NO Autoxidation Research Articles

GC-MS Studies on Nitric Oxide Autoxidation and S-Nitrosothiol Hydrolysis to Nitrite in pH-Neutral Aqueous Buffers: Definite Results Using 15N and 18O Isotopes.

Nitrite (O=N-O-, NO2-) and nitrate (O=N(O)-O-, NO3-) are ubiquitous in nature. In aerated aqueous solutions, nitrite is considered the major autoxidation product of nitric oxide (●NO). ●NO is an environmental gas but is also endogenously produced from the amino acid L-arginine by the catalytic action of ●NO synthases. It is considered that the autoxidation of ●NO in aqueous solutions and in O2-containing gas phase proceeds via different neutral (e.g., O=N-O-N=O) and radical (e.g., ONOO●) intermediates. In aqueous buffers, endogenous S-nitrosothiols (thionitrites, RSNO) from thiols (RSH) such as L-cysteine (i.e., S-nitroso-L-cysteine, CysSNO) and cysteine-containing peptides such as glutathione (GSH) (i.e., S-nitrosoglutathione, GSNO) may be formed during the autoxidation of ●NO in the presence of thiols and dioxygen (e.g., GSH + O=N-O-N=O → GSNO + O=N-O- + H+; pKaHONO, 3.24). The reaction products of thionitrites in aerated aqueous solutions may be different from those of ●NO. This work describes in vitro GC-MS studies on the reactions of unlabeled (14NO2-) and labeled nitrite (15NO2-) and RSNO (RS15NO, RS15N18O) performed in pH-neutral aqueous buffers of phosphate or tris(hydroxyethylamine) prepared in unlabeled (H216O) or labeled H2O (H218O). Unlabeled and stable-isotope-labeled nitrite and nitrate species were measured by gas chromatography-mass spectrometry (GC-MS) after derivatization with pentafluorobenzyl bromide and negative-ion chemical ionization. The study provides strong indication for the formation of O=N-O-N=O as an intermediate of ●NO autoxidation in pH-neutral aqueous buffers. In high molar excess, HgCl2 accelerates and increases RSNO hydrolysis to nitrite, thereby incorporating 18O from H218O into the SNO group. In aqueous buffers prepared in H218O, synthetic peroxynitrite (ONOO-) decomposes to nitrite without 18O incorporation, indicating water-independent decomposition of peroxynitrite to nitrite. Use of RS15NO and H218O in combination with GC-MS allows generation of definite results and elucidation of reaction mechanisms of oxidation of ●NO and hydrolysis of RSNO.

Acceleration of the autoxidation of nitric oxide by proteins

Lipoproteins and lipid membranes accelerate •NO autoxidation by increasing local concentration of •NO and O2. Although the idea that proteins could also accelerate this reaction was presented some time ago, it was largely criticized and dismissed. Herein the effect of proteins on •NO autoxidation rates was studied following •NO disappearance with a selective electrode. It was found that human serum albumin (HSA) accelerated •NO autoxidation by a factor of 9 per g/mL of protein, much less than previously suggested. The acceleration by HSA was sensitive to pH and significantly decreased at pH lower than 4.5 coincident with the acid structure transition of HSA to a partially unfolded and rigid conformation. Other proteins with different surface hydrophobicity also accelerated •NO autoxidation and it was found to depend mostly on the protein size and dynamics. Mathematical simulations were performed to assess the physiological importance of this acceleration. It was calculated that in plasma the autoxidation of •NO is accelerated 1.38 times by HSA relative to water alone, but this becomes of little relevance when whole blood is simulated because of the rapid rate of •NO consumption by red blood cells.

Low-Temperature Trapping of Intermediates in the Reaction of NO• with O2.

