignin peroxidase) in the presence of extract aliquots.
Assays were performed at pH 4 for o-dianisidine, 6 for guaiacol and 5 for ferulic acid oxidation, using extinction coefficients of, respectively, 11.3, 26.6 and 8.68 mM-1.cm-1. With o-dianisidine as the reducing substrate, apparent Km, Vmax and catalytic efficiency were, respectively, 0.65±0.06 mM, 0.17±0.007 mM.min-1.mg prot-1 and 0.27±0.01 min-1.mg prot-1 for o-dianisidine and 0.5±0.05 mM, 0.255±0.005 mM.min-1.mg prot-1 and 0.5 min-1.mg prot-1 for H2O2; with guaiacol, the kinetics parameters were, respectively, 4.5±3 mM, 0.6±0.06 mM.min-1. mg prot-1 and 0.13±0.002 min-1.mg prot-1 for guaiacol and 0.9±0.2 mM, 0.52±0.04 mM.min-1.mg prot-1 and 0.58±0.04 min-1.mg prot-1 for H2O2; with ferulic acid, kinetics parameters were, respectively, 0.07±0.01 mM, 0.9±0.09 mM.min-1. mg prot-1 and 13±1 min-1.mg prot-1 for ferulic acid and 0.025±0.005 mM, 0.7±0.01 mM.min-1.mg prot-1 and 28±3 min-1.mg prot-1 for H2O2. The peroxidatic activities were sensitive to KCN, with IC50 of 0.12 ± 0.02 μM for o-dianisidine, 0.75±0.05 μM for guaiacol and 0.6 ± 0.05 μM for ferulic acid.
Results point out the predominance of lignin peroxidase activity over o-dianisidine and guaiacol peroxidases.
In particular, the formation of reactive oxygen species and ensuing oxidative stress is a constant menace for any organism.
Plants are equipped with a battery of antioxidative stress enzymes, including peroxidases that fulfill a variety of functions while scavenging H2O2. In this study, peroxidase activities were identified in an extract obtained from Thymus citriodorus roots by monitoring spectrophotometrically the H2O2-mediated oxidation of either o-dianisidine (at 460 nm), guaiacol (at 470 nm) or ferulic acid (at 310 nm) (lignin peroxidase) in the presence of extract aliquots.
Assays were performed at pH 4 for o-dianisidine, 6 for guaiacol and 5 for ferulic acid oxidation, using extinction coefficients of, respectively, 11.3, 26.6 and 8.68 mM-1.cm-1. With o-dianisidine as the reducing substrate, apparent Km, Vmax and catalytic efficiency were, respectively, 0.65±0.06 mM, 0.17±0.007 mM.min-1.mg prot-1 and 0.27±0.01 min-1.mg prot-1 for o-dianisidine and 0.5±0.05 mM, 0.255±0.005 mM.min-1.mg prot-1 and 0.5 min-1.mg prot-1 for H2O2; with guaiacol, the kinetics parameters were, respectively, 4.5±3 mM, 0.6±0.06 mM.min-1. mg prot-1 and 0.13±0.002 min-1.mg prot-1 for guaiacol and 0.9±0.2 mM, 0.52±0.04 mM.min-1.mg prot-1 and 0.58±0.04 min-1.mg prot-1 for H2O2; with ferulic acid, kinetics parameters were, respectively, 0.07±0.01 mM, 0.9±0.09 mM.min-1. mg prot-1 and 13±1 min-1.mg prot-1 for ferulic acid and 0.025±0.005 mM, 0.7±0.01 mM.min-1.mg prot-1 and 28±3 min-1.mg prot-1 for H2O2. The peroxidatic activities were sensitive to KCN, with IC50 of 0.12 ± 0.02 μM for o-dianisidine, 0.75±0.05 μM for guaiacol and 0.6 ± 0.05 μM for ferulic acid.
Results point out the predominance of lignin peroxidase activity over o-dianisidine and guaiacol peroxidases.
Authors
J. Keyhani, E. Keyhani
Keywords
antioxidative stress enzymes, enzymatic activity, ferulic acid, guaiacol, Lamiaceae, o-dianisidine
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