Carbon Dioxide Enhancement of Peroxynitrite-Mediated Protein Tyrosine Nitration

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Vol. 333, No. 1, September 1, pp. 42–48, 1996 Article No. 0362

Carbon Dioxide Enhancement of Peroxynitrite-Mediated Protein Tyrosine Nitration Andrew Gow,* Daniel Duran,* Stephen R. Thom,*,† and Harry Ischiropoulos*,‡,1 *Institute for Environmental Medicine, †Department of Emergency Medicine and ‡Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Received March 29, 1996, and in revised form May 28, 1996

Production of reactive species has been associated with tissue injury in diverse human disorders and experimental models of disease. Peroxynitrite is a strong oxidant with multiple pathways of reactivity. One protein modification reaction that may be specific to peroxynitrite is the nitration of the ortho position of tyrosine residues and nitrotyrosine has been used as a marker for peroxynitrite-mediated oxidative stress. Nitrotyrosine was formed when peroxynitrite was reacted at physiological pH with fatty acid-free bovine serum albumin or with human plasma proteins. Nitrotyrosine was not formed when proteins were incubated with nitric oxide, nitrogen dioxide, or nitric oxide plus hydrogen peroxide in the presence of ferrous iron or ferrihorseradish peroxidase. Low-molecularweight molecules such as uric acid, ascorbate, and sulfhydryls inhibited protein tyrosine nitration in the absence of bicarbonate. Addition of bicarbonate catalytically enhanced the yield of nitration and overcame the inhibition of these antioxidants. Bicarbonate/CO2 enhanced the yield of protein nitrotyrosine in a concentration-dependent manner. Catalysis of nitration is achieved by the interaction of CO2 with the peroxynitrite anion. A mechanism is proposed involving an ONOO(O)CO0 intermediate, which readily nitrates tyrosine residues in a non-radical-dependent manner. Thus, peroxynitrite nitrates tyrosine residues by a mechanism that is catalyzed by CO2 under normal physiological conditions. q 1996 Academic Press, Inc.

Peroxynitrite2 is formed by the rapid reaction (k Å 6.7–4 1 109 M01 s01) of nitric oxide with superoxide (1, 1 To whom correspondence should be addressed at Institute for Environmental Medicine, University of Pennsylvania, 1 John Morgan Building, 3620 Hamilton Walk, Philadelphia, PA 19104-6068. Fax: (215) 898-0868. 2 The IUPAC recommended nomenclature for peroxynitrite is oxoperoxonitrate (10), for peroxynitrous acid is hydrogen oxoperoxonitrate, and for nitric oxide is nitrogen monoxide.

2). Rat alveolar macrophages, human neutrophils, and bovine aorta cells have been shown to generate peroxynitrite (3–6). Peroxynitrite is capable of oxidizing lipids, thiols, deoxyribose, and a number of organic molecules (7–10). We have previously shown that the major product from the spontaneous reaction of peroxynitrite with proteins is nitrotyrosine (11). Both the lowmolecular-weight metal catalyst, Fe3/ –EDTA, and metal-containing enzymes, such as Cu,Zn superoxide dismutase, catalyze the nitration of several phenolic compounds as well as protein tyrosine (11, 12). Using antibodies against nitrotyrosine and other analytical techniques, protein nitration has been detected in human coronary artery atherosclerotic lesions (13), neonatal lung injury (14, 15), 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine, NMDA, and 3-nitropropionic acidmediated neuronal toxicity (16, 17), multiple sclerosis (18), endotoxemia (19, 20), ischemic lung injury (21), and in inflammatory cell-mediated diseases (22–25). At physiological pH and 257C peroxynitrite becomes protonated to form peroxynitrous acid which rapidly isomerizes to form nitrate, k Å 1.3 s01 (26). Both the peroxynitrite anion and peroxynitrous acid are highly reactive with sulfhydryl groups (8), thioethers (27), and ascorbate (28). All of these reactions represent alternative pathways of reactivity for peroxynitrite in addition to nitration of tyrosine residues in the cellular environment. The purpose of this paper was to investigate the mechanism by which nitration of protein tyrosine residues occurs within the physiological environment. A major constituent of the physiological environment, which reacts with peroxynitrite, is bicarbonate/ CO2 . Because bicarbonate is present at a concentration of approximately 25 mM in plasma, its involvement in peroxynitrite-mediated reactions may be significant. Bicarbonate/CO2 has been shown to inhibit the bactericidal activity of peroxynitrite with a half-maximal effective concentration of 2.5 mM (29). The reaction of bicarbonate with peroxynitrite was also reported to en-

