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Detection and identification of oxidants formed during •NO/O2•? reaction: a multi-well plate CW-EPR spectroscopy combined with HPLC analyses.

ABSTRACT: New techniques and probes are routinely emerging for detecting short-lived free radicals such as superoxide radical anion (O?(•-)), nitric oxide ((•)NO), and transient oxidants derived from peroxynitrite (ONOO(-)/ONOOH). Recently, we reported the profiles of oxidation products (2-hydroxyethidium, ethidium, and various dimeric products) of the fluorogenic probe hydroethidine (HE) in the (•)NO/O?(•-) system (Zielonka et al. 2012). In this study, we used HPLC analyses of HE oxidation products in combination with continuous wave electron paramagnetic resonance (CW-EPR) spin trapping with 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) to define the identity of the oxidizing species formed in the (•)NO/O?(•-) system. EPR spin-trapping technique is still considered as the gold standard for characterization of free radicals and their intermediates. We monitored formation of BMPO-superoxide (BMPO-(•)OOH) and BMPO-hydroxyl (BMPO-(•)OH) radical adducts. Simultaneous analyses of results from EPR spin-trapping and HPLC measurements are helpful in the interpretation of the mechanism of formation of products of HE oxidation.

SUBMITTER: Koto T

PROVIDER: S-EPMC4780754 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Detection and identification of oxidants formed during •NO/O2•⁻ reaction: a multi-well plate CW-EPR spectroscopy combined with HPLC analyses.

Koto T T Michalski R R Zielonka J J Joseph J J Kalyanaraman B B

Free radical research 20140401 4

New techniques and probes are routinely emerging for detecting short-lived free radicals such as superoxide radical anion (O₂(•-)), nitric oxide ((•)NO), and transient oxidants derived from peroxynitrite (ONOO(-)/ONOOH). Recently, we reported the profiles of oxidation products (2-hydroxyethidium, ethidium, and various dimeric products) of the fluorogenic probe hydroethidine (HE) in the (•)NO/O₂(•-) system (Zielonka et al. 2012). In this study, we used HPLC analyses of HE oxidation products in co ...[more]

PMID: 24460755

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Project description:Recently, we showed that peroxynitrite (ONOO(-)) reacts directly and rapidly with aromatic and aliphatic boronic acids (k ? 10(6) M(-1)s(-1)). Product analyses and substrate consumption data indicated that ONOO(-) reacts stoichiometrically with boronates, yielding the corresponding phenols as the major product (?85-90%), and the remaining products (10-15%) were proposed to originate from free radical intermediates (phenyl and phenoxyl radicals). Here, we investigated in detail the minor, free radical pathway of boronate reaction with ONOO(-). The electron paramagnetic resonance (EPR) spin-trapping technique was used to characterize the free radical intermediates formed from the reaction between boronates and ONOO(-). Using 2-methyl-2-nitrosopropane (MNP) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) spin traps, phenyl radicals were trapped and detected. Although phenoxyl radicals were not detected, the positive effects of molecular oxygen, and inhibitory effects of hydrogen atom donors (acetonitrile, and 2-propanol) and general radical scavengers (GSH, NADH, ascorbic acid, and tyrosine) on the formation of phenoxyl radical-derived nitrated product, suggest that the phenoxyl radical was formed as the secondary species. We propose that the initial step of the reaction involves the addition of ONOO(-) to the boron atom in boronates. The anionic intermediate undergoes both heterolytic (major pathway) and homolytic (minor pathway) cleavage of the peroxy (O-O) bond to form phenol and nitrite as a major product (via a nonradical mechanism), or a radical pair PhB(OH)(2)O(•-)···(•)NO(2) as a minor product. It is conceivable that phenyl radicals are formed by the fragmentation of the PhB(OH)(2)O(•-) radical anion. According to the DFT quantum mechanical calculations, the energy barrier for the dissociation of PhB(OH)(2)O(•-) radical anion to form phenyl radical is only a few kcal/mol, suggesting rapid and spontaneous fragmentation of the PhB(OH)(2)O(•-) radical anion in aqueous media. Biological implications of the minor free radical pathway are discussed in the context of ONOO(-) detection, using the boronate probes.

Dataset Information

Detection and identification of oxidants formed during •NO/O2•? reaction: a multi-well plate CW-EPR spectroscopy combined with HPLC analyses.

Publications

Detection and identification of oxidants formed during •NO/O2•⁻ reaction: a multi-well plate CW-EPR spectroscopy combined with HPLC analyses.

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