An assay for pro-oxidant reactivity based on phenoxyl radicals generated by laccase

Abstract

A transient species may be detected with UV-vis and EPR spectroscopy during turnover of a laccase with quercetin; this species is assigned as a quercetin-derived radical, based on EPR spectra as well the observed UV-vis similarities (a 540nm centred band) with previously reported data. The rates of formation and decay of this species correlate well (r=0.9946) with the pro-oxidant reactivity manifested by flavonoids in the presence of laccase. An assay for the pro-oxidant reactivity of natural products is hence proposed based on the results reported here; its application is demonstrated for a series of pure compounds as well as for several propolis extracts. This assay has the advantages of using a biologically relevant process (haemoglobin oxidation), and not requiring the addition of oxidising agents such as peroxide or superoxide. Correlations, or the lack thereof, between the pro-oxidant parameters and the redox potentials, antioxidant capacities and lipophilicities, were analysed. The laccase employed in our study does display reactivity-related similarities to a range of other proteins, including human plasma ceruloplasmin.

Keywords: 2,2-diphenyl-1-picrylhydrazyl; 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); 2-amino-2-hydroxymethyl-propane-1,3-diol; 3-(N-morpholino)propanesulphonic acid; 5,5-dimethyl-1-pyrroline N-oxide; ABTS; APCI; AUC; CAT; DAD; DMPO; DPPH; EPR; Flavonoid; HRP; Hb; Laccase; MOPS; Met-haemoglobin; N-tris-(hydroxymethyl)-methyl-3-aminopropanesulphonic acid; NHE; PBS; PCA; Pro-oxidant, antioxidant; QF; Quercetin; ROS; RSD; Radical; SDS–PAGE; SE; SOD; TAPS; TRIS; area under the curve; atmospheric-pressure chemical ionisation; catalase from bovine liver; diode array detector; electron paramagnetic resonance; haemoglobin; horseradish peroxidase; normal hydrogen electrode; pQF; phosphate buffered saline; principal component analysis; pro-oxidant quercetin factor; quercetin factor; reactive oxygen species; relative standard deviation; sodium dodecyl sulphate–polyacrylamide gel electrophoresis; standard error; superoxide dismutase bovine.