nitrogen-dioxide and peroxynitric-acid

Reactions of linoleate (and presumably other unsaturated fatty acids) with reactive nitrogen species that form in biological systems from secondary reactions of .NO yield two main nitration product groups, LNO2 (formed by ONOO-, .NO2, or NO2+ reaction with linoleate), and LONO2 (formed by HONO reaction with 13(S)-HPODE, or .NO termination with LOO.). Comparison of HPLC retention times and m/z for lipid nitration products indicate that the mechanisms of nitrated product formation converge at several points: (i) The initial product of HONO attack on LOOH will be LOONO, which is identical to the initial termination product of LOO. reaction with .NO. (ii) Dissociation of LOONO to give LO. and .NO2 via caged radicals, which recombine to give LONO2 (m/z 340) will occur, regardless of how LOONO is formed (Fig. 7). (iii) In some experiments, the reaction of O2- (where oxidation is initiated by xanthine oxidase-derived O2- production and metal-dependent decomposition of H2O2) with .NO will result in generation of ONOO-. Nitration of unsaturated lipid by this species will yield a species demonstrated herein to be LNO2. Lipid oxidation leads to formation of bioactive products, including hydroxides, hydroperoxides, and isoprostanes. In vivo, nitrated lipids (LNO2, LONO2) may also possess bioactivity, for example through eicosanoid receptor binding activity, or by acting as antagonists/competitive inhibitors of eicosanoid receptor-ligand interactions. In addition, nitrated lipids could mediate signal transduction via direct .NO donation, transnitrosation, or following reductive metabolism. Similar bioactive products are formed following ONOO- reaction with glucose, glycerol, and other biomolecules.

Topics: Animals; Chromatography, High Pressure Liquid; Fatty Acids, Unsaturated; Free Radicals; Humans; Lipid Peroxidation; Nitrates; Nitric Oxide; Nitrogen Dioxide; Nitrous Acid

Nitric oxide (*NO) and nitrogen dioxide (*NO2) are hydrophobic gases. Therefore, lipid membranes and hydrophobic regions of proteins are potential sinks for these species. In these hydrophobic environments, reactive nitrogen species will exhibit different chemistry than in aqueous environments due to higher local concentrations and the lack of hydrolysis reactions. The peroxynitrite anion (ONOO-) and peroxynitrous acid (ONOOH) can freely pass through lipid membranes, making peroxynitrite-mediated reactions in a hydrophobic environment also of extreme relevance. The reactions observed by these reactive nitrogen species in a hydrophobic milieu include oxidation, nitration and even potent chain-breaking antioxidant reactions. The physiological and toxicological relevance of these reactions is discussed.

Topics: Animals; Cell Membrane; Free Radicals; Humans; Lipoproteins, LDL; Metals; Nitrates; Nitric Oxide; Nitrogen Dioxide; Oxidation-Reduction

