Mechanism of oxidative conversion of Amplex® Red to resorufin: Pulse radiolysis and enzymatic studies. Free Radic Biol Med 2016 Jun;95:323-32
Date
03/30/2016Pubmed ID
27021961Pubmed Central ID
PMC5697983DOI
10.1016/j.freeradbiomed.2016.03.027Scopus ID
2-s2.0-84963657120 (requires institutional sign-in at Scopus site) 134 CitationsAbstract
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(•) ((2)k=2.1·10(9)M(-1)s(-1), at pH=7.2) and CO3(•-) ((2)k=7.6·10(8)M(-1)s(-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.
Author List
Dębski D, Smulik R, Zielonka J, Michałowski B, Jakubowska M, Dębowska K, Adamus J, Marcinek A, Kalyanaraman B, Sikora AAuthors
Balaraman Kalyanaraman PhD Professor in the Biophysics department at Medical College of WisconsinJacek M. Zielonka PhD Assistant Professor in the Biophysics department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
CatalysisHorseradish Peroxidase
Hydrogen Peroxide
Oxazines
Oxidants
Oxidation-Reduction
Peroxynitrous Acid
Pulse Radiolysis
