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Previous studies indicated that bound sulfur species (BSS), including hydrogen polysulfide (H 2 S n ), have various physiological functions in mammalian cells. Although H 2 S n molecules have been considered as secondary metabolites derived from hydrogen sulfide (H 2 S) based on in vitro studies or predetermined reaction formula, the physiological form of BSS and their endogenous concentration remain unclear. In the present study, we aimed to improve the usual method using monobromobimane (mBB) followed by high performance liquid chromatographic (HPLC) analysis for HS － for simultaneous determination of H 2 S, H 2 S 2 , H 2 S 3 and cysteine persulfide in biological samples. We demonstrated that mBB derivatization of H 2 S and H 2 S n standards under alkaline conditions (pH 9.5) induced significant decreases in H 2 S 2 and H 2 S 3 levels and a significant increase in the H 2 S level in an incubation time-dependent manner. Conversely, the derivatization of mBB adducts of H 2 S 2 and H 2 S 3 were stable under neutral conditions (pH 7.0), which is physiologically relevant. Therefore, we re-examined the method using mBB and applied an improved method for the evaluation of H 2 S, H 2 S 2 , and H 2 S 3 in mouse brain under physiological pH conditions. The concentrations of H 2 S and H 2 S 2 were 0.030 ± 0.004 μmol/g protein and 0.026 ± 0.002 μmol/g protein, respectively. Although the level of H 2 S 3 was below the quantification limit of this method, H 2 S 3 was detected in mouse brain. Using the method established here, we reveal for the first time the existence of endogenous H 2 S 2 and H 2 S 3 in mammalian brain tissues. H 2 S 2 and H 2 S 3 exert anti-oxidant activity and anti-carbonyl stress effects through the regulation of redox balance in neuronal cells. Thus, our observations provide novel insights into the physiological functions of BSS in the brain and into neuronal diseases involved in redox imbalance. [ABSTRACT FROM AUTHOR]

Population studies suggest cardiovascular health benefits of consuming fruits and vegetables rich in polyphenolic compounds such as flavonoids. We reported previously that the flavonoid quercetin protects arteries from oxidant-induced endothelial dysfunction and attenuates atherosclerosis in apolipoprotein E gene knockout mice, with induction of heme oxygenase-1 (Hmox1) playing a critical role. The present study investigated the structural requirements of flavonoids to induce Hmox1 in human aortic endothelial cells (HAEC). We identified ortho-dihydroxyl groups and an α,β-unsaturated system attached to a catechol as the key structural requirements for Hmox1 induction. Active but not inactive flavonoids had a low oxidation potential and prevented ascorbate autoxidation, suggesting that Hmox1 inducers readily undergo oxidation and that oxidized, rather than reduced, flavonoids may be the biological inducer of Hmox1. To test this hypothesis, we synthesized stable derivatives of caffeic acid (3-(3,4-dihyroxyphenyl)-2-propenoic acid) containing either ortho-dihydroxy or ortho-dioxo groups. Compared with the dihydroxy compound, the quinone analog induced Hmox1 more potently in HAEC and also provided enhanced protection to arteries of wild type animals against oxidant-induced endothelial dysfunction. In contrast, the quinone analog failed to provide protection against oxidant-induced endothelial dysfunction in arteries of Hmox1 –/– mice, establishing a key role for Hmox1 in vascular protection. These results suggest that oxidized forms of dietary polyphenols are the likely inducers of Hmox1 and may explain in part the protective cardiovascular effects of diets rich in these compounds. [ABSTRACT FROM AUTHOR]

Epoxyeicosatrienoic acids (EETs) are metabolites of arachidonic acid (AA) oxidation that have important cardioprotective and signaling properties. AA is an ω-6 polyunsaturated fatty acid (PUFA) that is prone to autoxidation. Although hydroperoxides and isoprostanes are major autoxidation products of AA, EETs are also formed from the largely overlooked peroxyl radical addition mechanism. While autoxidation yields both cis - and trans -EETs, cytochrome P450 (CYP) epoxygenases have been shown to exclusively catalyze the formation of all regioisomer cis -EETs, on each of the double bonds. In plasma and red blood cell (RBC) membranes, cis - and trans -EETs have been observed, and both have multiple physiological functions. We developed a sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay that separates cis - and trans - isomers of EETs and applied it to determine the relative distribution of cis - vs. trans -EETs in reaction mixtures of AA subjected to free radical oxidation in benzene and liposomes in vitro . We also determined the in vivo distribution of EETs in several tissues, including human and mouse heart, and RBC membranes. We then measured EET levels in heart and RBC of young mice compared to old. Formation of EETs in free radical reactions of AA in benzene and in liposomes exhibited time- and AA concentration-dependent increase and trans -EET levels were higher than cis -EETs under both conditions. In contrast, cis -EET levels were overall higher in biological samples. In general, trans -EETs increased with mouse age more than cis -EETs. We propose a mechanism for the non-enzymatic formation of cis - and trans -EETs involving addition of the peroxyl radical to one of AA's double bonds followed by bond rotation and intramolecular homolytic substitution (S H i). Enzymatic formation of cis -EETs by cytochrome P450 most likely occurs via a one-step concerted mechanism that does not allow bond rotation. The ability to accurately measure circulating EETs resulting from autoxidation or enzymatic reactions in plasma and RBC membranes will allow for future studies investigating how these important signaling lipids correlate with heart disease outcomes. [ABSTRACT FROM AUTHOR]

Human serum albumin (HSA) has a single reduced cysteine residue, Cys34, whose acidity has been controversial. Three experimental approaches (pH-dependence of reactivity towards hydrogen peroxide, ultraviolet titration and infrared spectroscopy) are used to determine that the p K a value in delipidated HSA is 8.1±0.2 at 37 °C and 0.1 M ionic strength. Molecular dynamics simulations of HSA in the sub-microsecond timescale show that while sulfur exposure to solvent is limited and fluctuating in the thiol form, it increases in the thiolate, stabilized by a persistent hydrogen-bond (HB) network involving Tyr84 and bridging waters to Asp38 and Gln33 backbone. Insight into the mechanism of Cys34 oxidation by H 2 O 2 is provided by ONIOM(QM:MM) modeling including quantum water molecules. The reaction proceeds through a slightly asynchronous S N 2 transition state (TS) with calculated Δ ‡ G and Δ ‡ H barriers at 298 K of respectively 59 and 54 kJ mol −1 (the latter within chemical accuracy from the experimental value). A post-TS proton transfer leads to HSA–SO − and water as products. The structured reaction site cages H 2 O 2 , which donates a strong HB to the thiolate. Loss of this HB before reaching the TS modulates Cys34 nucleophilicity and contributes to destabilize H 2 O 2 . The lack of reaction-site features required for differential stabilization of the TS (positive charges, H 2 O 2 HB strengthening) explains the striking difference in kinetic efficiency for the same reaction in other proteins (e.g. peroxiredoxins). The structured HB network surrounding HSA–SH with sequestered waters carries an entropic penalty on the barrier height. These studies contribute to deepen the understanding of the reactivity of HSA–SH, the most abundant thiol in human plasma, and in a wider perspective, provide clues on the key aspects that modulate thiol reactivity against H 2 O 2 . [ABSTRACT FROM AUTHOR]