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hance luminol chemiluminescence possibly via the formation of a ONOO(CO20) intermediate (30). Recently, a rapid reaction between the peroxynitrite anion and CO2 with a rate constant of approximately 2.9 1 104 01 01 M s has been reported (31). This rate constant is high enough to imply that CO2 should effectively react with peroxynitrite anion under physiological conditions. In this paper we examined the reactive pathway of peroxynitrite with HCO30/CO2 and its role in protein tyrosine nitration.

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nitrotyrosine antibody at a concentration of 1 mg/ml in 1% gelatin for 3 hours when chromogenic substrates were used for visualization, or 0.5 mg/ml anti-nitrotyrosine antibody for luminol-enhanced chemiluminescence detection (ECL). For both detection methods an antirabbit goat IgG conjugated to horseradish peroxidase at 1:1000 and 1:5000 dilution in 1% gelatin in TBS, respectively, was incubated for 2 h and the blot was exposed to either 4-chloro-1-naphthol (Bio-Rad) or the chemiluminescent solution (Amersham, Arlington Heights, IL). The binding of the antibody was completely inhibited by 10 mM nitrotyrosine but not by 10 mM of aminotyrosine, phosphotyrosine, methyltyrosine, and tyrosine.

RESULTS MATERIALS AND METHODS Exposure of proteins and plasma to nitric oxide and peroxynitrite. Fatty acid-free bovine serum albumin (4 mg/ml) was reacted with nitric oxide or peroxynitrite in 100 mM potassium phosphate buffer, containing 0.9% NaCl and 100 mM of the metal chelator diethylenepentaacetic acid (dtpa),3 pH 7.4. We have previously shown that the dtpa–metal complex does not catalyze the peroxynitrite-mediated nitration of tyrosine and other phenolics (11). Fresh plasma was collected from adult volunteers in heparinized tubes. The plasma was centrifuged at 10,000g and diluted with 100 mM potassium phosphate buffer, pH 7.4, to obtain aliquots with 1 mg protein/ml. These samples were incubated with either nitric oxide or peroxynitrite. Nitric oxide gas was generated from the acidification of KNO2 in the presence of ferrous iron under a nitrogen atmosphere. The nitric oxide gas was passed through a gas washer containing 1 M NaOH and was collected under nitrogen in deionized water that had passed through a chelex 100 metal-chelating column. The concentration of nitric oxide in solution was measured by a nitric oxide-selective electrochemical sensor (World Precision Instruments, Sarasota, FL) as described previously (33). We also exposed BSA and plasma to nitric oxide donors diethylamine–NO, spermine–NO (Cayman Chemical Co., Ann Arbor, MI), and S-nitroso-N-acetylpenicillamine (Molecular Probes, Eugene, OR). Peroxynitrite was synthesized as described previously (3) or by utilizing 3-morpholinosydnonimine (SIN-1) which decomposes to release nitric oxide as well as superoxide (32). Protein and plasma sulfhydryl levels were measured as described previously (8). Nitrotyrosine was detected by its pH-dependent absorbance. At pH 11.5 the absorption maxima of 4 mg/ml BSA reacted with 2 mM peroxynitrite was at 430 nm (e430 nm Å 4400 M01 cm01) and at pH 6.0 the absorption maxima was at 348 nm. Absorption spectra were monitored using a UV–visible diode-array spectrophotometer (Hewlet–Packard, Wilmington, DE) equipped with a thermostat and stirring module. Bicarbonate was added to the reaction mixture, prior to the addition of peroxynitrite, from a 250 mM stock. Bicarbonate stock was prepared by adding solid NaHCO3 to water that had been passed through a chelex column and vigorously boiled for 10 min. The pH was adjusted by the addition of 3 N NaOH or 3 N HCl prior to the addition of peroxynitrite such that the pH after addition was as desired. Stopped-flow measurements were performed in a Hi-Tech Scientific spectrophotometer as described previously (11, 12, 32). Western blot analysis. Affinity-purified polyclonal anti-nitrotyrosine antibody was obtained from Dr. J. S. Beckman and Ya ZouYe, University of Alabama at Birmingham. Plasma proteins were separated using 10% SDS–PAGE and transferred overnight to nitrocellulose by electroblot. After blocking with 2% gelatin (Bio-Rad) in Tris-buffered saline (TBS), the nitrocellulose was exposed to the anti-

