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Rafael Radi - One of the best experts on this subject based on the ideXlab platform.
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Fluorescence and chemiluminescence approaches for Peroxynitrite detection.
Free radical biology & medicine, 2018Co-Authors: Carolina Prolo, Natalia Rios, Lucía Piacenza, María Noel Alvarez, Rafael RadiAbstract:In the last two decades, there has been a significant advance in understanding the biochemistry of Peroxynitrite, an endogenously-produced oxidant and nucleophile. Its relevance as a mediator in several pathologic states and the aging process together with its transient character and low steady-state concentration, motivated the development of a variety of techniques for its unambiguous detection and estimation. Among these, fluorescence and chemiluminescence approaches have represented important tools with enhanced sensitivity but usual limited specificity. In this review, we analyze selected examples of molecular probes that permit the detection of Peroxynitrite by fluorescence and chemiluminescence, disclosing their mechanism of reaction with either Peroxynitrite or Peroxynitrite-derived radicals. Indeed, probes have been divided into 1) redox probes that yield products by a free radical mechanism, and 2) electrophilic probes that evolve to products secondary to the nucleophilic attack by Peroxynitrite. Overall, boronate-based compounds are emerging as preferred probes for the sensitive and specific detection and quantitation. Moreover, novel strategies involving genetically-modified fluorescent proteins with the incorporation of unnatural amino acids have been recently described as Peroxynitrite sensors. This review analyzes the most commonly used fluorescence and chemiluminescence approaches for Peroxynitrite detection and provides some guidelines for appropriate experimental design and data interpretation, including how to estimate Peroxynitrite formation rates in cells.
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biochemistry of Peroxynitrite and protein tyrosine nitration
Chemical Reviews, 2018Co-Authors: Gerardo Ferrersueta, Beatriz Alvarez, Madia Trujillo, Nicolas Campolo, Silvina Bartesaghi, Sebastian Carballal, Natalia Romero, Rafael RadiAbstract:Peroxynitrite is a short-lived and reactive biological oxidant formed from the diffusion-controlled reaction of the free radicals superoxide (O2•–) and nitric oxide (•NO). In this review, we first analyze the biochemical evidence for the formation of Peroxynitrite in vivo and the reactions that lead to it. Then, we describe the principal reactions that Peroxynitrite undergoes with biological targets and provide kinetic and mechanistic details. In these reactions, Peroxynitrite has roles as (1) peroxide, (2) Lewis base, and (3) free radical generator. Physiological levels of CO2 can change the outcome of Peroxynitrite reactions. The second part of the review assesses the formation of protein 3-nitrotyrosine (NO2Tyr) by Peroxynitrite-dependent and -independent mechanisms, as one of the hallmarks of the actions of •NO-derived oxidants in biological systems. Moreover, tyrosine nitration impacts protein structure and function, tyrosine kinase signal transduction cascades and protein turnover. Overall, the revi...
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Peroxynitrite Formation and Detection in Living Cells
Nitric Oxide, 2017Co-Authors: Natalia Rios, Carolina Prolo, Lucía Piacenza, María Noel Alvarez, Rafael RadiAbstract:Abstract Different cell types produce Peroxynitrite under conditions of simultaneous generation of its precursor radicals, superoxide and nitric oxide. The detection of cell-derived Peroxynitrite is technically challenging due to its short biological half-life and low steady-state concentration and the shortage of specific methods. However, appropriate use of molecular footprints and probes together with a wise application of pharmacological and genetic approaches, allow for its unambiguous detection. Among the molecular footprints left by Peroxynitrite, the measurement and identification of tyrosine-nitrated proteins is of prime relevance. Regarding molecular probes, recent advances on the characterization of the reactivity of boronate-based molecules with Peroxynitrite have opened new and more specific ways for cellular detection. A critical analysis of the chemical basis and usefulness of the existing methods utilized for the cellular detection of Peroxynitrite will be performed. Also, the role of intracellular modulators of Peroxynitrite reactivity and levels (e.g., CO2, uric acid, peroxiredoxins) and how they can influence the detected levels will be assessed. The accurate cellular detection of Peroxynitrite in different cellular and extracellular compartments and the estimation of its formation rates, represent fundamental steps to understand how nitric oxide-derived oxidants affect biological processes, including mitochondrial dysfunction and cell death.
