Formation of Nitrating and Chlorinating Species by Reaction of Nitrite with Hypochlorous Acid

Detection of 3-nitrotyrosine has served as an in vivo marker for the production of the cytotoxic species peroxynitrite (ONOO−). We show here that reaction of nitrite (NO−2), the autoxidation product of nitric oxide (·NO), with hypochlorous acid (HOCl) forms reactive intermediate species that are also capable of nitrating phenolic substrates such as tyrosine and 4-hydroxyphenylacetic acid, with maximum yields obtained at physiological pH. Monitoring the reaction of NO−2 with HOCl by continuous flow photodiode array spectrophotometry indicates the formation of a transient species with spectral characteristics similar to those of nitryl chloride (Cl-NO2). Reaction of synthetic Cl-NO2 with N-acetyl-L-tyrosine results in the formation of 3-chlorotyrosine and 3-nitrotyrosine in ratios that are similar to those obtained by the NO−2/HOCl reaction (4:1). Tyrosine residues in bovine serum albumin are also nitrated and chlorinated by NO−2/HOCl and synthetic Cl-NO2. The reaction of N-acetyl-L-tyrosine with NO−2/HOCl or authentic Cl-NO2 also produces dityrosine, suggesting that free radical intermediates are involved in the reaction mechanism. Our data indicate that while chlorination reactions of Cl-NO2 are mediated by direct electrophilic addition to the aromatic ring, a free radical mechanism appears to be operative in nitrations mediated by NO−2/HOCl or Cl-NO2, probably involving the combination of nitrogen dioxide (·NO2) and tyrosyl radical. We propose that NO−2 reacts with HOCl by Cl+ transfer to form both cis- and trans-chlorine nitrite (Cl-ONO) and Cl-NO2 as intermediates that modify tyrosine by either direct reaction or after decomposition to reactive free and solvent-caged Cl· and ·NO2 as reactive species. Formation of Cl-NO2 and/or Cl-ONO in vivo may represent previously unrecognized mediators of inflammation-mediated protein modification and tissue injury, and offers an additional mechanism of tyrosine nitration independent of ONOO−. Detection of 3-nitrotyrosine has served as an in vivo marker for the production of the cytotoxic species peroxynitrite (ONOO−). We show here that reaction of nitrite (NO−2), the autoxidation product of nitric oxide (·NO), with hypochlorous acid (HOCl) forms reactive intermediate species that are also capable of nitrating phenolic substrates such as tyrosine and 4-hydroxyphenylacetic acid, with maximum yields obtained at physiological pH. Monitoring the reaction of NO−2 with HOCl by continuous flow photodiode array spectrophotometry indicates the formation of a transient species with spectral characteristics similar to those of nitryl chloride (Cl-NO2). Reaction of synthetic Cl-NO2 with N-acetyl-L-tyrosine results in the formation of 3-chlorotyrosine and 3-nitrotyrosine in ratios that are similar to those obtained by the NO−2/HOCl reaction (4:1). Tyrosine residues in bovine serum albumin are also nitrated and chlorinated by NO−2/HOCl and synthetic Cl-NO2. The reaction of N-acetyl-L-tyrosine with NO−2/HOCl or authentic Cl-NO2 also produces dityrosine, suggesting that free radical intermediates are involved in the reaction mechanism. Our data indicate that while chlorination reactions of Cl-NO2 are mediated by direct electrophilic addition to the aromatic ring, a free radical mechanism appears to be operative in nitrations mediated by NO−2/HOCl or Cl-NO2, probably involving the combination of nitrogen dioxide (·NO2) and tyrosyl radical. We propose that NO−2 reacts with HOCl by Cl+ transfer to form both cis- and trans-chlorine nitrite (Cl-ONO) and Cl-NO2 as intermediates that modify tyrosine by either direct reaction or after decomposition to reactive free and solvent-caged Cl· and ·NO2 as reactive species. Formation of Cl-NO2 and/or Cl-ONO in vivo may represent previously unrecognized mediators of inflammation-mediated protein modification and tissue injury, and offers an additional mechanism