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

Amplex (R) 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 (R) Red to resorufin. In this study we investigated the mechanism of one-electron oxidation of Amplex (R) 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 (N-3(center dot), CO3 center dot-, (NO2)-N-center dot and GS(center dot)). The rate constants for the Amplex (R) Red oxidation by N-3(center dot) ((2)k = 2.1 . 10(9) M-1 s(-1), at pH = 7.2) and CO3 center dot- ((2)k = 7.6 . 10(8) M(-1)s(-1), at pH = 10.3) were determined. Two intermediates formed during the conversion of Amplex (R) Red into resorufin have been characterized. Based on the results obtained, the mechanism of transformation of Amplex (R) Red into resorufin, involving disproportionation of the Amplex (R) Red-derived radical species, has been proposed. The results indicate that peroxynitrite-derived radicals, but not peroxynitrite itself, are capable to oxidize Amplex (R) Red to resorufin. We also demonstrate that horseradish peroxidase can catalyze oxidation of Amplex (R) Red not only by hydrogen peroxide, but also by peroxynitrite, which needs to be considered when employing the probe for hydrogen peroxide detection. (C) 2016 Elsevier Inc. All rights reserved.