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Peroxynitrite, a stealthy biological oxidant
The Journal of biological chemistry, 2013Co-Authors: Rafael RadiAbstract:Peroxynitrite is the product of the diffusion-controlled reaction of nitric oxide and superoxide radicals. Peroxynitrite, a reactive short-lived peroxide with a pKa of 6.8, is a good oxidant and nucleophile. It also yields secondary free radical intermediates such as nitrogen dioxide and carbonate radicals. Much of nitric oxide- and superoxide-dependent cytotoxicity resides on Peroxynitrite, which affects mitochondrial function and triggers cell death via oxidation and nitration reactions. Peroxynitrite is an endogenous toxicant but is also a cytotoxic effector against invading pathogens. The biological chemistry of Peroxynitrite is modulated by endogenous antioxidant mechanisms and neutralized by synthetic compounds with Peroxynitrite-scavenging capacity.
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Peroxynitrite detoxification and its biologic implications.
Antioxidants & redox signaling, 2008Co-Authors: Madia Trujillo, Gerardo Ferrer-sueta, Rafael RadiAbstract:Peroxynitrite is a cytotoxic oxidant formed in vivo from the diffusional-controlled reaction between nitric oxide and superoxide radicals. Increased Peroxynitrite formation has been related to the pathogenesis of multiple diseases, thus underlining the importance of understanding the mechanisms of its detoxification. In nature, different enzymatic routes for Peroxynitrite decomposition have evolved. Among them, peroxiredoxins catalytically reduce Peroxynitrite in vitro; modulation of their expression affects Peroxynitrite-mediated cytotoxicity, and their content changes in pathologic conditions associated with increased Peroxynitrite formation in vivo, thus indicating a physiologic role of these enzymes in Peroxynitrite reduction. Selenium-containing glutathione peroxidase also catalyzes Peroxynitrite reduction, but its role in vivo is still a matter of debate. In selected cellular systems, heme proteins also play a role in Peroxynitrite detoxification, such as its isomerization by oxyhemoglobin in red blood cells. Moreover, different pharmacologic approaches have been used to decrease the toxicity related to Peroxynitrite formation. Manganese or iron porphyrins catalyze Peroxynitrite decomposition, and their protective role in vivo has been confirmed in biologic systems. Glutathione peroxidase mimetics also rapidly reduce Peroxynitrite, but their biologic role is less well established. Flavonoids, nitroxides, and tyrosine-containing peptides decreased Peroxynitrite-mediated toxicity under different conditions, but their mechanism of action is indirect.
William A Pryor - One of the best experts on this subject based on the ideXlab platform.
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Distinguishing reactivities of Peroxynitrite and hydroxyl radical.
Methods in Enzymology, 2004Co-Authors: Giuseppe L. Squadrito, Rao M. Uppu, William A PryorAbstract:Publisher Summary This chapter discusses the distinguishing reactivities of Peroxynitrite and hydroxyl radical. The oxidations using Peroxynitrite prepared by independent methods afford the same yields of ethylene and methionine sulfoxide from methionine. The presence of metal ion chelators such as ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA) does not affect these yields, suggesting that trace amounts of metal ions are not involved in these processes when the Peroxynitrite is treated with MnO2 to destroy the hydrogen peroxide, or when a hydrogen peroxide free preparation is selected. Ethylene is formed from both the reactions of methionine with Peroxynitrite and the hydroxyl radical. The yield of ethylene from the reaction of methionine with Peroxynitrite is not affected by hydroxyl radical scavengers that do not react with ground-state peroxynitrous acid (HOONO). These data suggest that Peroxynitrite oxidations involve ground-state HOONO and/or excited-state peroxynitrous acid (HOONO*) but not hydroxyl radicals. The effects of metal ion chelators that either exacerbate or inhibit Fenton-type reactions on oxidations by Peroxynitrite are also reviewed in the chapter.
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Reaction of Uric Acid with Peroxynitrite and Implications for the Mechanism of Neuroprotection by Uric Acid
Archives of biochemistry and biophysics, 2000Co-Authors: Giuseppe L. Squadrito, Rao M. Uppu, Rafael Cueto, Andres E. Splenser, Athanasios Valavanidis, Houwen Zhang, William A PryorAbstract: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.
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The reaction of melatonin with Peroxynitrite: formation of melatonin radical cation and absence of stable nitrated products.
Biochemical and biophysical research communications, 1998Co-Authors: Houwen Zhang, Giuseppe L. Squadrito, William A PryorAbstract:Peroxynitrite is capable of hydroxylating and nitrating aromatic species. However, nitromelatonin is not found as a final product when melatonin was allowed to react with Peroxynitrite either in the presence or absence of added bicarbonate. In the absence of bicarbonate, the two major products formed are 6-hydroxymelatonin and 5-methoxy-2-hydro-pyrroloindole, and the latter is the only major product with excess bicarbonate. A transient purple intermediate with a maximum absorbance at about 520 nm is observed upon mixing solutions containing Peroxynitrite and melatonin. These observations indicate that the melatoninyl radical cation is formed in the Peroxynitrite/melatonin reaction, providing a direct evidence for the one-electron oxidation ability of Peroxynitrite. The melatoninyl radical cation also is observed with excess bicarbonate.