of tyrosine nitration independent of ONOO−. INTRODUCTIONNitrogen monoxide (nitric oxide, ·NO) 1The abbreviations used are: ·NOnitric oxideO2superoxideHOClhypochlorous acidNO−2nitriteONOO−peroxynitriteONOOHperoxynitrous acidNO−3nitrateNO2-Tyr3-nitrotyrosineCl-Tyr3-chlorotyrosineHPA4-hydroxyphenylacetic acidNO2-HPA3-nitro-4-hydroxyphenylacetic acidCl-HPA3-chloro-4-hydroxyphenylacetic acidCl-PhechlorophenylalanineNATN-acetyl-L-tyrosineNAPN-acetyl-L-phenylalanineMPA4-methoxyphenylacetic acidCl-NO2nitryl chlorideCl-ONOchlorine nitriteROSreactive oxygen speciesRNSreactive nitrogen speciesHPLChigh pressure liquid chromatographyPDAphotodiode array. is produced by a variety of cells through the activity of constitutive and inducible forms of nitric oxide synthase (1Knowles R.G. Moncada S. Biochem. J. 1994; 298: 249-258Google Scholar). ·NO is an important endogenous mediator in such diverse biochemical and physiological processes as neurotransmission, smooth muscle relaxation, platelet aggregation and adhesion, macrophage-mediated cytotoxicity, and learning and memory (2Moncada S. Palmer R.M.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142Google Scholar, 3Schmidt H.H. Walter U. Cell. 1994; 78: 919-925Google Scholar). Although basal levels of free ·NO are normally quite low (nanomolar), local ·NO concentrations have been shown to increase to levels ranging from 4 to 30 µM under pathologic conditions (4Hooper D.C. Ohnishi S.T. Kean R. Numagami Y. Dietzschold B. Koprowski H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5312-5316Google Scholar, 5Malinski T. Zhang Z.G. Chopp M. J. Cerebral Blood Flow Metab. 1993; 13: 355-358Google Scholar).·NO reacts at a near diffusion-controlled rate with superoxide (O2) (k = 6.7 × 109M−1 s−1) (6Huie R.E. Padmaja S. Free Rad. Res. Commun. 1993; 18: 195-199Google Scholar) to form the cytotoxic species peroxynitrite (ONOO−). The formation of ONOO− is thought to be responsible, at least in part, for the observed toxicity associated with ·NO (7Brunelli L. Crow J.P. Beckman J.S. Arch. Biochem. Biophys. 1995; 316: 327-334Google Scholar, 8Lipton S.A. Choi Y.-B. Pan Z.-H. Lei S.Z. Chen H.-S.V. Sucher N.J. Loscalzo J. Singel D.J. Stamler J.S. Nature. 1993; 364: 626-632Google Scholar). At physiological pH the protonated form of ONOO−, peroxynitrous acid (ONOOH) (pKa = 6.8), is highly unstable and rapidly decomposes to nitrate (NO−3). ONOOH is thought to 1) react directly with biological molecules via a vibrationally excited intermediate (ONOOH*), 2) decompose by homolytic dissociation to form nitrogen dioxide (·NO2) and the hydroxyl radical (·OH), or 3) by heterolytic dissociation to form the nitryl cation (nitronium ion, NO+2) (reviewed in Ref. 9Pryor W.A. Squadrito G.L. Am. J. Physiol. 1995; 268: L699-L722Google Scholar). ONOO−/ONOOH reacts with proteins, leading to the oxidation of cysteine, methionine, and tryptophan residues, and can induce protein carbonyl formation and nonspecific fragmentation (10Ischiropoulos H. Al-Mehdi A.B. FEBS Lett. 1995; 364: 279-282Google Scholar, 11Pryor W.A. Jin X. Squadrito G.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11173-11177Google Scholar, 12Radi R. Beckman J.S. Bush K.M. Freeman B.A. J. Biol. Chem. 1991; 266: 4244-4250Google Scholar). In addition, ONOO−/ONOOH can react readily with phenolic compounds to form nitrated, hydroxylated, and dimerized products (13Halfpenny E. Robinson P.L. J. Chem. Soc. (Lond.). 1952; : 939-946Google Scholar, 14Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1620-1624Google Scholar, 15Van der Vliet A. Eiserich J.P. O'Neill C.A. Halliwell B. Cross C.E. Arch. Biochem. Biophys. 1995; 319: 341-349Google Scholar, 16Beckman J.S. Ischiropoulos H. Zhu L. van der Woerd M. Smith C. Chen J. Harrison J. Martin J.C. Tsai M. Arch. Biochem. Biophys. 