Nitrogen dioxide ((*)NO(2)) participates in a variety of biological reactions. Of great interest are the reactions of (*)NO(2) with oxymyoglobin and oxyhemoglobin, which are the predominant hemeproteins in biological systems. Although these reactions occur rapidly during the nitrite-catalyzed autoxidation of hemeproteins, their roles in systems producing (*)NO(2) in the presence of these hemeproteins have been greatly underestimated. In the present study, we employed pulse radiolysis to study directly the kinetics and mechanism of the reaction of oxymyoglobin (MbFe(II)O(2)) with (*)NO(2). The rate constant of this reaction was determined to be (4.5 +/- 0.3) x 10(7) M(-1)s(-1), and is among the highest rate constants measured for (*)NO(2) with any biomolecule at pH 7.4. The interconversion among the various oxidation states of myoglobin that is prompted by nitrogen oxide species is remarkable. The reaction of MbFe(II)O(2) with (*)NO(2) forms MbFe(III)OONO(2), which undergoes rapid heterolysis along the O-O bond to yield MbFe(V)=O and NO(3-). The perferryl-myoglobin (MbFe(V)=O) transforms rapidly into the ferryl species that has a radical site on the globin ((*)MbFe(IV)=O). The latter oxidizes another oxymyoglobin (10(4) M(-1)s(-1) < k(17) < 10(7) M(-1)s(-1)) and generates equal amounts of ferrylmyoglobin and metmyoglobin. At much longer times, the ferrylmyoglobin disappears through a relatively slow comproportionation with oxymyoglobin (k(18) = 21.3 +/- 5.3 M(-1)s(-1)). Eventually, each (*)NO(2) radical converts three oxymyoglobin molecules into metmyoglobin. The same intermediate, namely MbFe(III)OONO(2), is also formed via the reaction peroxynitrate (O(2)NOO(-)/O(2)NOOH) with metmyoglobin (k(19) = (4.6 +/- 0.3) x 10(4) M(-1)s(-1)). The reaction of (*)NO(2) with ferrylmyoglobin (k(20) = (1.2 +/- 0.2) x 10(7) M(-1)s(-1)) yields MbFe(III)ONO(2), which in turn dissociates (k(21) = 190 +/- 20 s(-1)) into metmyoglobin and NO(3-). This rate constant was found to be the same as that measured for the decay of the intermediate formed in the reaction of MbFe(II)O(2) with (*)NO, which suggests that MbFe(III)ONO(2) is the intermediate observed in both processes. This conclusion is supported by thermokinetic arguments. The present results suggest that hemeproteins may detoxify (*)NO(2) and thus preempt deleterious processes, such as nitration of proteins. Such a possibility is substantiated by the observation that the reactions of (*)NO(2) with the various oxidation s

Topics: Animals; Free Radicals; Horses; Kinetics; Metmyoglobin; Myoglobin; Nitrates; Nitrogen Dioxide; Oxidation-Reduction; Pulse Radiolysis; Spectrophotometry

The second-order rate constants for the reactions between nitrogen monoxide and oxymyoglobin or oxyhemoglobin, determined by stopped-flow spectroscopy, increase with increasing pH. At pH 7.0 the rates are (43.6 +/- 0.5) x 10(6) M(-1) x s(-1) for oxymyoglobin and (89 +/- 3) x 10(6) M(-1) x s(-1) for oxyhemoglobin (per heme), whereas at pH 9.5 they are (97 +/- 3) x 10(6) M(-1) x s(-1) and (144 +/- 3) x 10(6) M(-1) x s(-1), respectively. The rate constants for the reaction between oxyhemoglobin and NO* depend neither on the association grade of the protein (dimer/tetramer) nor on the concentration of the phosphate buffer (100-1 mM). The nitrogen monoxide-mediated oxidations of oxymyoglobin and oxyhemoglobin proceed via intermediate peroxynitrito complexes which were characterized by rapid scan UV/vis spectroscopy. The two complexes MbFe(III)OONO and HbFe(III)OONO display very similar spectra with absorption maxima around 500 and 635 nm. These species can be observed at alkaline pH but rapidly decay to the met-form of the proteins under neutral or acidic conditions. The rate of decay of MbFe(III)OONO increases with decreasing pH and is significantly larger than those of the analogous complexes of the two subunits of hemoglobin. No free peroxynitrite is formed during these reactions, and nitrate is formed quantitatively, at both pH 7.0 and 9.0. This result indicates that, as confirmed from protein analysis after reacting the proteins with NO* for 10 times, when peroxynitrite is coordinated to the heme of myoglobin or hemoglobin it rapidly isomerizes to nitrate without nitrating the globins in physiologically significant amounts.

Topics: Animals; Chromatography, High Pressure Liquid; Horses; Humans; Hydrogen-Ion Concentration; Kinetics; Ligands; Metmyoglobin; Myoglobin; Nitrates; Nitric Oxide; Nitrogen Dioxide; Oxidation-Reduction; Oxyhemoglobins; Spectrophotometry, Ultraviolet; Spectrum Analysis