The autoxidation of NO• was studied in glass-like matrices of 2-methylbutane at 110 K and in a 8:3 v/v mixture of 2,2-dimethylbutane and n-pentane (rigisolve) at 80-90 K, by letting gaseous NO• diffuse into these solvents that were saturated with O2. In 2-methyllbutane, we observed a red compound. However, in rigisolve at 85-90 K, a bright yellow color appears that turns red when the sample is warmed by 10-20 K. The new yellow compound is a precursor of the red one and also diamagnetic. The UV-vis spectrum of the yellow compound contains a band which resembles that present in ONOO-. Because the red and yellow intermediates are not paramagnetic, we postulate that O═N-O-O• is in close contact with NO•, or with another O═N-O-O•. Diffusion of gaseous O2 into rigisolve saturated with NO• does not produce a color; however, a weak EPR signal (g = 2.010) is observed. This signal most likely indicates the presence of ONOO•. These findings complement our earlier observation of a red color at low temperatures and the presence of ONOO• in the gas phase (Galliker, B.; Kissner, R.; Nauser, T.; Koppenol, W. H. Chem. Eur. J. 2009, 15, 6161-6168), and they indicate that the termolecular autoxidation of nitrogen monoxide proceeds via the intermediate ONOO• and not via N2O2.

Analytical Expression for the NO Concentration Profile Following NONOate Decomposition in the Presence of Oxygen.

We have derived an analytical expression for the time dependent NO concentration from NONOate donors in the presence of oxygen for the process of NO release from NO donors following autoxidation. This analytical solution incorporates the kinetics of the releases with the autoxidation and is used to fit the simulated NO concentration profile to the experimental data. This allows one to determine the NO release rate constant, k 1, the NO release stoichiometric coefficient, v NO, and the NO autoxidation reaction rate constant, k 2. This analytical solution also allows us to predict the real NO concentration released from NO donors under aerobic conditions, while v NO is reportedly two under aerobic conditions, it falls to lower values in the presence of oxygen.

A review and discussion of platelet nitric oxide and nitric oxide synthase: do blood platelets produce nitric oxide from L-arginine or nitrite?

The NO/sGC/cGMP/PKG system is one of the most powerful mechanisms responsible for platelet inhibition. In numerous publications, expression of functional NO synthase (NOS) in human and mouse platelets has been reported. Constitutive and inducible NOS isoforms convert L-arginine to NO and L-citrulline. The importance of this pathway in platelets and in endothelial cells for the regulation of platelet function is discussed since decades. However, there are serious doubts in the literature concerning both expression and functionality of NOS in platelets. In this review, we aim to present and critically evaluate recent data concerning NOS expression and function in platelets, and to especially emphasise potential pitfalls of detection of NOS proteins and measurement of NOS activity. Prevailing analytical problems are probably the main sources of contradictory data on occurrence, activity and function of NOS in platelets. In this review we also address issues of how these problems can be resolved. NO donors including organic nitrites (RONO) and organic nitrate (RONO2) are inhibitors of platelet activation. Endogenous inorganic nitrite (NO2 (-)), the product of NO autoxidation, and exogenous inorganic nitrite are increasingly investigated as NO donors in the circulation. The role of platelets in the generation of NO from nitrite is also discussed.

Comparison of the chemical reactivity of synthetic peroxynitrite with that of the autoxidation products of nitroxyl or its anion

Donors of nitroxyl (HNO) exhibit pharmacological properties that are potentially favorable for treatment of a variety of diseases. To fully evaluate the pharmacological utility of HNO, it is therefore important to understand its chemistry, particularly involvement in deleterious biological reactions. Of particular note is the cytotoxic species formed from HNO autoxidation that is capable of inducing double strand DNA breaks. The identity of this species remains elusive, but a conceivable product is peroxynitrous acid. However, chemical comparison studies have demonstrated that HNO autoxidation leads to a unique reactive nitrogen oxide species to that of synthetic peroxynitrite. Here, we extend the analysis to include a new preparation of peroxynitrite formed via autoxidation of nitroxyl anion (NO−). Both peroxynitrite preparations exhibited similar chemical profiles, although autoxidation of NO− provided a more reliable sample of peroxynitrite. Furthermore, the observed dissimilarities to the HNO donor Angeli's salt substantiate that HNO autoxidation produces a unique intermediate from peroxynitrite.