1992; 298: 438-445Google Scholar, 17Van der Vliet A. O'Neill C.A. Halliwell B. Cross C.E. Kaur H. FEBS Lett. 1994; 339: 89-92Google Scholar), and nitration of free tyrosine, or tyrosine in proteins, has served as a “marker” and “index” of ONOO− formation in vivo.Based upon tyrosine nitration assays and the formation of “peroxynitrite-specific” luminescence, stimulated macrophages (18Ischiropoulos H. Zhu L. Beckman J.S. Arch. Biochem. Biophys. 1992; 298: 446-451Google Scholar), neutrophils (19Carreras M.C. Pargament G.A. Catz S.D. Poderoso J.J. Boveris A. FEBS Lett. 1994; 341: 65-68Google Scholar), and endothelial cells (20Kooy N.W. Royall J.A. Arch. Biochem. Biophys. 1994; 310: 352-359Google Scholar) have been proposed to form significant quantities of ONOO− in vitro. In fact, the detection of 3-nitrotyrosine (NO2-Tyr) in a variety of pathologic conditions in vivo, such as inflammatory lung disease (21Haddad I.Y. Pataki C. Beckman J.S. S. J. 1994; Scholar), J.S. Chen J. Biochem. 1994; Scholar), and H. Halliwell B. FEBS Lett. 1994; Scholar), has been to ONOO− in of direct for the production of ONOO− in biological is formation in vivo is G.L. W.A. Chem. Biol. 1995; inflammatory reactive oxygen species are produced from cells J. 1995; 43: Scholar). stimulated neutrophils and macrophages significant levels of superoxide (O2) and as a of the of the and Scholar). In the of of the that is produced under conditions is to the hypochlorous acid (HOCl) by the of as shown in Reaction produced from neutrophils has been shown to react with and and tyrosine to form and 3-chlorotyrosine J. Biol. Chem. 1995; Scholar, S.T. Scholar), the has been proposed to as a marker of HOCl production in vivo FEBS Lett. addition to macrophages R. J.S. Scholar) and neutrophils (19Carreras M.C. Pargament G.A. Catz S.D. Poderoso J.J. Boveris A. FEBS Lett. 1994; 341: 65-68Google Scholar) can also of ·NO through the of inducible nitric oxide the of neutrophils to ·NO is S. A. J. Biol. 1995; Scholar). ·NO can react with biological thought to and protein J.S. Singel D.J. Loscalzo J. 1992; Scholar, M. Y. J. Biol. Chem. 1994; Scholar, J.P. J. van der Vliet A. Cross C.E. Halliwell B. Biochem. J. 1995; 310: Scholar). ·NO can also react with in to nitrite via a mechanism thought to a variety of reactive nitrogen species ·NO2 and R.E. Proc. Natl. Acad. Sci. U. S. A. 1993; Scholar). In fact, NO−2 has been used as a marker of ·NO production in and in vivo and has been shown to concentrations of to 4 µM in from with Palmer R.M.J. Moncada S. 1992; Scholar) and as as µM in B. J. J. D.J. C. Singel D.J. Loscalzo J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1993; Scholar). produced an inflammatory react with a of to form species. the of HOCl with ·NO or NO−2 has been proposed to form species capable of and nitrating substrates FEBS Lett. 1994; Scholar, Y. Biochem. Biol. 1995; to the of with the inflammatory HOCl in an to the species that may be under physiological inflammatory Our results indicate that NO−2 reacts with HOCl to form an intermediate to be nitryl chloride and/or nitrite that is capable of and phenolic compounds We propose that the formation of Cl-NO2 and/or Cl-ONO by reaction a mechanism of inflammation-mediated biological and offers an additional or mechanism of tyrosine nitration independent of ONOO− the of and by inflammatory are in an of the of and that are to at of is to The show that the of and HOCl may be important under inflammatory conditions in We have shown that the autoxidation product of ·NO in biological reacts with HOCl to a species that can and phenolic compounds such as tyrosine, both free and The detection of in a variety of pathologic (21Haddad I.Y. Pataki C. Beckman J.S. S. J. 1994; Scholar, J.S. Chen J. Biochem. 