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oxidative chemistry of nitric oxide the roles of superoxide Peroxynitrite and carbon dioxide
Free Radical Biology and Medicine, 1998Co-Authors: Giuseppe L. Squadrito, William A PryorAbstract:Abstract The roles of superoxide (O2•−), Peroxynitrite, and carbon dioxide in the oxidative chemistry of nitric oxide ( NO) are reviewed. The formation of Peroxynitrite from NO and O2•− is controlled by superoxide dismutase (SOD), which can lower the concentration of superoxide ions. The concentration of CO2 in vivo is high (ca. 1 mM), and the rate constant for reaction of CO2 with −OONO is large (pH-independent k = 5.8 × 104 M−1s−1). Consequently, the rate of reaction of Peroxynitrite with CO2 is so fast that most commonly used scavengers would need to be present at very high, near toxic levels in order to compete with Peroxynitrite for CO2. Therefore, in the presence of physiological levels of bicarbonate, only a limited number of biotargets react directly with Peroxynitrite. These include heme-containing proteins such as hemoglobin, peroxidases such as myeloperoxidase, seleno-proteins such as glutathione peroxidase, proteins containing zinc-thiolate centers such as the DNA-binding transcription factors, and the synthetic antioxidant ebselen. The mechanism of the reaction of CO2 with −OONO produces metastable nitrating, nitrosating, and oxidizing species as intermediates. An analysis of the lifetimes of the possible intermediates and of the catalysis of Peroxynitrite decompositions suggests that the reactive intermediates responsible for reactions with a variety of substrates may be the free radicals NO2 and CO3•−. Biologically important reactions of these free radicals are, for example, the nitration of tyrosine residues. These nitrations can be pathological, but they also may play a signal transduction role, because nitration of tyrosine can modulate phosphorylation and thus control enzymatic activity. In principle, it might be possible to block the biological effects of Peroxynitrite by scavenging the free radicals NO2 and CO3•−. Because it is difficult to directly scavenge Peroxynitrite because of its fast reaction with CO2, scavenging of intermediates from the Peroxynitrite/CO2 reaction would provide an additional way of preventing Peroxynitrite-mediated cellular effects. The biological effects of Peroxynitrite also can be prevented by limiting the formation of Peroxynitrite from NO by lowering the concentration of O2•− using SOD or SOD mimics. Increased formation of Peroxynitrite has been linked to Alzheimer’s disease, rheumatoid arthritis, atherosclerosis, lung injury, amyotrophic lateral sclerosis, and other diseases.
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The catalytic role of carbon dioxide in the decomposition of Peroxynitrite.
Free radical biology & medicine, 1997Co-Authors: William A Pryor, Rao M. Uppu, Houwen Zhang, Jean-noel Lemercier, Giuseppe L. SquadritoAbstract:The fast reaction of Peroxynitrite with CO2 and the high concentration of dissolved CO2 in vivo (ca. 1 mM) suggest that CO2 modulates most of the reactions of Peroxynitrite in biological systems. The addition of Peroxynitrite to CO2 produces of the adduct ONOO-CO2- (1). The production of 1 greatly accelerates the decomposition of Peroxynitrite to give nitrate. We now show that the formation of 1 is followed by reformation of CO2 (rather than another carbonate species such as CO3 = or HCO3-). To show this, it is necessary to study systems with limiting concentrations of CO2. (When CO2 is present in excess, its concentration remains nearly constant during the decomposition of Peroxynitrite, and the recycling of CO2, although it occurs, can not be detected kinetically). We find that CO2 is a true catalyst of the decomposition of Peroxynitrite, and this fundamental insight into its action must be rationalized by any in vivo or in vitro reaction mechanism that is proposed. When the concentration of CO2 is lower than that of Peroxynitrite, the reformation of CO2 amplifies the fraction of Peroxynitrite that reacts with CO2. Even low concentrations of CO2 that result from the dissolution of ambient CO2 can have pronounced catalytic effects. These effects can cause deviations from predicted kinetic behavior in studies of Peroxynitrite in noncarbonate buffers in vitro, and since 1 and other intermediates derived from it are oxidants and/or nitrating agents, some of the reactions attributed to Peroxynitrite may depend on the availability of CO2.
Csaba Szabo - One of the best experts on this subject based on the ideXlab platform.