Nitrogen dioxide (*NO2) is commonly known as an indoor and outdoor air pollutant. Inhalation of *NO2 is associated with epithelial cell injury, inflammation, and the aggravation of asthma. *NO2 can also be formed during inflammation, by the metabolism of nitric oxide. We describe a gas-phase exposure system for in vitro exposure of lung epithelial cells to *NO2. Immunofluorescence revealed 3-nitrotyrosine immunoreactivity of rat alveolar type II epithelial cells exposed to 5 parts per million of *NO2 for 4 h. Comparative analysis of log-phase and confluent cultures demonstrated that cell death occurred extensively in log-phase cells, whereas minimal death was observed in confluent cultures. Peroxynitrite (ONOO-) or the ONOO- generator 3-morpholinosydnonimine (SIN-1) caused similar amounts of death. Further, exposure of wounded cell cultures to *NO(2) or SIN-1 revealed that death was restricted to cells repopulating a wounded area. Cycloheximide or actinomycin D, inhibitors or protein and messenger RNA synthesis, respectively, significantly reduced terminal transferase reactivity, suggesting that a new protein(s) may be required for cell death. These results suggest that during restitution after pulmonary injury, epithelium may be sensitive to cell death by reactive nitrogen species.

Topics: Cell Count; Cell Death; Cells, Cultured; Culture Media, Conditioned; DNA, Single-Stranded; In Situ Nick-End Labeling; Lung; Molsidomine; Nitrates; Nitric Oxide Donors; Nitrites; Nitrogen Dioxide; Oxidative Stress; Respiratory Mucosa

In a recent publication [Nauser et al. (2001) Chem. Res. Toxicol. 14, 248-350], the authors estimated a value of 14 +/- 3 kcal/mol for the standard Gibbs energy of formation of ONOO(-) and argued that the experimental value of 16.6 kcal/mol [Merényi, G., and Lind, J. (1998) Chem. Res. Toxicol. 11, 243-246] is in error. The lower value would suggest that the yield of free radicals during decomposition of ONOOH into nitrate is negligibly low, i.e., less than 0.5%, though within the large error limit given, the radical yield might vary between 0.003% and ca. 80%. The experimental value of 16.6 +/- 0.4 kcal/mol was based on the determination of the rate constant of the forward reaction in the equilibrium ONOO(-) <==> (*)NO and O2(*-) by use of C(NO2)4, an efficient scavenger of O2(*-) which yields C(NO2)3(-). Nauser et al. reported that addition of (*)NO has no significant effect on the rate of formation of C(NO2)3(-), and therefore the formation of C(NO2)3(-) is due to a process other then reduction of C(NO2)4 by O2(*-). In addition, they argued that Cu(II) nitrilotriacetate enhances the rate of peroxynitrite decomposition at pH 9.3 without reduction of Cu(II). In the present paper, we show that the formation of C(NO2)3(-) due to the presence peroxynitrite is completely blocked upon addition of (*)NO. Furthermore, the acceleration of the rate of peroxynitrite decomposition at pH 9 in the presence of catalytic concentrations of SOD ([ONOO(-)]/[SOD] > 30) results in the same rate constant as that obtained in the presence of C(NO(2))(4). These results can only be rationalized by assuming that ONOO(-) homolyses into (*)NO and O2(*-) with k = 0.02 s(-1) at 25 degrees C. Thus, the critical experiments suggested by Nauser et al. fully support the currently accepted thermodynamics as well as the mode of decomposition of the ONOOH/ONOO(-) system.

Topics: Kinetics; Nitrates; Nitric Oxide; Nitrogen Dioxide; Oxidants; Thermodynamics

Topics: Administration, Inhalation; Animals; Endothelium, Vascular; Epoprostenol; Hemoglobins; Humans; Hydroxyl Radical; Hypertension, Pulmonary; Nitrates; Nitric Oxide; Nitrogen Dioxide; Oxidation-Reduction; Pneumonia; Prostaglandin H2; Prostaglandins H; Signal Transduction; Superoxides; Tyrosine