1994; Scholar, H. Halliwell B. FEBS Lett. 1994; Scholar) has been used to indicate the formation of ONOO− in reaction of tyrosine with the products of the NO−2/HOCl reaction also forms results that be as a marker of ONOO− as a marker of of NO−2/HOCl has been thought M. H. J. Am. Chem. Soc. Scholar) that the reaction of NO−2 with HOCl a of an oxygen transfer reaction of mechanism the nitration and chlorination reactions observed in Our data a mechanism involving the formation of reactive nitrating and oxidation of NO−2 by the reactive radical species Cl· and ·NO2 is HOCl is a an in the of to at pH FEBS Lett. 1994; Scholar), is that a oxidation mechanism the for the is J. Chem. Ref. 18: Scholar). In HOCl is a = FEBS Lett. 1994; Scholar) and the of NO−2 to the nitryl cation or an species. In addition to a direct oxidation of NO−2 by a reaction be In fact, to the reaction mechanism previously M. H. J. Am. Chem. Soc. Scholar), and Chem. 1991; Scholar) have that HOCl reacts with NO−2 by Cl+ to the intermediate Cl-NO2, to of the product of the reaction NO−2 and HOCl to be similar to that of authentic Cl-NO2 The of the of the NO−2/HOCl reaction is of J. Chem. Soc. (Lond.). : Scholar) and also indicate the formation of a species. In fact, the transfer of Cl+ to the oxygen in NO−2 is and the transient intermediate species is that both reactions the to under conditions is Cl-ONO can as both the cis- and that the the is with the the J. Chem. 1994; Scholar). can be Cl-ONO and the cis- and is also to be Harrison Martin J.C. van der Woerd M. Beckman J.S. J. Am. Chem. Soc. 1994; Scholar). Cl-ONO can readily to Cl-NO2 J. Chem. Scholar). We propose that intermediate Cl-ONO can in to Cl-NO2 by at least 1) of involving of the to the nitrogen Cl-NO2, or 2) of the in Cl-ONO to form a of solvent-caged Cl· and to either Cl-ONO or by to form Cl-NO2 of the solvent-caged Cl· and ·NO2 can as and in part, the radical involved in the nitration reactions observed in the NO−2/HOCl Cl-NO2 is to be and in cis- and J. Chem. 1994; Scholar), the of Cl-ONO to Cl-NO2 is a that the Cl-NO2. of Cl-ONO to Cl-NO2 is probably the of the the of the to the nitrogen the of is probably the species that to Cl-NO2, to the decomposition of acid the of to nitric acid an of Cl-ONO produces highly reactive species (Cl-NO2). Cl-ONO and the product of Cl-NO2, may both be reactive with nitrating and of Cl-NO2 as have shown that the of the reaction NO−2 and authentic Cl-NO2, or the species react with tyrosine to form and Although of are formation of the of intermediate tyrosyl The nitration of aromatic compounds by is thought to be a electrophilic aromatic is transfer reactions and radical intermediates in J. Am. Chem. Soc. Scholar). reaction mechanism transfer from the aromatic to by radical and the detection of in are to a nitration mechanism involving ·NO2 or the formation of a in the characteristics of the reaction and the reactive nitrating species by the reaction of NO−2 with HOCl is in reactions with the of a of appears capable of nitrating both the of the NO−2/HOCl reaction and synthetic Cl-NO2 to the of NO−2/HOCl and Cl-NO2 to nitrate as the species involved in tyrosine is suggesting that the reaction of Cl-NO2 with and aromatic compounds homolytic processes free radical intermediates H. J. Am. Chem. Soc. 1952; Scholar), probably involving both Cl· and E.A. J. Chem. Soc. (Lond.). : Scholar) have that Cl-NO2 decomposes at by to form and ·NO2 as shown in Reaction decomposition products may be responsible, at least in part, for the and nitrating of Cl-NO2 in We that phenolic nitration mediated by the NO−2/HOCl reaction the nitration reactions observed to be chlorination of aromatic such as appears to be by electrophilic aromatic In chlorination of aromatic compounds by and has been shown to be mediated by an a free radical mechanism J. Chem. Scholar). The increase in the formation of the by reactions of with both NO−2/HOCl and Cl-NO2 the of a mechanism of involving species to HOCl and Cl-NO2 appears to be In fact, the formation of from HOCl and Cl-NO2 can be and the in HOCl is in with in as shown in Reaction The formation of from Cl-NO2 has been proposed to by 1) the of molecules of Cl-NO2 to form Cl· to form and 2) the reaction of Cl-NO2 with E.A. J. Chem. Soc. (Lond.). : Scholar) as shown in Reaction 4 an electrophilic mechanism for chlorination