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

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 540 nm 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.