Peroxynitrite Is the Major Species Formed from Different Flux Ratios of Co-generated Nitric Oxide and Superoxide

Abstract

There is much interest in the nitration. , Reaction 1,The early indication of the occurrence of this reaction in biological systems came from the report on the inhibitory effect of O 2 . on the activity of endothelium-derived relaxing factor (7).After endothelium-derived relaxing factor identity was established as ⅐ NO (8, 9), its scavenging by O 2 . was first proposed as a contributing factor to endothelial injury (10). Reaction 1 has great physiological significance as both ⅐ NO and hydrogen peroxide (H 2 O 2 , the product of dismutation of O 2 . ) act as important second messengers in redox cell signaling (11,12). In the absence of scavengers, ONOO Ϫ decomposes at neutral pH via protonation to peroxynitrous acid (pK a ϭ 6.7, Reaction 2) to yield nitrate (NO 3 Ϫ ) and free radical intermediates:hydroxyl radical ( ⅐ OH) and nitrogen dioxide ( ⅐ NO 2 ) (Reaction 3, k 3 ϭ 1.25 s Ϫ1 ) (2, 13).In most biological systems, carbon dioxide is a likely scavenger of ONOO Ϫ , yielding a short-lived nitrosoperoxycarbonate anion (ONOOCO 2 Ϫ , Reaction 4, k 4 ϭ 2.9 ϫ 10 4 M Ϫ1 s Ϫ1 (14)). During the decomposition of ONOOCO 2 Ϫ , nitrate and carbon dioxide are formed, as well as nitrogen dioxide radical and carbonate radical anion (Reaction 5) (2, 13, 15).Due to the occurrence of Reactions 4 and 5, as well as the scavenging by peroxiredoxins or oxyhemoglobin in specific subcellular compartments, the lifetime of ONOO Ϫ in biological systems is limited to only a few milliseconds (2, 13). The current methodologies for detection of ONOO Ϫ are based on the detection of radical species formed from ONOO Ϫ decomposition, i.e. ⅐ NO 2 and CO 3 . or ⅐ OH, using tyrosine that forms nitrotyrosine (TyrNO 2 ) as a marker product of intracellular ⅐ NO 2 and dihydrorhodamine 123 (DHR) 2 as a fluorogenic probe for oxidants ( ⅐ NO 2 , ⅐ OH, CO 3 . ). However, ⅐ NO 2 radical formed from the ONOO Ϫ -independent processes, e.g. via myeloperoxidasecatalyzed oxidation of nitrite by H 2 O 2 (16), could make data interpretation more tenuous (17, 18). Additional problems with this indirect approach may arise from alternate mechanisms through which TyrNO 2 can be formed without the involvement of ⅐ NO 2 radicals (19). DHR can be oxidized to the fluorescent rhodamine molecule by various one-electron oxidants, including compounds I and II of peroxidases (20,21 2 The abbreviations used are: DHR, dihydrorhodamine 123; CBA, coumarin-7-boronic acid; CBE, coumarin-7-boronic acid, pinacolate ester; COH, 7-hydroxycoumarin; PAPA-NONOate, (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate; X, xanthine; XO, xanthine oxidase; SOD, superoxide dismutase; DTPA, diethylenetriaminepentaacetic acid; HPLC, high pressure liquid chromatography.

Global Profiling of Reactive Oxygen and Nitrogen Species in Biological Systems

“…The standards 2-OH-E ϩ , E ϩ , and E ϩ -E ϩ were prepared as described previously (15,16). Coumarin-7-boronic acid (CBA) was synthesized as described earlier (9). Fluorescein dimethylamide boronate (FlAmBE) synthesis is described in supplemental Methods and Figs.…”

Peroxynitrite, a Stealthy Biological Oxidant

“…Peroxynitrite anion reacts directly via two-electron oxidation with boronate-based compounds to yield their corresponding hydroxyl derivatives (95). In the case of aryl boronates, the corresponding phenols are the major final products.…”

Kinetics of Nitrite Reduction and Peroxynitrite Formation by Ferrous Heme in Human Cystathionine β-Synthase

“…Reduction of Fe(III)-CBS was performed under a nitrogen atmosphere by the addition of known aliquots of sodium dithionite (Na 2 S 2 O 4 ) and confirmed by the appearance of the 449-nm Soret peak in the UV-visible absorption spectrum. The generation of peroxynitrite was determined using the boronate-based probe, CBA, and monitored by the increase in fluorescence intensity (excitation at 330 nm, emission at 450 nm) because of formation of the corresponding hydroxylated compound, 7-hydroxycoumarin (COH) (42). The procedure was calibrated using authentic peroxynitrite, and fluorescence spectroscopy was similar to that previously reported (42), with the intensity of the excitation and emission bands increasing linearly with peroxynitrite concentrations (data not shown).…”

“…The generation of peroxynitrite was determined using the boronate-based probe, CBA, and monitored by the increase in fluorescence intensity (excitation at 330 nm, emission at 450 nm) because of formation of the corresponding hydroxylated compound, 7-hydroxycoumarin (COH) (42). The procedure was calibrated using authentic peroxynitrite, and fluorescence spectroscopy was similar to that previously reported (42), with the intensity of the excitation and emission bands increasing linearly with peroxynitrite concentrations (data not shown). Typical reaction mixtures were prepared in the anaerobic chamber and contained Fe(III)-CBS (15 M) in phosphate buffer (0.1 M, pH 7.4, with 0.1 mM DTPA) to which dithionite (100 M) was added.…”

“…Peroxynitrite Formation by Fe(II)NO ⅐ -CBS Exposed to O 2 -The formation of peroxynitrite was evaluated using the boronate probe CBA, which reacts fast with peroxynitrite (k ϭ 1.1 ϫ 10 6 M Ϫ1 s Ϫ1 ) to yield COH as the major product, which can be followed by fluorescence spectroscopy (42). As shown in Ϫ3 s Ϫ1 ).…”