the of a mechanism involving the addition of Cl· to the aromatic be for reactions involving Cl-NO2 or of with of chlorination and nitration have involved species from the decomposition of either Cl-NO2 or as by the of and are Cl-NO2 or Cl-ONO are at In vivo, Cl-NO2 and Cl-ONO be to be produced at that may the direct reaction of either species with biological substrates that are in In Cl-NO2 has been shown to be an for the nitration of aromatic compounds of intermediate C.A. J. Am. Chem. Soc. Scholar). an increase either in the of the aromatic to or in the of the a in the nitrating of Cl-NO2 and a increase in the of chlorinated products J. Chem. Soc. (Lond.). : Scholar). In fact, A. H. A. J. Am. Chem. Soc. 1995; Scholar) the of a the of Cl-NO2, the characteristics of Cl-NO2 those of nitryl reactions involving aromatic substrates such as tyrosine with conditions increase aromatic chlorination by Cl-NO2, suggesting a from to a species with Our data that Cl-NO2 has significant Cl+ in and is of Cl-NO2 that propose that can react directly with tyrosine via transfer to an intermediate radical of to the formation of and NO−2 reaction and is the product by reaction mechanism is to the nitration of phenolic substrates by J. Am. Chem. Soc. Scholar). of the radical and oxidation of NO−2 by Cl· of = J. Chem. Ref. 18: results in the formation of tyrosyl radical and ·NO2 reaction radical and ·NO2 can rapidly to (k = × 109M−1 W.A. H. J. Arch. Biochem. Biophys. and formation can be by the combination of tyrosyl reactions and proposed mechanism that the yields of the tyrosine modification products be the of dityrosine, with the data The proposed reaction also the of radical intermediates in the nitration of phenolic compounds by Cl-NO2, as by for the direct reactions of tyrosine with Cl-NO2. The direct reaction of Cl-NO2 with tyrosine by transfer from tyrosine to in an intermediate radical to the formation of and NO−2 as of the from the Cl· to NO−2 to ·NO2 can with tyrosyl radical to formation can be by the combination of tyrosyl Cl-ONO is a transient intermediate in the formation of the reactive species Cl-NO2 of the of NO−2/HOCl may be to to a proposed mechanism of ONOOH W.A. Squadrito G.L. Am. J. Physiol. 1995; 268: L699-L722Google Scholar, J.J. W.A. Ischiropoulos H. Beckman J.S. Chem. Res. 1992; Scholar), a vibrationally excited intermediate from may be to Cl-NO2 and to nitration and chlorination of tyrosine by direct The reaction propose for Cl-NO2 and Cl-ONO are to those for both direct and reactions with substrates can S. Chem. 1995; Scholar). of the reaction and is in to of the proposed and and of neutrophils at of tissue injury, leading to the formation of HOCl and is an of Our data that the reaction of HOCl with from ·NO produced by R. J.S. Scholar), endothelial cells (2Moncada S. Palmer R.M.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142Google Scholar), or cells B. J. J. D.J. C. Singel D.J. Loscalzo J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1993; Scholar), may be a operative in tissue at of Cl-NO2 is in vivo and is capable of nitrating tyrosine residues, may a for reaction is in of inflammatory lung (21Haddad I.Y. Pataki C. Beckman J.S. S. J. 1994; Scholar), J.S. Chen J. Biochem. 1994; Scholar), and H. Halliwell B. FEBS Lett. 1994; Scholar), a previously to ONOO− In fact, levels of NO−2 have been observed in similar Palmer R.M.J. Moncada S. 1992; Scholar, B. Loscalzo J. Stamler J.S. Am. J. 1994; Scholar, Moncada S. S. 1990; Scholar) and the of in have that the that HOCl is a of from J. Scholar), as as inflammatory lung and of tissue A. J. 1994; Scholar), the of HOCl in the of of We that tyrosine nitration by Cl-ONO and/or Cl-NO2, by the reaction of NO−2 with an important and additional mechanism for inflammation-mediated tyrosine nitration in vivo, independent of ONOO− indicate that NO−2 may be an marker of ·NO production by neutrophils or at of is by reaction with produced of ·NO production