Peroxynitrite, a biological oxidant formed from the reaction of nitric oxide with the superoxide radical, is associated with many pathologies, including neurodegenerative diseases, such as multiple sclerosis (MS). Gout (hyperuricemic) and MS are almost mutually exclusive, and uric acid has therapeutic effects in mice with experimental allergic encephalomyelitis, an animal disease that models MS. This evidence suggests that uric acid may scavenge peroxynitrite and/or peroxynitrite-derived reactive species. Therefore, we studied the kinetics of the reactions of peroxynitrite with uric acid from pH 6.9 to 8.0. The data indicate that peroxynitrous acid (HOONO) reacts with the uric acid monoanion with k = 155 M(-1) s(-1) (T = 37 degrees C, pH 7.4) giving a pseudo-first-order rate constant in blood plasma k(U(rate))(/plasma) = 0.05 s(-1) (T = 37 degrees C, pH 7.4; assuming [uric acid](plasma) = 0.3 mM). Among the biological molecules in human plasma whose rates of reaction with peroxynitrite have been reported, CO(2) is one of the fastest with a pseudo-first-order rate constant k(CO(2))(/plasma) = 46 s(-1) (T = 37 degrees C, pH 7.4; assuming [CO(2)](plasma) = 1 mM). Thus peroxynitrite reacts with CO(2) in human blood plasma nearly 920 times faster than with uric acid. Therefore, uric acid does not directly scavenge peroxynitrite because uric acid can not compete for peroxynitrite with CO(2). The therapeutic effects of uric acid may be related to the scavenging of the radicals CO(*-)(3) and NO(*)(2) that are formed from the reaction of peroxynitrite with CO(2). We suggest that trapping secondary radicals that result from the fast reaction of peroxynitrite with CO(2) may represent a new and viable approach for ameliorating the adverse effects associated with peroxynitrite in many diseases.

Topics: Bicarbonates; Carbon Dioxide; Free Radical Scavengers; Free Radicals; Humans; Hydrogen-Ion Concentration; Kinetics; Models, Biological; Neuroprotective Agents; Nitrates; Nitrogen Dioxide; Nitrous Acid; Oxidants; Peroxynitrous Acid; Temperature; Uric Acid

In a recent article [Khan, A. U., Kovacic, D., Kolbanovsky, A., Desai, M., Frenkel, K. & Geacintov, N. E. (2000) Proc. Natl. Acad. Sci. USA 97, 2984-2989], the authors claimed that ONOO(-), after protonation to ONOOH, decomposes into (1)HNO and (1)O(2) according to a spin-conserved unimolecular mechanism. This claim was based partially on their observation that nitrosylhemoglobin is formed via the reaction of peroxynitrite with methemoglobin at neutral pH. However, thermochemical considerations show that the yields of (1)O(2) and (1)HNO are about 23 orders of magnitude lower than those of ( small middle dot)NO(2) and ( small middle dot)OH, which are formed via the homolysis of ONOOH. We also show that methemoglobin does not form with peroxynitrite any spectrally detectable product, but with contaminations of nitrite and H(2)O(2) present in the peroxynitrite sample. Thus, there is no need to modify the present view of the mechanism of ONOOH decomposition, according to which initial homolysis into a radical pair, [ONO( small middle dot) ( small middle dot)OH](cage), is followed by the diffusion of about 30% of the radicals out of the cage, while the rest recombines to nitric acid in the solvent cage.

Topics: Animals; Anions; Cattle; Free Radicals; Hydrogen Peroxide; Methemoglobin; Nitrates; Nitrites; Nitrogen Dioxide; Nitrogen Oxides; Oxygen; Singlet Oxygen; Spectrophotometry, Ultraviolet

Reactive nitrogen species derived from nitric oxide are potent oxidants formed during inflammation that can oxidize membrane and lipoprotein lipids in vivo. Herein, it is demonstrated that several of these species react with unsaturated fatty acid to yield nitrated oxidation products. Using HPLC coupled with both UV detection and electrospray ionization mass spectrometry, products of reaction of ONOO- with linoleic acid displayed mass/charge (m/z) characteristics of LNO2 (at least three products at m/z 324, negative ion mode). Further analysis by MS/MS gave a major fragment at m/z 46. Addition of a NO2 group was confirmed using [15N]ONOO- which gave a product at m/z 325, fragmenting to form a daughter ion at m/z 47. Formation of nitrated lipids was inhibited by bicarbonate, superoxide dismutase (SOD), and Fe3+-EDTA, while the yield of oxidation products was decreased by bicarbonate and SOD, but not by Fe3+-EDTA. Reaction of linoleic acid with both nitrogen dioxide (*NO2) or nitronium tetrafluoroborate (NO2BF4) also yielded nitrated lipid products (m/z 324), with HPLC retention times and MS/MS fragmentation patterns identical to the m/z 324 species formed by reaction of ONOO- with linoleic acid. Finally, reaction of HPODE, but not linoleate, with nitrous acid (HONO) or isobutyl nitrite (BuiONO) yielded a product at m/z 340, or 341 upon reacting with [15N]HONO. MS/MS analysis gave an NO2- fragment, and 15N NMR indicated that the product contained a nitro (RNO2) functional group, suggesting that the product was nitroepoxylinoleic acid [L(O)NO2]. This species could form via homolytic dissociation of LOONO to LO* and *NO2 and rearrangement of LO* to an epoxyallylic radical L(O)* followed by recombination of L(O)* with *NO2. Since unsaturated lipids of membranes and lipoproteins are critical targets of reactive oxygen and nitrogen species, these pathways lend insight into mechanisms for the formation of novel nitrogen-containing lipid products in vivo and provide synthetic strategies for further structural and functional studies.

