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• (2k = 2.1·109 M−1s−1, at pH = 7.2) and CO3•− (2k = 7.6·108 M−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.

Possibilities and Challenges for Quantitative Optical Sensing of Hydrogen Peroxide

“…A study by Votyakova et al [123] showed interference of this reaction by NADH and reduced glutathione, which also caused an increase in fluorescence. Additionally, peroxinitrite has been shown to oxidize Amplex ® Red in the presence of HRP as well [124]. Different studies have also shown H 2 O 2 -independent oxidation pathways for Amplex ® Red, with e.g., carboxylesterase [125] or peroxinitrite-derived radicals [124].…”

“…Additionally, peroxinitrite has been shown to oxidize Amplex ® Red in the presence of HRP as well [124]. Different studies have also shown H 2 O 2 -independent oxidation pathways for Amplex ® Red, with e.g., carboxylesterase [125] or peroxinitrite-derived radicals [124]. HRP has also been used in combination with dihydrorhodamine 6G to form the highly fluorescent rhodamine 6G [126].…”

Toward selective detection of reactive oxygen and nitrogen species with the use of fluorogenic probes – Limitations, progress, and perspectives

“…Additionally, NADH and reduced glutathione in the presence of HRP can interfere with the measurement [4,48]. Our results indicate that the addition of peroxynitrite can lead to the oxidation of Amplex® Red [44]. The mechanism of this reaction involves the formation of oxidizing radicals (i.e., CO 3 •− and • NO 2 ) from the decomposition of peroxynitrite.…”

“…AR-derived radicals can also undergo dismutation to form the secondary transient product. Further de- N -acetylation of that product leads to the formation of resorufin [44]. The Amplex® Red assay is assumed to be highly selective toward H 2 O 2 .…”

“…Pulse radiolysis experiments have indicated that those radicals are able to oxidize Amplex® Red. HRP can significantly increase the yield of resorufin from Amplex® Red oxidation by peroxynitrite [44]. …”