Suberoylanilide hydroxamic acid radiosensitizes tumor hypoxic cells in vitro through the oxidation of nitroxyl to nitric oxide

The pharmacological effects of hydroxamic acids are partially attributed to their ability to serve as HNO and/or NO donors under oxidative stress. Previously, it was concluded that oxidation of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) by the metmyoglobin/H2O2 reaction system releases NO, which was based on spin trapping of NO and accumulation of nitrite. Reinvestigation of this system demonstrates the accumulation of N2O, which is a marker of HNO formation, at similar rates under normoxia and anoxia. In addition, the yields of nitrite that accumulated in the absence and the presence of O2 did not differ, implying that the source of nitrite is other than autoxidation of NO. In this system metmyoglobin is instantaneously and continuously converted into compound II, leading to one-electron oxidation of SAHA to its respective transient nitroxide radical. Studies using pulse radiolysis show that one-electron oxidation of SAHA (pKa=9.56±0.04) yields the respective nitroxide radical (pKa=9.1±0.2), which under all experimental conditions decomposes bimolecularly to yield HNO. The proposed mechanism suggests that compound I oxidizes SAHA to the respective nitroxide radical, which decomposes bimolecularly in competition with its oxidation by compound II to form HNO. Compound II also oxidizes HNO to NO and NO to nitrite. Given that NO, but not HNO, is an efficient hypoxic cell radiosensitizer, we hypothesized that under an oxidizing environment SAHA might act as a NO donor and radiosensitize hypoxic cells. Preincubation of A549 and HT29 cells with 2.5μM SAHA for 24h resulted in a sensitizer enhancement ratio at 0.01 survival levels (SER0.01) of 1.33 and 1.59, respectively. Preincubation of A549 cells with oxidized SAHA had hardly any effect and, with 2mM valproic acid, which lacks the hydroxamate group, resulted in SER0.01=1.17. Preincubation of HT29 cells with SAHA and Tempol, which readily oxidizes HNO to NO, enhanced the radiosensitizing effect of SAHA. Pretreatment with SAHA blocked A549 cells at the G1 stage of the cell cycle and upregulated γ-H2AX after irradiation. Overall, we conclude that SAHA enhances tumor radioresponse by multiple mechanisms that might also involve its ability to serve as a NO donor under oxidizing environments.

Nitric Oxide Interaction with Oxy–Coboglobin Models Containing trans-Pyridine Ligand: Two Reaction Pathways

The oxy-cobolglobin models of the general formula (Py)Co(Por)(O2) (Por = meso-tetraphenyl- and meso-tetra-p-tolylporphyrinato dianions) were constructed by sequential low-temperature interaction of Py and dioxygen with microporous layers of Co-porphyrins. At cryogenic temperatures small increments of NO were introduced into the cryostat and the following reactions were monitored by the FTIR and UV-visible spectroscopy during slow warming. Similar to the recently studied (NH3)Co(Por)(O2) system (Kurtikyan et al. J. Am. Chem. Soc., 2012, 134, 13671-13680), this interaction leads to the nitric oxide dioxygenation reaction with the formation of thermally unstable nitrato complexes (Py)Co(Por)(η(1)-ONO2). The reaction proceeds through the formation of the six-coordinate peroxynitrite adducts (Py)Co(Por)(OONO), as was demonstrated by FTIR measurements with the use of isotopically labeled (18)O2, (15)NO, N(18)O, and (15)N(18)O species and DFT calculations. In contrast to the ammonia system, however, the binding of dioxygen in (Py)Co(Por)(O2) is weaker and the second reaction pathway takes place due to autoxidation of NO by rebound O2 that in NO excess gives N2O3 and N2O4 species adsorbed in the layer. This leads eventually to partial formation of (Py)Co(Por)(NO) and (Py)Co(Por)(NO2) as a result of NO and NO2 reactions with five-coordinate Co(Por)(Py) complexes that are present in the layer after the O2 has been released. The former is thermally unstable and at room temperature passes to the five-coordinate nitrosyl complex, while the latter is a stable compound. In these experiments at 210 K, the layer consists mostly of six-coordinate nitrato complexes and some minor quantities of six-coordinate nitro and nitrosyl species. Their relative quantities depend on the experimental conditions, and the yield of nitrato species is proportional to the relative quantity of peroxynitrite intermediate. Using differently labeled nitrogen oxide isotopomers in different stages of the process the formation of the caged radical pair after homolytic disruption of the O-O bond in peroxynitrite moiety is clearly shown. The composition of the layers upon farther warming to room temperature depends on the experimental conditions. In vacuo the six-coordinate nitrato complexes decompose to give nitrate anion and oxidized cationic complex Co(III)(Por)(Py)2. In the presence of NO excess, however, the nitro-pyridine complexes (Py)Co(Por)(NO2) are predominantly formed formally indicating the oxo-transfer reactivity of (Py)Co(Por)(η(1)-ONO2) with regard to NO. Using differently labeled nitrogen in nitric oxide and coordinated nitrate a plausible mechanism of this reaction is suggested based on the isotope distribution in the nitro complexes formed.