The peroxidative activity of horseradish peroxidase (HRP) undergoes progressive inactivation while catalyzing the oxidation of nitrite by H 2 O 2 . The extent of inactivation increases as the pH increases, [nitrite] decreases or [H 2 O 2 ] increases, and is accompanied by a loss of the Soret peak of HRP along with yellow-greenish coloration of the solution. HRP-catalyzed nitrite oxidation by H 2 O 2 involves not only the formation of compounds I and II as transient heme species, but also compound III, all of which in turn, oxidize nitrite yielding • NO 2 . The rate constant of nitrite oxidation by compound III is at least 10-fold higher than that by compound II, which is also reducible by • NO 2 where its reduction by nitrite is the rate-determining step of the catalytic cycle. The extent of the loss of the Soret peak of HRP is lower than the loss of its peroxidative activity implying that deterioration of the heme moiety leading to iron release only partially contributes toward heme inactivation. Cyclic stable nitroxide radicals, such as 2,2,6,6-tetramethyl-piperidine-N-oxyl (TPO), 4-OH-TPO and 4-NH 2 -TPO at µM concentrations detoxify • NO 2 thus protecting HRP against inactivation mediated by this radical. Hence, HRP inactivation proceeds via nitration of the porphyrin ring most probably through compound I reaction with • NO 2 , which partially leads to deterioration of the heme moiety. The nitroxide acts catalytically since its oxidation by • NO 2 yields the respective oxoammonium cation, which is readily reduced back to the nitroxide by H 2 O 2 , superoxide ion radical, and nitrite. In addition, the nitroxide catalytically inhibits tyrosine nitration mediated by HRP/H 2 O 2 /nitrite reactions system as it efficiently competes with tyrosyl radical for • NO 2 . The inhibition by nitroxides of tyrosine nitration is demonstrated also in the case of microperoxidase (MP-11) and cytochrome c revealing an additional role played by nitroxide antioxidants. [ABSTRACT FROM AUTHOR]

For a long time iodine has been used as an active dermal agent in the treatment of inflammatory, immune-mediated and infectious diseases. Moreover, topical iodine application has been reported to provide protection against sulfur-mustard-induced skin lesions, heat-induced and acid-induced skin burns in both haired guinea-pigs and mouse ear swelling models. However, the exact mechanism of action underlying these benefits of iodine has not yet been elucidated. In the current study, a novel mechanism of action by which iodine provides skin protection and relief, based on its electrophilic nature, is suggested. This study demonstrates that both iodine and iodide are capable of activating the Nrf2 pathway in human skin. As a result, skin protection against UVB-induced damage was acquired and the secretion of pro-inflammatory cytokines (IL-6, IL-8) from LPS-challenged skin was reduced. Iodide role in the enhanced activation of this pathway is demonstrated. The mode of action by which iodine and iodide activate the Nrf2 pathway is discussed. [ABSTRACT FROM AUTHOR]

The specific and sensitive detection of peroxynitrite (ONOO − /ONOOH) in biological systems is a great challenge due to its high reactivity towards several biomolecules. Herein, we validated the advantages of using fluorescein-boronate (Fl-B) as a highly sensitive fluorescent probe for the direct detection of peroxynitrite under biologically-relevant conditions in two different cell models. The synthesis of Fl-B was achieved by a very simply two-step conversion synthetic route with high purity (>99%) and overall yield (∼42%). Reactivity analysis of Fl-B with relevant biological oxidants including hydrogen peroxide (H 2 O 2 ), hypochlorous acid (HOCl) and peroxynitrite were performed. The rate constant for the reaction of peroxynitrite with Fl-B was 1.7×10 6 M −1 s −1 , a million times faster than the rate constant measured for H 2 O 2 (k=1.7 M −1 s −1 ) and 2,700 faster than HOCl (6.2×10 2 M −1 s −1 ) at 37 °C and pH 7.4. The reaction of Fl-B with peroxynitrite was significant even in the presence of physiological concentrations of CO 2 , a well-known peroxynitrite reactant. Experimental and simulated kinetic analyses confirm that the main oxidation process of Fl-B takes place with peroxynitrite itself via a direct bimolecular reaction and not with peroxynitrite-derived radicals. Fl-B was successfully applied for the detection of endogenously-generated peroxynitrite by endothelial cells and in macrophage-phagocyted parasites. Moreover, the generated data allowed estimating the actual intracellular flux of peroxynitrite. For instance, ionomycin-stimulated endothelial cells generated peroxynitrite at a rate of ∼ 0.1 μM s −1 , while immunostimulated macrophages do so in the order of ∼1 μM s −1 inside T. cruzi -infected phagosomes. Fl-B revealed not to be toxic in concentrations up to 1 mM for 24 h. Cellular peroxynitrite detection was achieved by conventional laboratory fluorescence-based methods including flow cytometry and epi-fluorescence microscopy. Fl-B was shown to be more sensitive than the coumarin boronate due to a higher molar absorption coefficient and quantum yield. Overall, our results show that Fl-B is a kinetically selective and highly sensitive probe for the direct detection of cell-derived peroxynitrite. [ABSTRACT FROM AUTHOR]

