Mechanism of oxidative conversion of Amplex® Red to resorufin: Pulse radiolysis and enzymatic studies

Amplex® Red (10-acetyl-3,7-dihydroxyphenoxazine) is a fluorogenic probe widely used to detect and quantify hydrogen peroxide in biological systems. Detection of hydrogen peroxide is based on peroxidase-catalyzed oxidation of Amplex® Red to resorufin. In this study we investigated the mechanism of one-electron oxidation of Amplex® Red and we present the spectroscopic characterization of transient species formed upon the oxidation. Oxidation process has been studied by a pulse radiolysis technique with one-electron oxidants (N3•, CO3•–,•NO2 and GS•). The rate constants for the Amplex® Red oxidation by N3• (2k=2.1·109M−1s−1, at pH=7.2) and CO3•– (2k=7.6·108M−1s−1, at pH=10.3) were determined. Two intermediates formed during the conversion of Amplex® Red into resorufin have been characterized. Based on the results obtained, the mechanism of transformation of Amplex® Red into resorufin, involving disproportionation of the Amplex® Red-derived radical species, has been proposed. The results indicate that peroxynitrite-derived radicals, but not peroxynitrite itself, are capable to oxidize Amplex® Red to resorufin. We also demonstrate that horseradish peroxidase can catalyze oxidation of Amplex® Red not only by hydrogen peroxide, but also by peroxynitrite, which needs to be considered when employing the probe for hydrogen peroxide detection. [Display omitted] •Mechanism of 1-electron oxidation of Amplex Red has been studied by pulse radiolysis.•One-electron oxidation of Amplex Red leads to the formation of fluorescent resorufin.•HRP catalyzes oxidation of Amplex Red by hydrogen peroxide and peroxynitrite.•In the absence of HRP peroxynitrite-derived radicals oxidize Amplex Red to resorufin.