Topics: Chromatography, High Pressure Liquid; Chromatography, Liquid; Fatty Acids, Unsaturated; Hydrogen-Ion Concentration; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Magnetic Resonance Spectroscopy; Mass Spectrometry; Nitrates; Nitric Oxide; Nitrogen Dioxide; Nitrous Acid; Oxidants; Oxidation-Reduction

To react with peptides, nitric oxide.NO has to be activated by oxidation, or by coupling with superoxide (O.-2) thereby producing peroxynitrite. In the course of.NO oxidation,.NO2 free radicals and N2O3 may be formed. Using gamma-irradiation methods, we characterized the products formed by these nitrogen oxides with angiotensin II. Angiotensin II is specifically nitrated at its tyrosinyl residue by.NO2 or peroxynitrite. Equimolecular amounts of each reagent in K+/Pi solutions at pH 7.4 led to 56% and 5% nitration yields, respectively. Nitrogen oxides produced by autoxidation of.NO, as well as.NO2 under.NO, reacted only with the arginine residue, giving a mixture of peptides containing citrulline, a N-(hydroxylamino-cyanamido-) instead of guanido group, and a conjugated diene derived from an arginine side-chain. However, nitrosation reactions by N2O3 occurred only when the initial concentration of.NO2 was 10 times that able to react with angiotensin II. Thus, in this case.NO appears to protect against.NO2 action.

Topics: Angiotensin II; Chromatography, High Pressure Liquid; Free Radicals; Humans; In Vitro Techniques; Mass Spectrometry; Nitrates; Nitric Oxide; Nitrogen Dioxide

Recent thermodynamic calculations of Merenyi and Lind [(1997) Chem. Res. Toxicol. 10, 1216-1220] suggest that O=NOOH can undergo homolysis to form the hydroxyl radical and nitrogen dioxide. This result is based in part on our statement that the enthalpy of ionization of O=NOOH is close to zero [Koppenol et al. (1992) Chem. Res. Toxicol. 5, 834-842]. As the ionization of O=NOOH is sensitive to the milieu and the rate of isomerization (to nitrate) to the total concentration of O=NOOH and O=NOO- [Kissner et al. (1997) Chem. Res. Toxicol. 10, 1285-1292], we reinvestigated the temperature dependence of the ionization constant and determined a deltaHo of 4+/-2 kcal mol(-1). This results in a standard Gibbs energy of homolysis of 16 kcal mol(-1) and a rate of homolysis of 1 x 10(-2) s[-1]. Given the uncertainty in the Gibbs energy of homolysis, upper and lower rates are 1 x 10(-4) and 0.6 s(-1), slower than the rate of isomerization, 1.2 s(-1) at 25 degrees C. The recombination of the homolysis products NO2. and HO. is known to lead to mainly peroxynitrous acid. If one assumes that a few percent of the recombinations lead to nitrate instead, then the rate of homolysis must be much higher than the rate of isomerization. We conclude therefore that homolysis is unlikely.