S-nitrosothiols (SNO) perform many important functions in biological systems, but the mechanism by which they are generated in vivo remains a contentious issue. Nitric oxide (NO) reacts with thiols to form SNO only in the presence of a molecule that will accept an electron from either NO or the thiol. In this study, we present evidence that ferriheme accepts an electron from NO or glutathione (GSH) to generate S-nitrosoglutathione (GSNO) in vitro under anaerobic or hypoxic (2% O 2 ) conditions. Ferriheme formed charge transfer-stable complexes with NO to form ferriheme-NO (heme-Fe(II)-NO + ) and with GSH to form ferriheme-GS (heme-Fe(II)-GS • ) under anaerobic conditions. The reaction between GSH and the heme-Fe(II)-NO + complex or between NO and the heme-Fe(II)-GS • complex resulted in simultaneous reductive ferriheme nitrosylation (heme-Fe(II)NO) and the generation of GSNO. Thus, ferriheme is readily reduced to ferroheme in the presence of NO and GSH together, but not with either individually. The reaction between NO and the heme-Fe(II)-GS • complex to generate GSNO occurred more rapidly than NO was consumed by endothelial cells, but not red blood cells. In addition, pretreatment of endothelial cells with ferriheme or the ferriheme-GS complex generated SNO upon addition of NO under hypoxic conditions. The results of this study raise the possibility that in vivo, ferriheme can complex with GSH to form ferriheme-GS complex (heme-Fe(II)-GS • ), which rapidly reacts with NO to generate GSNO under intracellular oxygen levels. The GSNO formation by this mechanism is more efficient than any other in vitro mechanism(s) reported so far. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis ( M. tuberculosis ) is the intracellular bacterium responsible for tuberculosis disease (TD). Inside the phagosomes of activated macrophages, M. tuberculosis is exposed to cytotoxic hydroperoxides such as hydrogen peroxide, fatty acid hydroperoxides and peroxynitrite. Thus, the characterization of the bacterial antioxidant systems could facilitate novel drug developments. In this work, we characterized the product of the gene Rv1608c from M. tuberculosis, which according to sequence homology had been annotated as a putative peroxiredoxin of the peroxiredoxin Q subfamily (PrxQ B from M. tuberculosis or Mt PrxQ B). The protein has been reported to be essential for M. tuberculosis growth in cholesterol-rich medium. We demonstrated the M. tuberculosis thioredoxin B/C-dependent peroxidase activity of Mt PrxQ B, which acted as a two-cysteine peroxiredoxin that could function, although less efficiently, using a one-cysteine mechanism. Through steady-state and competition kinetic analysis, we proved that the net forward rate constant of Mt PrxQ B reaction was 3 orders of magnitude faster for fatty acid hydroperoxides than for hydrogen peroxide (3×10 6 vs 6×10 3 M − 1 s − 1 , respectively), while the rate constant of peroxynitrite reduction was (0.6−1.4) ×10 6 M − 1 s − 1 at pH 7.4. The enzyme lacked activity towards cholesterol hydroperoxides solubilized in sodium deoxycholate. Both thioredoxin B and C rapidly reduced the oxidized form of Mt PrxQ B, with rates constants of 0.5×10 6 and 1×10 6 M − 1 s − 1 , respectively. Our data indicated that Mt PrxQ B is monomeric in solution both under reduced and oxidized states. In spite of the similar hydrodynamic behavior the reduced and oxidized forms of the protein showed important structural differences that were reflected in the protein circular dichroism spectra. [ABSTRACT FROM AUTHOR]

Stable nitroxide radicals have multiple biological effects, although the mechanisms underlying them are not fully understood. Their protective effect against oxidative damage has been mainly attributed to scavenging deleterious radicals, oxidizing reduced metal ions and reducing oxyferryl centers of heme proteins. Yet, the potential of nitroxides to protect heme proteins against inactivation while suppressing or enhancing their catalytic activities has been largely overlooked. We have studied the effect of nitroxides, including TPO (2,2,6,6-tetramethylpiperidin-N-oxyl), 4-OH-TPO, 4-oxo-TPO and 3-carbamoyl proxyl, on the peroxidase-like activity of metmyoglobin (MbFe III ) and methemoglobin (HbFe III ) using nitrite as an electron donor by following heme absorption, H 2 O 2 consumption, O 2 evolution and nitrite oxidation. The results demonstrate that the peroxidase-like activity is accompanied by a progressive heme inactivation where MbFe III is far more resistant than HbFe III . Nitroxides convert the peroxidase-like activity into catalase-like activity while inhibiting heme inactivation and nitrite oxidation in a dose-dependent manner. The nitroxide facilitates H 2 O 2 dismutation, yet none of its reactions with any of the intermediates formed in these systems is rate-determining, and therefore its effect on the rate of the catalysis is hardly dependent on the kind of the nitroxide derivative and its concentration. The nitroxide at µM concentrations range catalytically inhibits nitrite oxidation, and consequently prevents tyrosine nitration induced by heme protein/H 2 O 2 /nitrite due to its fast oxidation by • NO 2 forming the respective oxoammonium cation, which is reduced back to the nitroxide by H 2 O 2 and by superoxide radical. The nitroxides are superior over common antioxidants, which their reaction with • NO 2 always yields secondary radicals leading eventually to consumption of the antioxidant. A mechanism is proposed, and the kinetic simulations fit very well the experimental data in the case of MbFe III where most of the rate constants of the reactions involved are independently known. [ABSTRACT FROM AUTHOR]

Obesity-induced changes in lipid metabolism are mechanistically associated with the development of insulin resistance and prediabetes. Recent studies have focused on the extent to which obesity-induced insulin resistance is mediated through oxylipins, derived from enzymatic and nonenzymatic lipid peroxidation. Vitamin E and vitamin C are widely used antioxidant supplements, but conflicting data exist as to whether supplementation with vitamins E and C reduces insulin resistance. The purpose of this work is (1) to test the hypothesis that supplementation with vitamin E and vitamin C prevents the development of insulin resistance and (2) to determine the extent to which antioxidant supplementation modifies obesity-induced changes in hepatic oxylipins. Using obesity-prone Sprague–Dawley rats fed a high-fat, hypercaloric diet, we found that vitamin E and C supplementation did not block the development of insulin resistance, despite increased plasma levels of these antioxidants and decreased hepatic F 2 -isoprostane (F 2 -IsoP) concentrations. The obese phenotype was associated with increased hepatic concentrations of cytochrome P450 (CYP450)-dependent linoleic acid and α-linolenic acid-derived epoxides. Antioxidant supplementation, but not obesity, decreased levels of the lipoxygenase (LOX)-dependent, arachidonic acid-derived products lipoxin A4 (LXA 4 ), 8,15-dihydroxtetraenoate (8,15-DiHETE), and 5,15-DiHETE. Our data demonstrate that antioxidant supplementation and obesity impact hepatic LOX- and CYP450-dependent oxylipin metabolism. [ABSTRACT FROM AUTHOR]