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pathophysiological roles of Peroxynitrite in circulatory shock
Shock, 2010Co-Authors: Csaba Szabo, Katalin ModisAbstract:Peroxynitrite is a reactive oxidant produced from nitric oxide (NO) and superoxide, which reacts with proteins, lipids and DNA and promotes cytotoxic and pro-inflammatory responses. Here we overview the role of Peroxynitrite in various forms of circulatory shock. Immunohistochemical and biochemical evidence demonstrate the production of Peroxynitrite in various experimental models of endotoxic and hemorrhagic shock, both in rodents and in large animals. In addition, biological markers of Peroxynitrite have been identified in human tissues after circulatory shock. Peroxynitrite can initiate toxic oxidative reactions in vitro and in vivo. Initiation of lipid peroxidation, direct inhibition of mitochondrial respiratory chain enzymes, inactivation of glyceraldehyde-3-phosphate dehydrogenase, inhibition of membrane Na+/K+ ATP-ase activity, inactivation of membrane sodium channels, and other oxidative protein modifications contribute to the cytotoxic effect of Peroxynitrite. In addition, Peroxynitrite is a potent trigger of DNA strand breakage, with subsequent activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP), which promotes cellular energetic collapse and cellular necrosis. Additional actions of Peroxynitrite that contribute to the pathogenesis of shock include inactivation of catecholamines and catecholamine receptors (leading to vascular failure), endothelial and epithelial injury (leading to endothelial and epithelial hyper-permeability and barrier dysfunction) as well as myocyte injury (contributing to loss of cardiac contractile function). Neutralization of Peroxynitrite with potent Peroxynitrite decomposition catalysts provides cytoprotective and beneficial effects in rodent and large animal models of circulatory shock.
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Multiple pathways of Peroxynitrite cytotoxicity.
Toxicology letters, 2003Co-Authors: Csaba SzaboAbstract:Peroxynitrite is a reactive oxidant produced from nitric oxide (NO) and superoxide, which reacts with a variety of biomolecules including proteins, lipids and DNA. Peroxynitrite is produced by the body in response to a variety of toxicologically relevant molecules including environmental toxins. It is also produced by the body in response to environmental toxins, as well as in reperfusion injury and inflammation. Here we overview the multiple pathways of Peroxynitrite cytotoxicity. Initiation of lipid peroxidation, direct inhibition of mitochondrial respiratory chain enzymes, inactivation of glyceraldehyde-3-phosphate dehydrogenase, inhibition of membrane Na(+)/K(+) ATP-ase activity, inactivation of membrane sodium channels, and other oxidative protein modifications contribute to the cytotoxic effect of Peroxynitrite. In addition, Peroxynitrite is a potent trigger of DNA strand breakage, with subsequent activation of the nuclear enzyme poly-ADP ribosyl synthetase or polymerase (PARP), with eventual severe energy depletion and necrosis of the cells. Studies conducted with Peroxynitrite decomposition catalysts suggest that neutralization of Peroxynitrite is of significant therapeutic benefit after exposure to various environmental toxins as well as in a variety of inflammatory and reperfusion disease conditions.
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Part I: Pathogenetic Role of Peroxynitrite in the Development of Diabetes and Diabetic Vascular Complications: Studies With FP15, A Novel Potent Peroxynitrite Decomposition Catalyst
Molecular Medicine, 2002Co-Authors: Csaba Szabo, Jon G. Mabley, Suzanne M. Moeller, Roman Shimanovich, Francisco G. Soriano, John H. Van Duzer, William Williams, Laszlo Virag, Pal Pacher, Andrew L. SalzmanAbstract:Background Peroxynitrite is a cytotoxic oxidant formed from nitric oxide (NO) and superoxide. Tyrosine nitration, a footprint of Peroxynitrite, has been demonstrated in the pancreatic islets as well as in the cardiovascular system of diabetic subjects. Delineation of the pathogenetic role of Peroxynitrite in disease conditions requires the use of potent, in vivo active Peroxynitrite decomposition catalysts. The aim of the current work was to produce a potent Peroxynitrite decomposition catalyst and to test its effects in rodent models of diabetes and its complications. Methods FP15 was synthesized and analyzed using standard chemical methods. Diabetes was triggered by the administration of streptozotocin. Tyrosine nitration was measured immunohistochemically. Cardiovascular and vascular measurements were conducted according to standard physiologic methods. Results FP15, a potent porphyrinic Peroxynitrite decomposition catalyst, potently inhibited tyrosine nitration and Peroxynitrite-induced cytotoxicity in vitro and in vivo. FP15 treatment (3–10 mg/kg/d) dose dependently and reduced the incidence and severity of diabetes mellitus in rats subjected to multiple low doses of streptozotocin, as well as in nonobese mice developing spontaneous autoimmune diabetes. Furthermore, treatment with FP15 protected against the development of vascular dysfunction (loss of endothelium-dependent relaxations) and the cardiac dysfunction (loss of my-ocardial contractility) in diabetic mice. FP15 treatment reduced tyrosine nitration in the diabetic pancreatic islets. Conclusions The current results demonstrate the importance of endogenous Peroxynitrite generation in the pathogenesis of autoimmune diabetes and diabetic cardiovascular complications. Peroxynitrite decomposition catalysts may be of therapeutic utility in diabetes and other pathophysiologic conditions.