Efficient nitrosation of glutathione by nitric oxide

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.

Nitrogen dioxide oxidizes mitochondrial cytochrome c

We previously reported that high micromolar concentrations of nitric oxide were able to oxidize mitochondrial cytochrome c at physiological pH, producing nitroxyl anion (Sharpe and Cooper, 1998 Biochem. J. 332, 9–19). However, the subsequent re-evaluation of the redox potential of the NO/NO- couple suggests that this reaction is thermodynamically unfavored. We now show that the oxidation is oxygen-concentration dependent and non stoichiometric. We conclude that the effect is due to an oxidant species produced during the aerobic decay of nitric oxide to nitrite and nitrate. The species is most probably nitrogen dioxide, NO2• a well-known biologically active oxidant. A simple kinetic model of NO autoxidation is able to explain the extent of cytochrome c oxidation assuming a rate constant of 3×106M-1s-1 for the reaction of NO2• with ferrocytochrome c. The importance of NO2• was confirmed by the addition of scavengers such as urate and ferrocyanide. These convert NO2• into products (urate radical and ferricyanide) that rapidly oxidize cytochrome c and hence greatly enhance the extent of oxidation observed. The present study does not support the previous hypothesis that NO and cytochrome c can generate appreciable amounts of nitroxyl ions (NO- or HNO) or of peroxynitrite.

Chemical model systems for cellular nitros(yl)ation reactions

S-nitros(yl)ation belongs to the redox-based posttranslational modifications of proteins but the underlying chemistry is controversial. In contrast to current concepts involving the autoxidation of nitric oxide ( NO, nitrogen monoxide), we and others have proposed the formation of peroxynitrite (oxoperoxonitrate (1−)) as an essential intermediate. This requires low cellular fluxes of NO and superoxide ( O 2 −), for which model systems have been introduced. We here propose two new systems for nitros(yl)ation that avoid the shortcomings of previous models. Based on the thermal decomposition of 3-morpholinosydnonimine, equal fluxes of NO and O 2 − were generated and modulated by the addition of NO donors or Cu,Zn-superoxide dismutase. As reactants for S-nitros(yl)ation, NADP +-dependent isocitrate dehydrogenase and glutathione were employed, for which optimal S-nitros(yl)ation was observed at nanomolar fluxes of NO and O 2 − at a ratio of about 3:1. The previously used reactants phenol and diaminonaphthalene (C- and N-nitrosation) demonstrated potential participation of multiple pathways for nitros(yl)ation. According to our data, neither peroxynitrite nor autoxidation of NO was as efficient as the 3 NO/1 O 2 − system in mediating S-nitros(yl)ation. In theory this could lead to an elusive nitrosonium (nitrosyl cation)-like species in the first step and to N 2O 3 in the subsequent reaction. Which of these two species or whether both together will participate in biological S-nitros(yl)ation remains to be elucidated. Finally, we developed several hypothetical scenarios to which the described NO/ O 2 − flux model could apply, providing conditions that allow either direct electrophilic substitution at a thiolate or S-nitros(yl)ation via transnitrosation from S-nitrosoglutathione.