Apigenin, a natural flavone, is emerging as a promising compound for the treatment of several diseases. One of the hallmarks of apigenin is the generation of intracellular reactive oxygen species (ROS), as judged by the oxidation of reduced dichlorofluorescein derivatives seen in many cell types. This study aimed to reveal some mechanisms by which apigenin can be oxidized and how apigenin-derived radicals affect the oxidation of 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein (H 2 DCF), a probe usually employed to detect intracellular ROS. Apigenin induced a rapid oxidation of H 2 DCF in two different immortalized cell lines derived from rat and human hepatic stellate cells. However, apigenin did not generate ROS in these cells, as judged by dihydroethidium oxidation and extracellular hydrogen peroxide production. In cell-free experiments we found that oxidation of apigenin leads to the generation of a phenoxyl radical, which directly oxidizes H 2 DCF with catalytic amounts of hydrogen peroxide. The net balance of the reaction was the oxidation of the probe by molecular oxygen due to redox cycling of apigenin. This flavonoid was also able to deplete NADH and glutathione by a similar mechanism. Interestingly, H 2 DCF oxidation was significantly accelerated by apigenin in the presence of horseradish peroxidase and xanthine oxidase, but not with other enzymes showing peroxidase-like activity, such as cytochrome c or catalase. We conclude that in cells treated with apigenin oxidation of reduced dichlorofluorescein derivatives does not measure intracellular ROS and that pro- and antioxidant effects of flavonoids deduced from these experiments are inconclusive and must be confirmed by other techniques. [ABSTRACT FROM AUTHOR]

Because 5-year survival rates for patients with metastatic melanoma remain below 25%, there is continued need for new therapeutic approaches. For some tumors, pharmacologic ascorbate treatment may have a beneficial antitumor effect and may work synergistically with standard chemotherapeutics. To investigate this possibility in melanoma, we examined the effect of pharmacologic ascorbate on B16-F10 cells. Murine models were employed to compare tumor size following treatment with ascorbate, and the chemotherapeutic agents dacarbazine or valproic acid, alone or in combination with ascorbate. Results indicated that nearly all melanoma cell lines were susceptible to ascorbate-mediated cytotoxicity. Compared to saline controls, pharmacologic ascorbate decreased tumor size in both C57BL/6 ( P <0.0001) and NOD- scid tumor bearing mice ( P <0.0001). Pharmacologic ascorbate was superior or equivalent to dacarbazine as an antitumor agent. Synergy was not apparent when ascorbate was combined with either dacarbazine or valproic acid; the latter combination may have additional toxicities. Pharmacologic ascorbate induced DNA damage in melanoma cells, as evidenced by increased phosphorylation of the histone variant, H2A.X. Differences were not evident in tumor samples from C57BL/6 mice treated with pharmacologic ascorbate compared to tumors from saline-treated controls. Together, these results suggest that pharmacologic ascorbate has a cytotoxic effect against melanoma that is largely independent of lymphocytic immune functions and that continued investigation of pharmacologic ascorbate in cancer treatment is warranted. [ABSTRACT FROM AUTHOR]

Pharmacological ascorbate has been shown to induce toxicity in a wide range of cancer cell lines. Pharmacological ascorbate in animal models has shown promise for use in cancer treatment. At pharmacological concentrations the oxidation of ascorbate produces a high flux of H 2 O 2 via the formation of ascorbate radical (Asc •- ). The rate of oxidation of ascorbate is principally a function of the level of catalytically active metals. Iron in cell culture media contributes significantly to the rate of H 2 O 2 generation. We hypothesized that increasing intracellular iron would enhance ascorbate-induced cytotoxicity and that iron chelators could modulate the catalytic efficiency with respect to ascorbate oxidation. Treatment of cells with the iron-chelators deferoxamine (DFO) or dipyridyl (DPD) in the presence of 2 mM ascorbate decreased the flux of H 2 O 2 generated by pharmacological ascorbate and reversed ascorbate-induced toxicity. Conversely, increasing the level of intracellular iron by preincubating cells with Fe-hydroxyquinoline (HQ) increased ascorbate toxicity and decreased clonogenic survival. These findings indicate that redox metal metals, e.g., Fe 3+ /Fe 2+ , have an important role in ascorbate-induced cytotoxicity. Approaches that increase catalytic iron could potentially enhance the cytotoxicity of pharmacological ascorbate in vivo . [ABSTRACT FROM AUTHOR]

The Rac1 GTPase is an essential and ubiquitous protein that signals through numerous pathways to control critical cellular processes, including cell growth, morphology, and motility. Rac1 deletion is embryonic lethal, and its dysregulation or mutation can promote cancer, arthritis, cardiovascular disease, and neurological disorders. Rac1 activity is highly regulated by modulatory proteins and posttranslational modifications. Whereas much attention has been devoted to guanine nucleotide exchange factors that act on Rac1 to promote GTP loading and Rac1 activation, cellular oxidants may also regulate Rac1 activation by promoting guanine nucleotide exchange. Herein, we show that Rac1 contains a redox-sensitive cysteine (Cys 18 ) that can be selectively oxidized at physiological pH because of its lowered p K a . Consistent with these observations, we show that Rac1 is glutathiolated in primary chondrocytes. Oxidation of Cys 18 by glutathione greatly perturbs Rac1 guanine nucleotide binding and promotes nucleotide exchange. As aspartate substitutions have been previously used to mimic cysteine oxidation, we characterized the biochemical properties of Rac1 C18D . We also evaluated Rac1 C18S as a redox-insensitive variant and found that it retains structural and biochemical properties similar to those of Rac1 WT but is resistant to thiol oxidation. In addition, Rac1 C18D , but not Rac1 C18S , shows greatly enhanced nucleotide exchange, similar to that observed for Rac1 oxidation by glutathione. We employed Rac1 C18D in cell-based studies to assess whether this fast-cycling variant, which mimics Rac1 oxidation by glutathione, affects Rac1 activity and function. Expression of Rac1 C18D in Swiss 3T3 cells showed greatly enhanced GTP-bound Rac1 relative to Rac1 WT and the redox-insensitive Rac1 C18S variant. Moreover, expression of Rac1 C18D in HEK-293T cells greatly promoted lamellipodia formation. Our results suggest that Rac1 oxidation at Cys 18 is a novel posttranslational modification that upregulates Rac1 activity. [ABSTRACT FROM AUTHOR]