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CRUCIAL ROLE OF APOPAIN IN THE Peroxynitrite-INDUCED APOPTOTIC DNA FRAGMENTATION
Free Radical Biology and Medicine, 1998Co-Authors: Laszlo Virag, Daniel Marmer, Csaba SzaboAbstract:Abstract Peroxynitrite, a cytotoxic oxidant formed in the reaction of superoxide and nitric oxide is known to cause programmed cell death. However, the mechanisms of Peroxynitrite-induced apoptosis are poorly defined. The present study was designed to characterize the molecular mechanisms by which Peroxynitrite induces apoptosis in HL-60 cells, with special emphasis on the role of caspases. Peroxynitrite induced the activation of apopain/caspase-3, but not ICE/caspase-1 as measured by the cleavage of fluorogenic peptides. Considering the short half-life of Peroxynitrite and the kinetics of caspase-3 activation (starting 3–4 h after Peroxynitrite treatment), the enzyme is not likely to become activated directly by the oxidant. Caspase-3 activation proved to be essential for DNA fragmentation, because pretreatment of the cells with the specific tetrapeptide inhibitor DEVD-fmk completely blocked Peroxynitrite-induced DNA fragmentation. Peroxynitrite-induced cytotoxicity was also significantly altered by the inhibition of caspase-3, whereas phosphatidylserine exposure was unaffected by DEVD-fmk treatment. Because many of the effects of Peroxynitrite are mediated by poly(ADP-ribose) synthetase (PARS) activation, we have also investigated the effect of PARS-inhibition on Peroxynitrite-induced apoptosis. We have found that PARS-inhibition modulates Peroxynitrite-induced apoptotic DNA fragmentation in the HL-60 cells. The effect of the PARS inhibitors, 3-aminobenzamide and 5-iodo-6-amino-1,2-benzopyrone were dependent on the concentration of Peroxynitrite used. While PARS-inhibition resulted in increased DNA-fragmentation at low doses (15 μM) of Peroxynitrite, a decreased DNA-fragmentation was found at high doses (60 μM) of Peroxynitrite. PARS inhibition negatively affected viability as determined by flow cytometry. These data demonstrate the crucial role of caspase-3 in mediating apoptotic DNA fragmentation in HL-60 cells exposed to Peroxynitrite.
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Mercaptoethylguanidine and Guanidine Inhibitors of Nitric-oxide Synthase React with Peroxynitrite and Protect against Peroxynitrite-induced Oxidative Damage
The Journal of biological chemistry, 1997Co-Authors: Csaba Szabo, Andrew L. Salzman, Gerardo Ferrer-sueta, Basilia Zingarelli, Garry J. Southan, Rafael RadiAbstract:Abstract Nitric oxide (NO) produced by the inducible nitric-oxide synthase (iNOS) is responsible for some of the pathophysiological alterations during inflammation. Part of NO-related cytotoxicity is mediated by Peroxynitrite, an oxidant species produced from NO and superoxide. Aminoguanidine and mercaptoethylguanidine (MEG) are inhibitors of iNOS and have anti-inflammatory properties. Here we demonstrate that MEG and related compounds are scavengers of Peroxynitrite. MEG caused a dose-dependent inhibition of the Peroxynitrite-induced oxidation of cytochrome c2+, hydroxylation of benzoate, and nitration of 4-hydroxyphenylacetic acid. MEG reacts with Peroxynitrite with a second-order rate constant of 1900 ± 64 M−1 s−1 at 37°C. In cultured macrophages, MEG reduced the suppression of mitochondrial respiration and DNA single strand breakage in response to Peroxynitrite. MEG also reduced the degree of vascular hyporeactivity in rat thoracic aortic rings exposed to Peroxynitrite. The free thiol plays an important role in the scavenging effect of MEG. Aminoguanidine neither affected the oxidation of cytochrome c2+ nor reacted with ground state Peroxynitrite, but inhibited the Peroxynitrite-induced benzoate hydroxylation and 4-hydroxyphenylacetic acid nitration, indicating that it reacts with activated peroxynitrous acid or nitrogen dioxide. Compounds that act both as iNOS inhibitors and Peroxynitrite scavengers may be useful anti-inflammatory agents.
Giuseppe L. Squadrito - One of the best experts on this subject based on the ideXlab platform.
