title: Mechanism of oxidative conversion of Amplex® Red to resorufin: Pulse radiolysis and enzymatic studies.
abstract: 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.
authors.affiliation: Institute of Applied Radiation Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland.
authors.forename: Dawid
authors.initials: D
authors.lastname: Dębski
authors.affiliation: Institute of Applied Radiation Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland.
authors.forename: Renata
authors.initials: R
authors.lastname: Smulik
authors.affiliation: Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226, United States. Electronic address: jzielonk@mcw.edu.
authors.forename: Jacek
authors.initials: J
authors.lastname: Zielonka
authors.affiliation: Institute of Applied Radiation Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland.
authors.forename: Bartosz
authors.initials: B
authors.lastname: Michałowski
authors.affiliation: Institute of Applied Radiation Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland.
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Jakubowska
authors.affiliation: Institute of Applied Radiation Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland.
authors.forename: Karolina
authors.initials: K
authors.lastname: Dębowska
authors.affiliation: Institute of Applied Radiation Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland.
authors.forename: Jan
authors.initials: J
authors.lastname: Adamus
authors.affiliation: Institute of Applied Radiation Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Marcinek
authors.affiliation: Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226, United States.
authors.forename: Balaraman
authors.initials: B
authors.lastname: Kalyanaraman
authors.affiliation: Institute of Applied Radiation Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland. Electronic address: adam.sikora@p.lodz.pl.
authors.forename: Adam
authors.initials: A
authors.lastname: Sikora
mesh_terms: D002384:Catalysis; D006735:Horseradish Peroxidase / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000302:isolation & purification* / Q000378:metabolism; D010078:Oxazines / Q000737:chemistry*; D016877:Oxidants / Q000737:chemistry*; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000302:isolation & purification* / Q000378:metabolism; D011675:Pulse Radiolysis
chemical_list: D010078:Oxazines; D016877:Oxidants; C470430:Amplex Red; D030421:Peroxynitrous Acid; C014180:resorufin; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase
keywords: Amplex® Red; Coumarin boronic acid; Hydrogen peroxide; Peroxynitrite; Pulse radiolysis