Topics: Entropy; Hydroxyl Radical; Nitrates; Nitrogen Dioxide; Protons; Thermodynamics

To determine whether asbestos inhalation induces the formation of reactive nitrogen species, three groups of rats were exposed intermittently over 2 wk to either filtered room air (sham-exposed) or to chrysotile or crocidolite asbestos fibers. The rats were killed at 1 or 6 wk after exposure. At 1 wk, significantly greater numbers of alveolar and pleural macrophages from asbestos-exposed rats than from sham-exposed rats demonstrated inducible nitric oxide synthase protein immunoreactivity. Alveolar macrophages from asbestos-exposed rats also generated significantly greater nitrite formation than did macrophages from sham-exposed rats. Strong immunoreactivity for nitrotyrosine, a marker of peroxynitrite formation, was evident in lungs from chrysotile- and crocidolite-exposed rats at 1 and 6 wk. Staining was most evident at alveolar duct bifurcations and within bronchiolar epithelium, alveolar macrophages, and the visceral and parietal pleural mesothelium. Lungs from sham-exposed rats demonstrated minimal immunoreactivity for nitrotyrosine. Significantly greater quantities of nitrotyrosine were detected by ELISA in lung extracts from asbestos-exposed rats than from sham-exposed rats. These findings suggest that asbestos inhalation can induce inducible nitric oxide synthase activation and peroxynitrite formation in vivo, and provide evidence of a possible alternative mechanism of asbestos-induced injury to that thought to be induced by Fenton reactions.

Topics: Animals; Asbestos, Crocidolite; Asbestos, Serpentine; Bronchoalveolar Lavage; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Inhalation Exposure; Lung; Macrophages; Macrophages, Alveolar; Male; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrogen Dioxide; Pleura; Pleural Effusion; Rats; Rats, Inbred F344; Reactive Oxygen Species; Tyrosine

The peroxynitrite anion and the nitrogen dioxide (radical) are important toxic species which can arise in vivo from nitric oxide. Both in vivo and in vitro cell protection is demonstrated for beta-carotene in the presence of vitamin E and vitamin C. A synergistic protection is observed compared to the individual anti-oxidants and this is explained in terms of an electron transfer reaction in which the beta-carotene radical is repaired by vitamin C.

Topics: Antioxidants; Ascorbic Acid; beta Carotene; Cell Survival; Drug Synergism; Free Radicals; Humans; Jurkat Cells; Lymphocytes; Nitrates; Nitrogen Dioxide; Oxidants; Vitamin E

A new sensitive and specific assay was developed and applied for the quantitative determination of 3-nitrotyrosine in proteins of human platelets. 3-Nitrotyrosine was quantitatively converted into a new pentafluorobenzyl derivative in a single step and detected as an abundant carboxylate anion at m/z 595 using negative ion chemical ionization gas chromatography/mass spectrometry. The internal standard, [13C6]-3-nitrotyrosine, was prepared via a new and efficient method using nitronium borofluorate dissolved in hydrochloric acid. The assay showed excellent linearity and sensitivity. Intact human platelets contained 1.4+/-0.6 ng of 3-nitrotyrosine per milligram of protein. Peroxynitrite increased 3-nitrotyrosine levels 4- to 535-fold at the concentration range of 10 to 300 microM. Decomposed peroxynitrite was without the effect. Nitrogen dioxide (43 microM) was also a potent tyrosine nitrating molecule, increasing the levels of 3-nitrotyrosine 153-fold. HOCl (50 microM) in the presence of nitrite (50 microM) increased the 3-nitrotyrosine levels 3-fold. Exposure of platelets to nitric oxide, nitrite, thrombin, adenosine diphosphate, platelet activating factor, and arachidonic acid had no effect on platelet 3-nitrotyrosine levels.

Topics: Blood Platelets; Fluorobenzenes; Gas Chromatography-Mass Spectrometry; Humans; Nitrates; Nitrogen Dioxide; Sensitivity and Specificity; Tyrosine

beta-Carotene absorbed 2 equimolar amounts of NO2 accompanying the complete destruction of beta-carotene. Electron spin resonance study using 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl revealed that no significant amounts of NO were released by the interaction. Nitrogen atoms derived from NO2 were tightly bound to the beta-carotene molecules. Destruction of beta-carotene was inhibited little by alpha-tocopherol and polyunsaturated fatty esters, and slightly by ascorbyl palmitate, indicating that beta-carotene was a more effective scavenger of NO2. ONOOH/ONOO- and 3-morpholinosydononimine similarly destroyed beta-carotene. The results suggest that beta-carotene contributes to the prevention of cytotoxicity and genotoxicity of NO2 and ONOOH/ONOO- derived from NO.