Membrane “Lens” Effect: Focusing the Formation of Reactive Nitrogen Oxides from the •NO/O2 Reaction

It was previously observed that lipid membranes accelerate *NO disappearance (Liu et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 2175), and here, we demonstrate that this translates into increased rates of *NO2 production and nitrosative chemistry. Not only the phospholipid membranes but also the atherosclerosis-related low-density lipoprotein (LDL) were able to accelerate the formation of *NO2, studied by stopped-flow spectrophotometry using ABTS as a probe. In addition, membranes, LDL, and Triton X-100 micelles significantly accelerated S-nitrosation of glutathione and captopril. It is shown here that autoxidation of *NO occurs 30 times more rapidly within the hydrophobic interior of these particles than in an equal volume of water, approximately 1 order of magnitude less than previous reports. This acceleration can be explained by the approximately 3 times higher solubility of *NO and O2 into these hydrophobic phases relative to water, which results in a higher local concentration of reactants ("lens effect") and, therefore, a higher rate of reaction. It is predicted that 50% of the oxidizing and nitrosating species derived from *NO autoxidation in cells will be formed in the small volume comprising cellular membranes (3% of the total); thus, biomolecules near the membranes will be exposed to fluxes of reactive nitrogen species 30-fold higher than their cytosolic counterparts.

Acceleration of nitric oxide autoxidation and nitrosation by membranes

The reaction between nitric oxide ((.)NO) and oxygen yields reactive species capable of oxidizing and nitrosating proteins, as well as deaminating DNA bases. Although this reaction is considered too slow to be biologically relevant, it has been shown that membranes, lipoproteins, mitochondria and possibly proteins can accelerate this reaction. This effect stems from the higher solubility of both (.)NO and O(2)in the hydrophobic phase of these biological particles, leading to a concentration of both reagents and so a higher rate of reaction. It has been determined that this reaction occurs from 30 to 300 times more rapidly within the membrane, while even higher values have been suggested for proteins. The autoxidation of (.)NO in membranes is not the main route for cellular (.)NO consumption but an important consequence of this phenomenon is to focus the generation of significant amounts of oxidizing and nitrosating molecules (nitrogen dioxide and dinitrogen trioxide) in the small volume comprised by cellular membranes. Even so, these reactive species are diffusible and their ultimate fate will depend on the reactivity towards available substrates rather than on physical barriers. The acceleration of (.)NO autoxidation by biological hydrophobic phases may thus be a general phenomenon that increases in importance in cases of (.)NO overproduction.

Mechanisms of ferriheme reduction by nitric oxide: nitrite and general base catalysis.

The reductive nitrosylation (Fe(III)(P) + 2NO + H(2)O = Fe(II)(P)(NO) + NO(2)(-) + 2H(+)) of the ferriheme model Fe(III)(TPPS) (TPPS = tetra(4-sulfonatophenyl)porphyrinato) has been investigated in moderately acidic solution. In the absence of added or adventitious nitrite, this reaction displays general base catalysis with several buffers in aqueous solutions. It was also found that the nitrite ion, NO(2)(-), is a catalyst for this reaction. Similar nitrite catalysis was demonstrated for another ferriheme model system Fe(III)(TMPy) (TMPy = meso-tetrakis(N-methyl-4-pyridyl)porphyrinato), and for ferriheme proteins met-hemoglobin (metHb) and met-myoglobin (metMb) in aqueous buffer solutions. Thus, it appears that such catalysis is a general mechanistic route to the reductive nitrosylation products. Two nitrite catalysis mechanisms are proposed. In the first, NO(2)(-) is visualized as operating via nucleophilic addition to the Fe(III)-coordinated NO in a manner similar to the reactions proposed for Fe(III) reduction promoted by other nucleophiles. This would give a labile N(2)O(3) ligand that hydrolyzes to nitrous acid, regenerating the original nitrite. The other proposal is that Fe(III) reduction is effected by direct outer-sphere electron transfer from NO(2)(-) to Fe(III)(P)(NO) to give nitrogen dioxide plus the ferrous nitrosyl complex Fe(II)(P)(NO). The NO(2) thus generated would be trapped by excess NO to give N(2)O(3) and, subsequently, nitrite. It is found that the nitrite catalysis rates are markedly sensitive to the respective Fe(III)(P)(NO) reduction potentials, which is consistent with the behavior expected for an outer-sphere electron-transfer mechanism. Nitrite is the product of NO autoxidation in aqueous solution and is a ubiquitous impurity in experiments where aqueous NO is added to an aerobic system to study biological effects. The present results demonstrate that such an impurity should not be assumed to be innocuous, especially in the context of recent reports that endogenous nitrite may play physiological roles relevant to the interactions of NO and ferriheme proteins.