in and of with and as by is a NO−2 has been shown to the activity of mechanism mediated by direct reaction of species Y. Biochem. Biol. 1995; Scholar, Free Biol. 1993; Scholar). Our that the reaction Cl-NO2, is a species that may as an in reaction acid, a product by oxidation of by S.T. S. Scholar), and NO−2 and/or can be In fact, Reaction may represent a mechanism by react with NO−2 to species capable of biological reaction represent an important mechanism and a for inflammation-mediated tissue here that NO−2 and HOCl react to form the reactive intermediates Cl-NO2 and/or species that are capable of and phenolic compounds such as Our data that is a marker of ONOO− formation in vivo and that Cl-NO2 and Cl-ONO may be important and previously produced at of in of nitration of to nitration by the nitryl under the of an species to nitration observed with or in INTRODUCTIONNitrogen monoxide (nitric oxide, ·NO) 1The abbreviations used are: ·NOnitric oxideO2superoxideHOClhypochlorous acidNO−2nitriteONOO−peroxynitriteONOOHperoxynitrous acidNO−3nitrateNO2-Tyr3-nitrotyrosineCl-Tyr3-chlorotyrosineHPA4-hydroxyphenylacetic acidNO2-HPA3-nitro-4-hydroxyphenylacetic acidCl-HPA3-chloro-4-hydroxyphenylacetic acidCl-PhechlorophenylalanineNATN-acetyl-L-tyrosineNAPN-acetyl-L-phenylalanineMPA4-methoxyphenylacetic acidCl-NO2nitryl chlorideCl-ONOchlorine nitriteROSreactive oxygen speciesRNSreactive nitrogen speciesHPLChigh pressure liquid chromatographyPDAphotodiode array. is produced by a variety of cells through the activity of constitutive and inducible forms of nitric oxide synthase (1Knowles R.G. Moncada S. Biochem. J. 1994; 298: 249-258Google Scholar). ·NO is an important endogenous mediator in such diverse biochemical and physiological processes as neurotransmission, smooth muscle relaxation, platelet aggregation and adhesion, macrophage-mediated cytotoxicity, and learning and memory (2Moncada S. Palmer R.M.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142Google Scholar, 3Schmidt H.H. Walter U. Cell. 1994; 78: 919-925Google Scholar). Although basal levels of free ·NO are normally quite low (nanomolar), local ·NO concentrations have been shown to increase to levels ranging from 4 to 30 µM under pathologic conditions (4Hooper D.C. Ohnishi S.T. Kean R. Numagami Y. Dietzschold B. Koprowski H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5312-5316Google Scholar, 5Malinski T. Zhang Z.G. Chopp M. J. Cerebral Blood Flow Metab. 1993; 13: 355-358Google Scholar).·NO reacts at a near diffusion-controlled rate with superoxide (O2) (k = 6.7 × 109M−1 s−1) (6Huie R.E. Padmaja S. Free Rad. Res. Commun. 1993; 18: 195-199Google Scholar) to form the cytotoxic species peroxynitrite (ONOO−). The formation of ONOO− is thought to be responsible, at least in part, for the observed toxicity associated with ·NO (7Brunelli L. Crow J.P. Beckman J.S. Arch. Biochem. Biophys. 1995; 316: 327-334Google Scholar, 8Lipton S.A. Choi Y.-B. Pan Z.-H. Lei S.Z. Chen H.-S.V. Sucher N.J. Loscalzo J. Singel D.J. Stamler J.S. Nature. 1993; 364: 626-632Google Scholar). At physiological pH the protonated form of ONOO−, peroxynitrous acid (ONOOH) (pKa = 6.8), is highly unstable and rapidly decomposes to nitrate (NO−3). ONOOH is thought to 1) react directly with biological molecules via a vibrationally excited intermediate (ONOOH*), 2) decompose by homolytic dissociation to form nitrogen dioxide (·NO2) and the hydroxyl radical (·OH), or 3) by heterolytic dissociation to form the nitryl cation (nitronium ion, NO+2) (reviewed in Ref. 9Pryor W.A. Squadrito G.L. Am. J. Physiol. 1995; 268: L699-L722Google Scholar). ONOO−/ONOOH reacts with proteins, leading to the oxidation of cysteine, methionine, and tryptophan residues, and can induce protein carbonyl formation and nonspecific fragmentation (10Ischiropoulos H. Al-Mehdi A.B. FEBS Lett. 1995; 364: 279-282Google Scholar, 11Pryor W.A. Jin X. Squadrito G.