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Distinguishing reactivities of Peroxynitrite and hydroxyl radical.
Methods in Enzymology, 2004Co-Authors: Giuseppe L. Squadrito, Rao M. Uppu, William A PryorAbstract:Publisher Summary This chapter discusses the distinguishing reactivities of Peroxynitrite and hydroxyl radical. The oxidations using Peroxynitrite prepared by independent methods afford the same yields of ethylene and methionine sulfoxide from methionine. The presence of metal ion chelators such as ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA) does not affect these yields, suggesting that trace amounts of metal ions are not involved in these processes when the Peroxynitrite is treated with MnO2 to destroy the hydrogen peroxide, or when a hydrogen peroxide free preparation is selected. Ethylene is formed from both the reactions of methionine with Peroxynitrite and the hydroxyl radical. The yield of ethylene from the reaction of methionine with Peroxynitrite is not affected by hydroxyl radical scavengers that do not react with ground-state peroxynitrous acid (HOONO). These data suggest that Peroxynitrite oxidations involve ground-state HOONO and/or excited-state peroxynitrous acid (HOONO*) but not hydroxyl radicals. The effects of metal ion chelators that either exacerbate or inhibit Fenton-type reactions on oxidations by Peroxynitrite are also reviewed in the chapter.
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Reaction of Uric Acid with Peroxynitrite and Implications for the Mechanism of Neuroprotection by Uric Acid
Archives of biochemistry and biophysics, 2000Co-Authors: Giuseppe L. Squadrito, Rao M. Uppu, Rafael Cueto, Andres E. Splenser, Athanasios Valavanidis, Houwen Zhang, William A PryorAbstract: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.
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The reaction of melatonin with Peroxynitrite: formation of melatonin radical cation and absence of stable nitrated products.
Biochemical and biophysical research communications, 1998Co-Authors: Houwen Zhang, Giuseppe L. Squadrito, William A PryorAbstract:Peroxynitrite is capable of hydroxylating and nitrating aromatic species. However, nitromelatonin is not found as a final product when melatonin was allowed to react with Peroxynitrite either in the presence or absence of added bicarbonate. In the absence of bicarbonate, the two major products formed are 6-hydroxymelatonin and 5-methoxy-2-hydro-pyrroloindole, and the latter is the only major product with excess bicarbonate. A transient purple intermediate with a maximum absorbance at about 520 nm is observed upon mixing solutions containing Peroxynitrite and melatonin. These observations indicate that the melatoninyl radical cation is formed in the Peroxynitrite/melatonin reaction, providing a direct evidence for the one-electron oxidation ability of Peroxynitrite. The melatoninyl radical cation also is observed with excess bicarbonate.
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oxidative chemistry of nitric oxide the roles of superoxide Peroxynitrite and carbon dioxide
Free Radical Biology and Medicine, 1998Co-Authors: Giuseppe L. Squadrito, William A PryorAbstract:Abstract The roles of superoxide (O2•−), Peroxynitrite, and carbon dioxide in the oxidative chemistry of nitric oxide ( NO) are reviewed. The formation of Peroxynitrite from NO and O2•− is controlled by superoxide dismutase (SOD), which can lower the concentration of superoxide ions. The concentration of CO2 in vivo is high (ca. 1 mM), and the rate constant for reaction of CO2 with −OONO is large (pH-independent k = 5.8 × 104 M−1s−1). Consequently, the rate of reaction of Peroxynitrite with CO2 is so fast that most commonly used scavengers would need to be present at very high, near toxic levels in order to compete with Peroxynitrite for CO2. Therefore, in the presence of physiological levels of bicarbonate, only a limited number of biotargets react directly with Peroxynitrite. These include heme-containing proteins such as hemoglobin, peroxidases such as myeloperoxidase, seleno-proteins such as glutathione peroxidase, proteins containing zinc-thiolate centers such as the DNA-binding transcription factors, and the synthetic antioxidant ebselen. The mechanism of the reaction of CO2 with −OONO produces metastable nitrating, nitrosating, and oxidizing species as intermediates. An analysis of the lifetimes of the possible intermediates and of the catalysis of Peroxynitrite decompositions suggests that the reactive intermediates responsible for reactions with a variety of substrates may be the free radicals NO2 and CO3•−. Biologically important reactions of these free radicals are, for example, the nitration of tyrosine residues. These nitrations can be pathological, but they also may play a signal transduction role, because nitration of tyrosine can modulate phosphorylation and thus control enzymatic activity. In principle, it might be possible to block the biological effects of Peroxynitrite by scavenging the free radicals NO2 and CO3•−. Because it is difficult to directly scavenge Peroxynitrite because of its fast reaction with CO2, scavenging of intermediates from the Peroxynitrite/CO2 reaction would provide an additional way of preventing Peroxynitrite-mediated cellular effects. The biological effects of Peroxynitrite also can be prevented by limiting the formation of Peroxynitrite from NO by lowering the concentration of O2•− using SOD or SOD mimics. Increased formation of Peroxynitrite has been linked to Alzheimer’s disease, rheumatoid arthritis, atherosclerosis, lung injury, amyotrophic lateral sclerosis, and other diseases.