The diterpenoid, adenanthin, represses tumor growth and prolongs survival in mouse promyelocytic leukemia models (Liu et al., Nat. Chem. Biol. 8, 486, 2012). It was proposed that this was done by inactivating peroxiredoxins (Prxs) 1 and 2 through the formation of an adduct specifically on the resolving Cys residue. We confirmed that adenanthin underwent Michael addition to isolated Prx2, thereby inhibiting oxidation to a disulfide-linked dimer. However, contrary to the original report, both the peroxidatic and the resolving Cys residues could be derivatized. Glutathione also formed an adenanthin adduct, reacting with a second-order rate constant of 25±5 M –1 s –1 . With 50 µM adenanthin, the peroxidatic and resolving Cys of Prx2 reacted with half-times of 7 and 40 min, respectively, compared with 10 min for GSH. When erythrocytes or Jurkat T cells were treated with adenanthin, we saw no evidence for a reaction with Prxs 1 or 2. Instead, adenanthin caused time- and concentration-dependent loss of GSH followed by dimerization of the Prxs. Prxs undergo continuous oxidation in cells and are normally recycled by thioredoxin reductase and thioredoxin. Our results indicate that Prx reduction was inhibited. We observed rapid inhibition of purified thioredoxin reductase (half-time 5 min with 2 µM adenanthin) and in cells, thioredoxin reductase was much more sensitive than GSH and loss of both preceded accumulation of oxidized Prxs. Thus, adenanthin is not a specific Prx inhibitor, and its reported antitumor and anti-inflammatory effects are more likely to involve more general inhibition of thioredoxin and/or glutathione redox pathways. [ABSTRACT FROM AUTHOR]

In this study, we examined the mechanisms and kinetic profiles of intracellular nitrosative processes using diaminofluorescein (DAF-2) as a target in RAW 264.7 cells. The intracellular formation of the fluorescent, nitrosated product diaminofluorescein triazol (DAFT) from both endogenous and exogenous nitric oxide (NO ) was prevented by deoxygenation and by cell membrane-permeable superoxide (O 2 − ) scavengers but not by extracellular bovine Cu,Zn-SOD. In addition, the DAFT formation rate decreased in the presence of cell membrane-permeable Mn porphyrins that are known to scavenge peroxynitrite (ONOO − ) but was enhanced by HCO 3 − /CO 2 . Together, these results indicate that nitrosative processes in RAW 264.7 cells depend on endogenous intracellular O 2 − and are stimulated by ONOO − /CO 2 -derived radical oxidants. The N 2 O 3 scavenger sodium azide (NaN 3 ) only partially attenuated the DAFT formation rate and only with high NO (>120 nM), suggesting that DAFT formation occurs by nitrosation (azide-susceptible DAFT formation) and predominantly by oxidative nitrosylation (azide-resistant DAFT formation). Interestingly, the DAFT formation rate increased linearly with NO concentrations of up to 120–140 nM but thereafter underwent a sharp transition and became insensitive to NO . This behavior indicates the sudden exhaustion of an endogenous cell substrate that reacts rapidly with NO and induces nitrosative processes, consistent with the involvement of intracellular O 2 − . On the other hand, intracellular DAFT formation stimulated by a fixed flux of xanthine oxidase-derived extracellular O 2 − that also occurs by nitrosation and oxidative nitrosylation increased, peaked, and then decreased with increasing NO , as previously observed. Thus, our findings complementarily show that intra- and extracellular O 2 − -dependent nitrosative processes occurring by the same chemical mechanisms do not necessarily depend on NO concentration and exhibit different unusual kinetic profiles with NO dynamics, depending on the biological compartment in which NO and O 2 − interact. [ABSTRACT FROM AUTHOR]

Although different routes for the S-nitrosation of cysteinyl residues have been proposed, the main in vivo pathway is unknown. We recently demonstrated that direct (as opposed to autoxidation-mediated) aerobic nitrosation of glutathione is surprisingly efficient, especially in the presence of Mg 2+ . In the present study we investigated this reaction in greater detail. From the rates of NO decay and the yields of nitrosoglutathione (GSNO) we estimated values for the apparent rate constants of 8.9±0.4 and 0.55±0.06 M −1 s −1 in the presence and absence of Mg 2+ . The maximum yield of GSNO was close to 100% in the presence of Mg 2+ but only about half as high in its absence. From this observation we conclude that, in the absence of Mg 2+ , nitrosation starts by formation of a complex between NO and O 2 , which then reacts with the thiol. Omission of superoxide dismutase (SOD) reduced by half the GSNO yield in the absence of Mg 2+ , demonstrating O 2 − formation. The reaction in the presence of Mg 2+ seems to involve formation of a Mg 2+ •glutathione (GSH) complex. SOD did not affect Mg 2+ -stimulated nitrosation, suggesting that no O 2 − is formed in that reaction. Replacing GSH with other thiols revealed that reaction rates increased with the p K a of the thiol, suggesting that the nucleophilicity of the thiol is crucial for the reaction, but that the thiol need not be deprotonated. We propose that in cells Mg 2+ -stimulated NO/O 2 -induced nitrosothiol formation may be a physiologically relevant reaction. [ABSTRACT FROM AUTHOR]

Abstract: Nitrosothiols are increasingly regarded as important participants in a range of physiological processes, yet little is known about their biological generation. Nitrosothiols can be formed from the corresponding thiols by nitric oxide in a reaction that requires the presence of oxygen and is mediated by reactive intermediates (NO2 or N2O3) formed in the course of NO autoxidation. Because the autoxidation of NO is second order in NO, it is extremely slow at submicromolar NO concentrations, casting doubt on its physiological relevance. In this paper we present evidence that at submicromolar NO concentrations the aerobic nitrosation of glutathione does not involve NO autoxidation but a reaction that is first order in NO. We show that this reaction produces nitrosoglutathione efficiently in a reaction that is strongly stimulated by physiological concentrations of Mg2+. These observations suggest that direct aerobic nitrosation may represent a physiologically relevant pathway of nitrosothiol formation. [Copyright &y& Elsevier]