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11173-11177Google Scholar, 12Radi R. Beckman J.S. Bush K.M. Freeman B.A. J. Biol. Chem. 1991; 266: 4244-4250Google Scholar). In addition, ONOO−/ONOOH can react readily with phenolic compounds to form nitrated, hydroxylated, and dimerized products (13Halfpenny E. Robinson P.L. J. Chem. Soc. (Lond.). 1952; : 939-946Google Scholar, 14Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1620-1624Google Scholar, 15Van der Vliet A. Eiserich J.P. O'Neill C.A. Halliwell B. Cross C.E. Arch. Biochem. Biophys. 1995; 319: 341-349Google Scholar, 16Beckman J.S. Ischiropoulos H. Zhu L. van der Woerd M. Smith C. Chen J. Harrison J. Martin J.C. Tsai M. Arch. Biochem. Biophys. 1992; 298: 438-445Google Scholar, 17Van der Vliet A. O'Neill C.A. Halliwell B. Cross C.E. Kaur H. FEBS Lett. 1994; 339: 89-92Google Scholar), and nitration of free tyrosine, or tyrosine in proteins, has served as a “marker” and “index” of ONOO− formation in vivo.Based upon tyrosine nitration assays and the formation of “peroxynitrite-specific” luminescence, stimulated macrophages (18Ischiropoulos H. Zhu L. Beckman J.S. Arch. Biochem. Biophys. 1992; 298: 446-451Google Scholar), neutrophils (19Carreras M.C. Pargament G.A. Catz S.D. Poderoso J.J. Boveris A. FEBS Lett. 1994; 341: 65-68Google Scholar), and endothelial cells (20Kooy N.W. Royall J.A. Arch. Biochem. Biophys. 1994; 310: 352-359Google Scholar) have been proposed to form significant quantities of ONOO− in vitro. In fact, the detection of 3-nitrotyrosine (NO2-Tyr) in a variety of pathologic conditions in vivo, such as inflammatory lung disease (21Haddad I.Y. Pataki C. Beckman J.S. S. J. 1994; Scholar), J.S. Chen J. Biochem. 1994; Scholar), and H. Halliwell B. FEBS Lett. 1994; Scholar), has been to ONOO− in of direct for the production of ONOO− in biological is formation in vivo is G.L. W.A. Chem. Biol. 1995; inflammatory reactive oxygen species are produced from cells J. 1995; 43: Scholar). stimulated neutrophils and macrophages significant levels of superoxide (O2) and as a of the of the and Scholar). In the of of the that is produced under conditions is to the hypochlorous acid (HOCl) by the of as shown in Reaction produced from neutrophils has been shown to react with and and tyrosine to form and 3-chlorotyrosine J. Biol. Chem. 1995; Scholar, S.T. Scholar), the has been proposed to as a marker of HOCl production in vivo FEBS Lett. addition to macrophages R. J.S. Scholar) and neutrophils (19Carreras M.C. Pargament G.A. Catz S.D. Poderoso J.J. Boveris A. FEBS Lett. 1994; 341: 65-68Google Scholar) can also of ·NO through the of inducible nitric oxide the of neutrophils to ·NO is S. A. J. Biol. 1995; Scholar). ·NO can react with biological thought to and protein J.S. Singel D.J. Loscalzo J. 1992; Scholar, M. Y. J. Biol. Chem. 1994; Scholar, J.P. J. van der Vliet A. Cross C.E. Halliwell B. Biochem. J. 1995; 310: Scholar). ·NO can also react with in to nitrite via a mechanism thought to a variety of reactive nitrogen species ·NO2 and R.E. Proc. Natl. Acad. Sci. U. S. A. 1993; Scholar). In fact, NO−2 has been used as a marker of ·NO production in and in vivo and has been shown to concentrations of to 4 µM in from with Palmer R.M.J. Moncada S. 1992; Scholar) and as as µM in B. J. J. D.J. C. Singel D.J. Loscalzo J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1993; Scholar). produced an inflammatory react with a of to form species. the of HOCl with ·NO or NO−2 has been proposed to form species capable of and nitrating substrates FEBS Lett. 1994; Scholar, Y. Biochem. Biol. 1995; to the of with the inflammatory HOCl in an to the species that may be under physiological inflammatory Our results indicate that NO−2 reacts with HOCl to form an intermediate to be nitryl chloride and/or nitrite that is capable of and phenolic compounds We propose that the formation of Cl-NO2 and/or Cl-ONO by reaction a mechanism of inflammation-mediated biological and offers an additional or mechanism of tyrosine nitration independent of ONOO−