Topics: Amiloride; Ascorbic Acid; beta Carotene; Docosahexaenoic Acids; Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Nitrates; Nitrogen Dioxide; Vitamin E

The involvement of nitric oxide (NO) and its reactive intermediates such as nitrogen dioxide (NO2) and peroxynitrite (ONOO-) in the activation of matrix metallo-proteinase was investigated. The human neutrophil procollagenase (matrix metalloproteinase-8) (M(r), 85 kDa) was purified to homogeneity from human neutrophils by using column chromatography. After incubation of human neutrophil procollagenase with various nitrogen oxide-generating systems, collagenolytic activity in each reaction system was measured. In addition, neutrophil collagenase activity was determined by assessment of proteolysis of human alpha 1-protease inhibitor. NO was formed by the propylamine NONOate, and NO2 was generated by oxidation of NO with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO). NO2, formed by NONOate and carboxy-PTIO, and the synthetic ONOO- exhibited strong activation of the procollagenase at 1-20 microM. Significant activation of the procollagenase was observed with use of authentic NO2 gas as well. Constant flux infusion of ONOO- into the procollagenase solution resulted in stronger procollagenase activation than did a bolus addition of ONOO- to the reaction mixture. However, NO showed only weak activating potential under the aerobic (ambient) condition; an NO concentration of more than 10 mM was needed for appreciable activation of the procollagenase. Of considerable importance was the fact that NO participates in activation of the neutrophil collagenase through its conversion to NO2 or ONOO- in human neutrophils. These results suggest that NO2 and ONOO- may be potent activators of human neutrophil procollagenase.

Topics: alpha 1-Antitrypsin; Benzoates; Collagen; Collagenases; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Enzyme Precursors; Humans; Imidazoles; Leukocyte Elastase; Matrix Metalloproteinase 8; Methemoglobin; Neutrophils; Nitrates; Nitric Oxide; Nitrogen Dioxide; Oxyhemoglobins; Phenanthrolines; Tetradecanoylphorbol Acetate; Triazines

Peroxynitrite (ONOO-) is a 'reactive nitrogen species' that can be formed (among other reactions) by combination of superoxide (O2.-) and nitric oxide (NO.) radicals. It is being increasingly proposed as a contributor to tissue injury in several human diseases. The evidence presented for peroxynitrite participation usually includes the demonstration of increased nitrotyrosine levels in the injured tissue. Indeed, this is often the only evidence presented: the assumption is that formation of nitrotyrosine is a biomarker specifically diagnostic of ONOO- production. The present article examines this assumption and concludes that nitrotyrosine is a biomarker for 'nitrating species' rather than being specific for ONOO-.

Topics: Biomarkers; Free Radicals; Humans; Molecular Structure; Nitrates; Nitric Oxide; Nitrogen Dioxide; Nitrous Acid; Peroxidase; Superoxides; Tyrosine

The equilibrium constant, K3, of aqueous homolysis of peroxynitrous acid into hydroxyl and nitrogen dioxide free radicals was estimated to be 5 x 10(-10) M. This value was derived from a thermodynamic cycle by use of the experimentally known delta fH degree(ONOO-,aq) = -10.8 kcal/mol and the enthalpy of ionic dissociation of ONOOH(aq), delta H degree 1 = 0 kcal/mol, as well as of the entropy of gaseous ONOOH, S degree(ONOOH,g) = 72 eu. Furthermore we assumed the entropy of hydration of ONOOH, delta S degree 2, to be -25 eu, a value closely bracketed by the hydration entropies of analogous substances. The rate constant of radical recombination of OH. with NO2. to yield ONOOH, k-3, was resimulated from experimental data and found to be ca. 5 x 10(9) M-1 s-1. Together with the estimated K3, this yields the homolysis rate constant k3 = 2.5 s-1. This value is close to 0.5 s-1, the rate constant of formation of a reactive intermediate during the isomerization of peroxynitrous acid to nitrate. Our thermodynamic estimate is therefore consistent with substantial amounts of OH. and NO2. free radicals being formed in this process. The thermodynamic implications for the carbon dioxide/peroxynitrite system are also discussed.