Abstract: Hydrogen sulfide (H2S) is a ubiquitous gaseous signaling molecule that plays a vital role in numerous cellular functions and has become the focus of many research endeavors, including pharmacotherapeutic manipulation. Among the challenges facing the field is the accurate measurement of biologically active H2S. We have recently reported that the typically used methylene blue method and its associated results are invalid and do not measure bona fide H2S. The complexity of analytical H2S measurement reflects the fact that hydrogen sulfide is a volatile gas and exists in the body in various forms, including a free form, an acid-labile pool, and bound as sulfane sulfur. Here we describe a new protocol to discretely measure specific H2S pools using the monobromobimane method coupled with RP-HPLC. This new protocol involves selective liberation, trapping, and derivatization of H2S. Acid-labile H2S is released by incubating the sample in an acidic solution (pH 2.6) of 100mM phosphate buffer with 0.1mM diethylenetriaminepentaacetic acid (DTPA), in an enclosed system to contain volatilized H2S. Volatilized H2S is then trapped in 100mM Tris–HCl (pH 9.5, 0.1mM DTPA) and then reacted with excess monobromobimane. In a separate aliquot, the contribution of the bound sulfane sulfur pool was measured by incubating the sample with 1mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride), a reducing agent, to reduce disulfide bonds, in 100mM phosphate buffer (pH 2.6, 0.1mM DTPA), and H2S measurement was performed in a manner analogous to the one described above. The acid-labile pool was determined by subtracting the free hydrogen sulfide value from the value obtained by the acid-liberation protocol. The bound sulfane sulfur pool was determined by subtracting the H2S measurement from the acid-liberation protocol alone compared to that of TCEP plus acidic conditions. In summary, our new method allows very sensitive and accurate measurement of the three primary biological pools of H2S, including free, acid-labile, and bound sulfane sulfur, in various biological specimens. [Copyright &y& Elsevier]

Abstract: Inflammatory diseases are associated with the accumulation of activated inflammatory cells, particularly polymorphonuclear neutrophils (PMNs), which release reactive oxygen species (ROS) to eradicate foreign bodies and microorganisms. To assess the location and extent of localized inflammatory responses, L-012, a highly sensitive chemiluminescent probe, was employed to noninvasively monitor the production of ROS. We found that L-012-associated chemiluminescence imaging can be used to identify and to quantify the extent of inflammatory responses. Furthermore, regardless of differences among animal models, there is a good linear relationship between chemiluminescence intensity and PMN numbers surrounding inflamed tissue. Depletion of PMNs substantially diminished L-012-associated chemiluminescence in vivo. Finally, L-012-associated chemiluminescence imaging was found to be a powerful tool for assessing implant-mediated inflammatory responses by measuring chemiluminescence intensity at the implantation sites. These results support the use of L-012 for monitoring the kinetics of inflammatory responses in vivo via the detection and quantification of ROS production. [Copyright &y& Elsevier]

Abstract: It is well established that nitric oxide (•NO) reacts with cellular iron and thiols to form dinitrosyliron complexes (DNIC). Little is known, however, regarding their formation and biological fate. Our quantitative measurements reveal that cellular concentrations of DNIC are proportionally the largest of all •NO-derived adducts (900pmol/mg protein, or 45–90μM). Using murine macrophages (RAW 264.7), we measured the amounts, and kinetics, of DNIC assembly and disappearance from endogenous and exogenous sources of •NO in relation to iron and O2 concentration. Amounts of DNIC were equal to or greater than measured amounts of chelatable iron and depended on the dose and duration of •NO exposure. DNIC formation paralleled the upregulation of iNOS and occurred at low physiologic •NO concentrations (50–500nM). Decreasing the O2 concentration reduced the rate of enzymatic •NO synthesis without affecting the amount of DNIC formed. Temporal measurements revealed that DNIC disappeared in an oxygen-independent manner (t 1/2 =80min) and remained detectable long after the •NO source was removed (>24h). These results demonstrate that DNIC will be formed under all cellular settings of •NO production and that the contribution of DNIC to the multitude of observed effects of •NO must always be considered. [Copyright &y& Elsevier]

Abstract: Alkyl hydroperoxide reductase E (AhpE), a novel subgroup of the peroxiredoxin family, comprises Mycobacterium tuberculosis AhpE (MtAhpE) and AhpE-like proteins present in many bacteria and archaea, for which functional characterization is scarce. We previously reported that MtAhpE reacted ~103 times faster with peroxynitrite than with hydrogen peroxide, but the molecular reasons for that remained unknown. Herein, we investigated the oxidizing substrate specificity and the oxidative inactivation of the enzyme. In most cases, both peroxidatic thiol oxidation and sulfenic acid overoxidation followed a trend in which those peroxides with the lower leaving-group pK a reacted faster than others. These data are in agreement with the accepted mechanisms of thiol oxidation and support that overoxidation occurs through sulfenate anion reaction with the protonated peroxide. However, MtAhpE oxidation and overoxidation by fatty acid-derived hydroperoxides (~108 and 105 M−1 s−1, respectively, at pH 7.4 and 25°C) were much faster than expected according to the Brønsted relationship with leaving-group pK a. A stoichiometric reduction of the arachidonic acid hydroperoxide 15-HpETE to its corresponding alcohol was confirmed. Interactions of fatty acid hydroperoxides with a hydrophobic groove present on the reduced MtAhpE surface could be the basis of their surprisingly fast reactivity. [Copyright &y& Elsevier]

Abstract: Proteins containing reactive cysteine residues (protein-Cys) are receiving increased attention as mediators of hydrogen peroxide signaling. These proteins are mainly identified by mining the thiol proteomes of oxidized protein-Cys in cells and tissues. However, it is difficult to determine if oxidation occurs through a direct reaction with hydrogen peroxide or by thiol–disulfide exchange reactions. Kinetic studies with purified proteins provide invaluable information about the reactivity of protein-Cys residues with hydrogen peroxide. Previously, we showed that the characteristic UV–Vis spectrum of horseradish peroxidase compound I, produced from the oxidation of horseradish peroxidase by hydrogen peroxide, is a simple, reliable, and useful tool to determine the second-order rate constant of the reaction of reactive protein-Cys with hydrogen peroxide and peroxynitrite. Here, the method is fully described and extended to quantify reactive protein-Cys residues and micromolar concentrations of hydrogen peroxide. Members of the peroxiredoxin family were selected for the demonstration and validation of this methodology. In particular, we determined the pK a of the peroxidatic thiol of rPrx6 (5.2) and the second-order rate constant of its reactions with hydrogen peroxide ((3.4±0.2)×107 M−1 s−1) and peroxynitrite ((3.7±0.4)×105 M−1 s−1) at pH 7.4 and 25°C. [Copyright &y& Elsevier]