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The catalytic role of carbon dioxide in the decomposition of Peroxynitrite.
Free radical biology & medicine, 1997Co-Authors: William A Pryor, Rao M. Uppu, Houwen Zhang, Jean-noel Lemercier, Giuseppe L. SquadritoAbstract:The fast reaction of Peroxynitrite with CO2 and the high concentration of dissolved CO2 in vivo (ca. 1 mM) suggest that CO2 modulates most of the reactions of Peroxynitrite in biological systems. The addition of Peroxynitrite to CO2 produces of the adduct ONOO-CO2- (1). The production of 1 greatly accelerates the decomposition of Peroxynitrite to give nitrate. We now show that the formation of 1 is followed by reformation of CO2 (rather than another carbonate species such as CO3 = or HCO3-). To show this, it is necessary to study systems with limiting concentrations of CO2. (When CO2 is present in excess, its concentration remains nearly constant during the decomposition of Peroxynitrite, and the recycling of CO2, although it occurs, can not be detected kinetically). We find that CO2 is a true catalyst of the decomposition of Peroxynitrite, and this fundamental insight into its action must be rationalized by any in vivo or in vitro reaction mechanism that is proposed. When the concentration of CO2 is lower than that of Peroxynitrite, the reformation of CO2 amplifies the fraction of Peroxynitrite that reacts with CO2. Even low concentrations of CO2 that result from the dissolution of ambient CO2 can have pronounced catalytic effects. These effects can cause deviations from predicted kinetic behavior in studies of Peroxynitrite in noncarbonate buffers in vitro, and since 1 and other intermediates derived from it are oxidants and/or nitrating agents, some of the reactions attributed to Peroxynitrite may depend on the availability of CO2.
Beatriz Alvarez - One of the best experts on this subject based on the ideXlab platform.
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biochemistry of Peroxynitrite and protein tyrosine nitration
Chemical Reviews, 2018Co-Authors: Gerardo Ferrersueta, Beatriz Alvarez, Madia Trujillo, Nicolas Campolo, Silvina Bartesaghi, Sebastian Carballal, Natalia Romero, Rafael RadiAbstract:Peroxynitrite is a short-lived and reactive biological oxidant formed from the diffusion-controlled reaction of the free radicals superoxide (O2•–) and nitric oxide (•NO). In this review, we first analyze the biochemical evidence for the formation of Peroxynitrite in vivo and the reactions that lead to it. Then, we describe the principal reactions that Peroxynitrite undergoes with biological targets and provide kinetic and mechanistic details. In these reactions, Peroxynitrite has roles as (1) peroxide, (2) Lewis base, and (3) free radical generator. Physiological levels of CO2 can change the outcome of Peroxynitrite reactions. The second part of the review assesses the formation of protein 3-nitrotyrosine (NO2Tyr) by Peroxynitrite-dependent and -independent mechanisms, as one of the hallmarks of the actions of •NO-derived oxidants in biological systems. Moreover, tyrosine nitration impacts protein structure and function, tyrosine kinase signal transduction cascades and protein turnover. Overall, the revi...
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Peroxynitrite reactivity with amino acids and proteins.
Amino Acids, 2003Co-Authors: Beatriz Alvarez, Rafael RadiAbstract:Peroxynitrite, the product of the fast reaction between nitric oxide ((*)NO) and superoxide O(2)(*-) radicals, is an oxidizing and nitrating agent which is able to traverse biological membranes. The reaction of Peroxynitrite with proteins occurs through three possible pathways. First, Peroxynitrite reacts directly with cysteine, methionine and tryptophan residues. Second, Peroxynitrite reacts fast with transition metal centers and selenium-containing amino acids. Third, secondary free radicals arising from Peroxynitrite homolysis such as hydroxyl and nitrogen dioxide, and the carbonate radical formed in the presence of carbon dioxide, react with protein moieties too. Nitration of tyrosine residues is being recognized as a marker of the contribution of nitric oxide to oxidative damage. Peroxynitrite-dependent tyrosine nitration is likely to occur through the initial reaction of Peroxynitrite with carbon dioxide or metal centers leading to secondary nitrating species. The preferential protein targets of Peroxynitrite and the role of proteins in Peroxynitrite detoxifying pathways are discussed.