Kinetics of the reaction between nitric oxide and glutathione: implications for thiol depletion in cells

Nitric oxide in the absence of oxygen was suggested to react with 5–50 mM glutathione (GSH) over many minutes when [NO ] ≪ [GSH] (N. Hogg et al., FEBS Lett. 382:223–228; 1996). However, Aravindakumar et al. ( J. Chem. Soc. Perkin Trans. 2:663–669; 2002) provided data suggesting ∼200-fold higher reactivity under conditions of [NO ] ≫ [GSH]. To help resolve these differences, the rate of loss of NO (∼9 μM) in aqueous solutions of GSH (2.5–20 mM) was measured by chemiluminescence. An apparent second-order rate constant of 0.080 ± 0.008 M −1 s −1 at pH 7.4, 37°C, was calculated based on the total [GSH] and “pseudo-first-order” kinetics; thiolate anion was much more reactive than undissociated thiol. These data imply a half-life of ∼30 min for low concentrations of NO with 5 mM GSH, 37°C, pH 7.4, in the absence of oxygen. Possible kinetic schemes that can partially explain the divergent literature reports are discussed, notably an equilibrium in the reaction between NO and GSH. Human breast carcinoma MCF-7 cells were exposed to NO (initially ∼18 μM) in alidded six well plate in an anaerobic chamber in vitro; intracellular GSH levels decreased by half in ∼60 min. Aerobic exposure depletes GSH in cells in vitro much faster because of autoxidation of NO to NO 2 , >10 8 times more reactive toward GSH.

Nitric oxide is consumed, rather than conserved, by reaction with oxyhemoglobin under physiological conditions.

Although irreversible reaction of NO with the oxyheme of hemoglobin (producing nitrate and methemoglobin) is extremely rapid, it has been proposed that, under normoxic conditions, NO binds preferentially to the minority deoxyheme to subsequently form S-nitrosohemoglobin (SNOHb). Thus, the primary reaction would be conservation, rather than consumption, of nitrogen oxide. Data supporting this conclusion were generated by using addition of a small volume of a concentrated aqueous solution of NO to a normoxic hemoglobin solution. Under these conditions, however, extremely rapid reactions can occur before mixing. We have thus compared bolus NO addition to NO generated homogeneously throughout solution by using NO donors, a more physiologically relevant condition. With bolus addition, multiple hemoglobin species are formed (as judged by visible spectroscopy) as well as both nitrite and nitrate. With donor, only nitrate and methemoglobin are formed, stoichiometric with the amount of NO liberated from the donor. Studies with increasing hemoglobin concentrations reveal that the nitrite-forming reaction (which may be NO autoxidation under these conditions) competes with reaction with hemoglobin. SNOHb formation is detectable with either bolus or donor; however, the amounts formed are much smaller than the amount of NO added (less than 1%). We conclude that the reaction of NO with hemoglobin under normoxic conditions results in consumption, rather than conservation, of NO.

Distinction between Nitrosating Mechanisms within Human Cells and Aqueous Solution

The quintessential nitrosating species produced during NO autoxidation is N(2)O(3). Nitrosation of amine, thiol, and hydroxyl residues can modulate critical cell functions. The biological mechanisms that control reactivity of nitrogen oxide species formed during autoxidation of nano- to micromolar levels of NO were examined using the synthetic donor NaEt(2)NN(O)NO (DEA/NO), human tumor cells, and 4,5-diaminofluorescein (DAF). Both the disappearance of NO and formation of nitrosated product from DAF in aerobic aqueous buffer followed second order processes; however, consumption of NO and nitrosation within intact cells were exponential. An optimal ratio of DEA/NO and 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (PTIO) was used to form N(2)O(3) through the intermediacy of NO(2). This route was found to be most reflective of the nitrosative mechanism within intact cells and was distinct from the process that occurred during autoxidation of NO in aqueous media. Manipulation of the endogenous scavengers ascorbate and glutathione indicated that the location, affinity, and concentration of these substances were key determinants in dictating nitrosative susceptibility of molecular targets. Taken together, these findings suggest that the functional effects of nitrosation may be organized to occur within discrete domains or compartments. Nitrosative stress may develop when scavengers are depleted and this architecture becomes compromised. Although NO(2) was not a component of aqueous NO autoxidation, the results suggest that the intermediacy of this species may be a significant factor in the advent of either nitrosation or oxidation chemistry in biological systems.