Abstract: Unlike intermolecular disulfide bonds, other protein cross-links arising from oxidative modifications cannot be reversed and are presumably more toxic to cells because they may accumulate and induce protein aggregation. However, most of these irreversible protein cross-links remain poorly characterized. For instance, the antioxidant enzyme human superoxide dismutase 1 (hSod1) has been reported to undergo non-disulfide covalent dimerization and further oligomerization during its bicarbonate-dependent peroxidase activity. The dimerization was shown to be dependent on the oxidation of the single, solvent-exposed Trp32 residue of hSod1, but the covalent dimer was not isolated nor was its structure determined. In this work, the hSod1 covalent dimer was isolated, digested with trypsin in H2O and H2 18O, and analyzed by UV–Vis spectroscopy and mass spectrometry (MS). The results demonstrate that the covalent dimer consists of two hSod1 subunits cross-linked by a ditryptophan, which contains a bond between C3 and N1 of the respective Trp32 residues. We further demonstrate that the cross-link cleaves under usual MS/MS conditions leading to apparently unmodified Trp32, partially hinders proteolysis, and provides a mechanism to explain the formation of hSod1 covalent trimers and tetramers. This characterization of the covalent hSod1 dimer identifies a novel oxidative modification of protein Trp residues and provides clues for studying its occurrence in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Peroxynitrite is a reactive oxidant produced in vivo in response to oxidative and other stress by the diffusion-limited reaction of nitric oxide and superoxide. This article is focused on the identification of free radical intermediates of uric acid formed during its reaction with peroxynitrite. The experimental approach included the ESR spin trapping of the radical generated from the reaction between uric acid and peroxynitrite at pH 7.4 and mass spectrometry studies of the trapped radicals. Using PBN (N-tert-butyl-α-phenylnitrone) as the spin trapping agent, a six-line ESR spectrum was obtained and its hyperfine coupling constants, a N =15.6G and a H =4.4G, revealed the presence of carbon-based radicals. Further structural identification of the PBN–radical adducts was carried out using liquid chromatography–mass spectrometry. After comparison with the control reactions, two species were identified that correspond to the protonated molecules (M+1) at m/z 352 and 223, respectively. The ions of m/z 352 were characterized as the PBN–triuretcarbonyl radical adduct and the m/z 223 ion was identified as the PBN–aminocarbonyl radical adduct. Their mechanism of formation is discussed. [Copyright &y& Elsevier]

Abstract: The reactions of nitrite with deoxygenated human erythrocytes were examined using membrane inlet mass spectrometry to detect the accumulation of NO in an extracellular solution. In this method an inlet utilizing a silicon rubber membrane is submerged in cell suspensions and allows NO to pass from the extracellular solution into the mass spectrometer. This provides a direct, continuous, and quantitative determination of nitric oxide concentrations over long periods without the necessity of purging the suspension with inert gas. We have not observed accumulation of NO compared with controls on a physiologically relevant time scale and conclude that, within the limitations of the mass spectrometric method and our experimental conditions, erythrocytes do not generate a net efflux of NO after the addition of millimolar concentrations of nitrite. Moreover, there was no evidence at the mass spectrometer of the accumulation of a peak at mass 76 that would indicate N2O3, an intermediate that decays into NO and NO2. Inhibition of red cell membrane anion exchangers and aquaporins did not affect these processes. [Copyright &y& Elsevier]

Abstract: The reaction between NO, thiols, and oxygen has been studied in some detail in vitro due to its perceived importance in the mechanism of NO-dependent signal transduction. The formation of S-nitrosothiols and thiol disulfides from this chemistry has been suggested to be an important component of the biological chemistry of NO, and such subsequent thiol modifications may result in changes in cellular function and phenotype. In this study we have reinvestigated this reaction using both experiment and simulation and conclude that: (i) S-nitrosation through radical and nonradical pathways is occurring simultaneously, (ii) S-nitrosation through direct addition of NO to thiol does not occur to any meaningful extent, and (iii) protein hydrophobic environments do not catalyze or enhance S-nitrosation of either themselves or of glutathione. We conclude that S-nitrosation and disulfide formation in this system occur only after the initial reaction between NO and oxygen to form nitrogen dioxide, and that hydrophobic protein environments are unlikely to play any role in enhancing and targeting S-nitrosothiol formation. [Copyright &y& Elsevier]

Abstract: In this study, we show that boronates, a class of synthetic organic compounds, react rapidly and stoichiometrically with peroxynitrite (ONOO−) to form stable hydroxy derivatives as major products. Using a stopped-flow kinetic technique, we measured the second-order rate constants for the reaction with ONOO−, hypochlorous acid (HOCl), and hydrogen peroxide (H2O2) and found that ONOO− reacts with 4-acetylphenylboronic acid nearly a million times (k =1.6×106 M−1 s−1) faster than does H2O2 (k =2.2 M−1 s−1) and over 200 times faster than does HOCl (k =6.2×103 M−1 s−1). Nitric oxide and superoxide together, but not alone, oxidized boronates to the same phenolic products. Similar reaction profiles were obtained with other boronates. Results from this study may be helpful in developing a novel class of fluorescent probes for the detection and imaging of ONOO− formed in cellular and cell-free systems. [Copyright &y& Elsevier]