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Oxidation of ubiquinol by Peroxynitrite: implications for protection of mitochondria against nitrosative damage
Biochemical Journal, 2000Co-Authors: Francisco SchÖpfer, Maria Cecilia Carreras, A. Boveris, Enrique Cadenas, Natalia RiobÓ, Beatriz Alvarez, Rafael Radi, Juan José PoderosoAbstract:A major pathway of nitric oxide utilization in mitochondria is its conversion to Peroxynitrite, a species involved in biomolecule damage via oxidation, hydroxylation and nitration reactions. In the present study the potential role of mitochondrial ubiquinol in protecting against Peroxynitrite-mediated damage is examined and the requirements of the mitochondrial redox status that support this function of ubiquinol are established. (1) Absorption and EPR spectroscopy studies revealed that the reactions involved in the ubiquinol/Peroxynitrite interaction were first-order in Peroxynitrite and zero-order in ubiquinol, in agreement with the rate-limiting formation of a reactive intermediate formed during the isomerization of Peroxynitrite to nitrate. Ubiquinol oxidation occurred in one-electron transfer steps as indicated by the formation of ubisemiquinone. (2) Peroxynitrite promoted, in a concentration-dependent manner, the formation of superoxide anion by mitochondrial membranes. (3) Ubiquinol protected against Peroxynitrite-mediated nitration of tyrosine residues in albumin and mitochondrial membranes, as suggested by experimental models, entailing either addition of ubiquinol or expansion of the mitochondrial ubiquinol pool caused by selective inhibitors of complexes III and IV. (4) Increase in membrane-bound ubiquinol partially prevented the loss of mitochondrial respiratory function induced by Peroxynitrite. These findings are analysed in terms of the redox transitions of ubiquinone linked to both nitrogen-centred radical scavenging and oxygen-centred radical production. It may be concluded that the reaction of mitochondrial ubiquinol with Peroxynitrite is part of a complex regulatory mechanism with implications for mitochondrial function and integrity.
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The Biological Chemistry of Peroxynitrite
Nitric Oxide, 2000Co-Authors: Rafael Radi, Beatriz Alvarez, Gerardo Ferrer-sueta, Ana Denicola, Homero RubboAbstract:Publisher Summary This chapter provides a comprehensive overview of the physical and biological chemistry of Peroxynitrite. A foundation is provided to rationalize the biological fate and actions of Peroxynitrite and the strategies for preventing Peroxynitrite-dependent biological damage and pathology. Peroxynitrite anion is formed in vivo as a result of the diffusion controlled reaction between nitric oxide (NO) and superoxide anion radicals. The anion and its conjugated acid, peroxynitrous acid, are strong oxidant species that cause molecular damage in a variety of pathophysiological conditions. Peroxynitrite reacts fast with a number of biological targets, including thiols, metalloproteins, and carbon dioxide, or more slowly decomposes to hydroxyl and nitrogen dioxide radicals by proton-catalyzed homolysis. Carbon dioxide accounts for a significant fraction of Peroxynitrite consumption and leads to the secondary formation of carbonate and nitrogen dioxide radicals. At the molecular level, the predominant outcome of Peroxynitrite reactions in vivo is one or two electron oxidations and nitrations. Peroxynitrite can diffuse through tissue compartments, being able to cross biomembranes by both passive diffusion and anion channels. Thus, although the biological half-life of Peroxynitrite is short, it is sufficient for Peroxynitrite to diffuse a couple of cell diameters and cause biological effects distant from its site of production.
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Peroxynitrite-dependent tryptophan nitration.
Chemical research in toxicology, 1996Co-Authors: Beatriz Alvarez, Bruce A. Freeman, Homero Rubbo, Marion Kirk, Stephen Barnes, Rafael RadiAbstract:Peroxynitrite (ONOO-), the reaction product of superoxide (O2•-) and nitric oxide (•NO), nitrates tyrosine and other phenolics. We report herein that tryptophan is also nitrated by Peroxynitrite in the absence of transition metals to one predominant isomer of nitrotryptophan, as determined from spectral characteristics and liquid chromatography−mass spectrometry analysis. At high Peroxynitrite to tryptophan ratios, other oxidation products were detected as well. The amount of nitrotryptophan formed from Peroxynitrite increased at acidic pH, with an apparent pKa of 7.8. High concentrations of Fe3+-EDTA were required to enhance Peroxynitrite-induced nitrotryptophan formation, while addition of up to 15 μM Cu/Zn superoxide dismutase had a minimal effect on tryptophan nitration. Cysteine, ascorbate, and methionine decreased nitrotryptophan yield to an extent similar to that predicted by their reaction rates with ground-state Peroxynitrite, and typical hydroxyl radical scavengers partially inhibited nitration....