Release of nitric oxide from donors with known half-life: a mathematical model for calculating nitric oxide concentrations in aerobic solutions.

The NO concentrations released from donor compounds are difficult to predict as they are determined by formation and inactivation reactions. To calculate the concentrations of NO over time, we have developed a mathematical model which is based on a system of two differential equations describing the first order decomposition of the NO donor in association with the third order reaction of NO with oxygen. Although there is no closed formula for the solution, it can be easily computed by any standard numerical differential solver or simulation software with the following input parameters: initial concentration and decomposition rate constant of the NO donor, O2 concentration, and rate constant for NO autoxidation. The model was validated by monitoring NO release from 2,2-diethyl-1-nitroso-oxyhydrazine (DEA/NO) with a Clark-type NO-sensitive electrode at two different temperatures (25 and 37 degrees C) and DEA/NO concentrations ranging from 1 to 10 microM. Under all conditions, there was an excellent agreement between experimental and calculated data. In addition to the computer modeling, we present graphical plots which allow a rough but very easy estimation of the actual NO concentrations if appropriate computer software should not be available.

Nitric oxide induces oxidative damage in addition to deamination in macrophage DNA.

Inflammatory cells such as phagocytes, neutrophils, and macrophages have been implicated in the pathogenesis of several forms of clinical and experimental tumor development. It is hypothesized that this process is mediated by the production of reactive species including NO., O2.-, H2O2, and ONOO- which inflict DNA damage. In this study, the role of NO. in combination with oxygen radicals in DNA damage was investigated. DNA deamination (xanthine) and oxidation [5-(hydroxymethyl)uracil (5HMU), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FAPY-G), and 8-oxoguanine (8oxoG)] products were identified in the DNA of macrophages (RAW264.7) activated with Escherichia coli lipopolysaccharide (LPS) and mouse gamma-interferon (INF-gamma). The formation of these products was inhibited by N-methyl-L-arginine (NMA), a nitric oxide synthase inhibitor. NMA inhibited only the production of nitric oxide and had no effect on superoxide production. These results demonstrate that NO. plays a dual role in damaging the DNA of activated macrophages. Autoxidation of NO. leads to nitrosating species which cause deamination of bases. Reaction of NO. with O2.- leads to DNA oxidative damage due to the formation of peroxynitrite which may have HO.-like oxidizing potential. Another possible mechanism of oxidative damage by NO. could be the mobilization of free iron by NO. which could ultimately cause Fenton-type reactions. Therefore, nitric oxide not only leads to deamination of DNA bases but is also an obligatory factor in oxidative damage to DNA.

Kinetic and mechanistic aspects of the NO oxidation by O2 in aqueous phase

AbstractThe oxidation kinetics of NO by O2 in aqueous solution was observed using a stopped flow apparatus. The kinetics follows a third order rate law of the form k · [NO]2 · [O2] in analogy to gas‐phase results. The rate constant at 296 K was measured as (6.4 ± 0.8) · 106 M−2 s−1 with an activation energy of 2.3 kcal/mol and a preexponential factor of (4.0 ± 0.5) · 108 M−2 s−1. The rate constant displays a very slight pH dependence corresponding to less than a factor of three over the range 0 to 12. The system NO/O2 in aqueous solution is an efficient nitrosating agent which has been tested using phenol as a substrate over the pH range 0 to 12. The rate limiting step leading to formation of 4‐nitrosophenol is the formation of the reactive intermediate whose competitive hydrolysis yields HONO or NO2−. The absence of NO3− in the autoxidation of NO, the exclusive presence of NO2− as a product of the nitrosation reaction of phenol, and the kinetic results of the N3− trapping experiments point towards N2O3 as the reactive intermediate. © 1994 John Wiley & Sons, Inc.