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

Publication date: Available online 26 March 2016
Source:Free Radical Biology and Medicine
Author(s): Dawid Dębski, Renata Smulik, Jacek Zielonka, Bartosz Michałowski, Małgorzata Jakubowska, Karolina Dębowska, Jan Adamus, Andrzej Marcinek, Balaraman Kalyanaraman, Adam Sikora
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.

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