Carbonate radicals (CO3˙−) can be formed biologically by the reaction of OH with bicarbonate, the decomposition of the peroxynitrite–carbon dioxide adduct (ONOOCO2˙−), and enzymatic activities, i.e., peroxidase activity of CuZnSOD and xanthine oxidase turnover in the presence of bicarbonate. It has been reported that the spin-trap DMPO reacts with CO3˙− to yield transient species to yield finally the DMPO–OH spin adduct. In this study, the kinetics of reaction of CO3˙− with DMPO were studied by pulse radiolysis, yielding a second-order rate constant of 2.5 × 106 M− 1 s− 1. A Fenton system, composed of FeII–DTPA plus H2O2, generated OH that was trapped by DMPO; the presence of 50–500 mM bicarbonate, expected to convert ˙OH to CO3˙−, markedly inhibited DMPO–OH formation. This was demonstrated to be due mainly to a fast reaction of CO3˙− with FeII–DTPA (k = 6.1×108M−1 s−1), supported by kinetic analysis. Generation of CO3− by the Fenton system was further proved by analysis of tyrosine oxidation products: the presence of bicarbonate caused a dose-dependent inhibition of 3,4-dihydroxiphenylalanine with a concomitant increase of 3,3′-dityrosine yields, and the presence of DMPO inhibited tyrosine oxidation, in agreement with the rate constants with OH or CO3˙−. Similarly, the formation of CO3˙− by CuZnSOD/H2O2/bicarbonate and peroxynitrite–carbon dioxide was supported by DMPO hydroxylation and kinetic competition data. Finally, the reaction of CO3˙− with DMPO to yield DMPO–OH was shown in peroxynitrite-forming macrophages. In conclusion, CO3˙− reacts quite rapidly with DMPO and may contribute to DMPO–OH yields in chemical and cellular systems; in turn, the extent of oxidation of other target molecules (such as tyrosine) by CO3˙− will be sensitive to the presence of DMPO. [Copyright &y& Elsevier]

Abstract: H2O2 produced by stimulation of the macrophage NADPH oxidase is involved both in bacterial killing and as a second messenger in these cells. Protein tyrosine phosphatases (PTPs) are targets for H2O2 signaling through oxidation of their catalytic cysteine, resulting in inhibition of their activity. Here, we show that, in the rat alveolar macrophage NR8383 cell line, H2O2 produced through the ADP-stimulated respiratory burst induces the formation of a disulfide bond between PTP1B and GSH that was detectable with an antibody to glutathione–protein complexes and was reversed by DTT addition. PTP1B glutathionylation was dependent on H2O2 as the presence of catalase at the time of ADP stimulation inhibited the formation of the conjugate. Interestingly, other PTPs, i.e., SHP-1 and SHP-2, did not undergo glutathionylation in response to ADP stimulation of the respiratory burst, although glutathionylation of these proteins could be shown by reaction with 25 mM glutathione disulfide in vitro. While previous studies have suggested the reversible oxidation of PTP1B during signaling or showed PTP1B glutathionylation in vitro, the present study directly demonstrates that physiological stimulation of H2O2 production results in PTP1B glutathionylation in intact cells, which may affect downstream signaling. [Copyright &y& Elsevier]

Abstract: The observation that antioxidant vitamins fail to confer protective benefits in large, well-designed randomized clinical trials has led many to question the role of oxidative stress in the pathogenesis of disease. However, there is little evidence that proposed antioxidants actually scavenge reactive intermediates in vivo. Ascorbate reacts rapidly with oxidants produced by activated neutrophils in vitro, and neutrophils markedly increase their oxidant production when mice are infected intraperitoneally with the gram-negative bacterium Klebsiella pneumoniae. To explore the antioxidant properties of ascorbate in vivo, we therefore used K. pneumoniae infection as a model of oxidative stress. When mice deficient in l-gulono-γ-lactone oxidase (Gulo −/−), the rate-limiting enzyme in ascorbate synthesis, were depleted of ascorbate and infected with K. pneumoniae, they were three times as likely as ascorbate-replete Gulo −/−mice to die from infection. Mass spectrometric analysis of peritoneal lavage fluid revealed a marked increase in the levels of oxidized amino acids and of F2-isoprostanes (sensitive and specific markers of lipid oxidation) in infected animals. Surprisingly, there were no significant differences in the levels of the oxidation products in the ascorbate-deficient and -replete Gulo −/−mice. Our observations suggest that ascorbate plays a previously unappreciated role in host defense mechanisms against invading pathogens but that the vitamin does not protect amino acids and lipids from oxidative damage during acute inflammation. To examine the oxidation hypothesis of disease, optimal antioxidant regimens that block oxidative reactions in animals and humans need to be identified. [Copyright &y& Elsevier]

Ascorbic acid (AA) is thought to be an important antioxidant in the respiratory tract, whose regulation is yet to be fully characterized. We investigated whether AA in respiratory tract lining fluids (RTLFs) can be augmented by oral supplementation with AA. Plasma, nasal lavage fluids (NLFs), induced sputum (IS), and saliva were analyzed for AA immediately before and 2 h after ingestion of 2 g of AA in 13 healthy subjects. Concentrations of AA (median and range) were 52.5 (16.0–88.5), 2.4 (0.18–4.66), 2.4 (0.18–6.00), and 0.55 (0.18–18.90) μmol/l, respectively. Two hours after ingestion of AA, plasma AA increased 2-fold (p = .004), NLF AA increased 3-fold (p = .039), but IS and saliva AA did not increase. As AA concentrations in saliva and tracheobronchial secretions were low compared with other common extracellular components (such as urate), we evaluated the fate of AA in these fluids. Addition of AA to freshly obtained saliva or IS resulted in rapid depletion, which could be largely prevented or reversed by sodium azide or dithiothreitol. These findings suggest that oxidant-producing systems in saliva and airway secretions, such as heme peroxidases and other oxidizing substances, rapidly consume AA. Whereas oral supplementation resulted in detectable increases of AA in NLFs, its levels in tracheobronchial lining fluid, as measured by IS, were unaffected and remained relatively low, suggesting that AA may play a less significant antioxidant role in this compartment as compared with most other extracellular compartments. [Copyright &y& Elsevier]

The reaction of NO and NO2− with hemoglobin (Hb) is of pivotal importance to blood vessel function. Both species show at least two different reactions with Fe2+Hb: one with deoxygenated Hb, in which the biological properties of NO are preserved, and another with oxygenated hemoglobin (oxyHb), in which both species are oxidizes to NO3−. In this study we compared the oxidative reactions of NO and NO2− and, in particular, the radical intermediates formed during transformation to NO3−. The reaction of NO2− with oxyHb was accelerated at high heme concentrations and produced stoichiometric amounts of NO3−. Direct EPR and spin trapping studies showed that NO2−, but not NO, induced the formation of globin Tyr-, Trp-, and Cys-centered radicals. MS studies provided evidence of the formation of ∼2% nitrotyrosine in both the α and β subunits, suggesting that NO2 diffuses in part away from the heme and reacts with Tyr radicals. No nitrotyrosines were detected in the reaction of NO with oxyHb. Collectively, these results indicate that NO2− reaction with oxyHb causes an oxidative challenge not observed with NO. The differences in oxidation mechanisms of NO and NO2− are discussed. [Copyright &y& Elsevier]