Topics: Carbon Dioxide; Entropy; Free Radicals; Hydroxyl Radical; Nitrates; Nitrogen Dioxide; Oxidants; Oxidation-Reduction; Thermodynamics

Oxymyoglobin under argon reacts with NO2- and NO2 (N2O4) to produce metmyoglobin in a spectrally clean process with clear isosbestic points. In both cases, the reactant is NO2-. The second-order rate constant for NO2- or N2O4 is the same: d(Mb+)/dt = k(MbO2)(NO2-) where k = 0.21 +/- 0.02 L mol-1 S-1. The reaction of MbO2 with NO under argon is a complex process and entails the generation of Mb+ and OONO- (peroxynitrite) in the first step. The latter (lambda MAX, 302 nm) was poorly resolved from more intense protein absorption in the 300-nm region. However, at pH 9, the change in absorbance corresponded exactly to a quantitative production of the OONO-ion. Hydroxy radicals from it were trapped with ethylene-1, 2-(13) C. The initial step is followed in sequence by the rapid formation of MbNO+. The iron(III)-nitrosyl adduct hydrolyzes slowly to MbII and NO2- (k = 8.0 +/- 0.8 x 10(-5) S-1. MbII then rapidly associates with NO, and MbNO is the final product of this reaction. Oxymyoglobin is inert to NO3-. In contrast to the results under argon, in air the reactions of MbO2 with NO2-, NO, and NO2 (N2O4) all proceed in the same autocatalytic fashion with kAVE (for the autocatalytic rates) approximately equal to 9 +/- 5 L mol-1 s-1. Nitrite is the initial reactant in all cases. Isosbestic points are not observed in the visible spectrum, and additional porphyrin iron-ligated species are intermediates. Based upon work with iron porphyrins.

Topics: Air; Animals; Argon; Horses; Kinetics; Myocardium; Myoglobin; Nitrates; Nitrogen Dioxide; Nitrous Oxide

Topics: Free Radicals; Hydrogen-Ion Concentration; Hydroxyl Radical; Hydroxylation; Kinetics; Mannitol; Nitrates; Nitric Oxide; Nitrogen Dioxide; Nitrogen Oxides; Oxidants; Phenylalanine; Tyrosine

Peroxynitrite is a strong oxidant formed by macrophages and potentially by other cells that produce nitric oxide and superoxide. Peroxynitrite was highly bactericidal, killing Escherichia coli in direct proportion to its concentration with an LD50 of 250 microM at 37 degrees C in potassium phosphate, pH 7.4. The apparent bactericidal activity of a given concentration peroxynitrite at acidic pH was less than that at neutral and alkaline pH. However, after taking the rapid pH-dependent decomposition of peroxynitrite into account, the rate of the killing was not significantly different at pH 5 compared to pH 7.4. Metal chelators did not decrease peroxynitrite-mediated killing, indicating that exogenous transition metals were not required for toxicity. The hydroxyl radical scavengers mannitol, ethanol, and benzoate did not significantly affect toxicity while dimethyl sulfoxide enhanced peroxynitrite-mediated killing. Dimethyl sulfoxide is a more efficient hydroxyl radical scavenger than the other three scavengers and increased the formation of nitrogen dioxide from peroxynitrite. In the presence of 100 mM dimethyl sulfoxide, 60.0 +/- 0.3 microM nitrogen dioxide was formed from 250 microM peroxynitrite as compared to 2.0 +/- 0.1 microM in buffer alone. Thus, formation of nitrogen dioxide may have enhanced the toxicity of peroxynitrite decomposing in the presence of dimethyl sulfoxide.

Topics: Anti-Bacterial Agents; Benzoates; Benzoic Acid; Dimethyl Sulfoxide; Escherichia coli; Ethanol; Free Radical Scavengers; Hydrogen-Ion Concentration; Kinetics; Luminescent Measurements; Mannitol; Nitrates; Nitrogen Dioxide