3 Abbreviations used: dtpa, diethylenepentaacetic acid; BSA, bovine serum albumin; TBS, Tris-buffered saline; ECL, luminol-enhanced chemiluminescence detection; SIN-1, 3-morpholinosydnonimine; HRP, ferrihorseradish peroxidase.

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Detection of nitrotyrosine in peroxynitrite-reacted plasma proteins. Nitrotyrosine was detected immunochemically using an affinity-purified polyclonal antibody directed against nitrotyrosine. Human plasma from three different individuals was reacted with either chemically synthesized peroxynitrite (300 mM), or superoxide and nitric oxide generated simultaneously by 1 mM SIN-1 for 3 h. The rate of peroxynitrite production by 1 mM SIN-1, as measured by dihydrorhodamine 123 oxidation, was approximately 10 mM min01. As shown in Fig. 1, nitrotyrosine was formed in nearly the same plasma proteins in all samples reacted with either peroxynitrite or peroxynitrite generated by SIN-1. Nitrotyrosine is a specific product of peroxynitrite reaction with proteins in plasma. Although tyrosine nitration requires the presence of nitric oxide, nitric oxide or nitrogen oxides are not responsible for the nitration of plasma proteins. Nitrotyrosine was not detected when plasma was exposed to a flux of nitric oxide at 17 mM min01 for 3 h at 377C, or to a bolus addition of up to 0.5 mM. Nitrotyrosine was also not produced when plasma samples were incubated with nitrogen dioxide or other nitrogen oxides formed during the aerobic oxidation of nitric oxide, or when plasma proteins were incubated with combinations of authentic nitric oxide, or nitric oxide donors, plus 100 mM H2O2 , 100 mM H2O2 with 10 mM Fe2/, or 100 mM H2O2 with 25 mg ferrihorseradish peroxidase (HRP) (Fig. 1). Using the ECL method (lower limit of detection is 10 pg nitrotyrosine), nitrotyrosine was absent in any of the above conditions except after samples were exposed to peroxynitrite or to SIN-1. Furthermore, addition of 100 mM H2O2 in the presence of 30 mM nitrite at physiologic pH did not result in nitrotyrosine formation in plasma proteins (not shown). These results are consistent with the previous observation that exposure to nitric oxide or nitrogen dioxide at concentrations between 1.5 and 4 mM did not form nitrotyrosine in Cu,Zn superoxide dismutase (11). Inhibition of nitration by plasma antioxidants. Plasma contains several molecules which can react with peroxynitrite and therefore influence the forma-

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FIG. 1. Nitration of plasma proteins by peroxynitrite. Plasma from three volunteers, designated as plasma-1, -2, and -3, were exposed at 1 mg/ml protein to: Lanes 2, 7, and 15, 300 mM peroxynitrite; Lanes 3, 5, and 13, nitric oxide and superoxide generated simultaneously by 1 mM SIN-1; Lanes 6 and 14, reverse order of addition for peroxynitrite; Lanes 4 and 12, 1 mM inactive SIN-1; Lane 8, nitric oxide at 17 mM/min generated by 1 mM spermine–NO; Lane 9, same as 8 plus 100 mM H2O2 ; Lane 10, same as 9 plus 10 mM Fe2/; Lane 11, same as 9 plus 25 mg HRP; Lane 12, same as 8 plus 10 mM Fe2/. After transfer to nitrocellulose the blot was developed with affinity-purified antinitrotyrosine polyclonal IgG and visualized with an anti-rabbit IgG conjugated to HRP using 4-chloro-1-naphthol as a HRP substrate. The same results were repeated for all three different plasma.

tion of nitrotyrosine (8, 27, 28, 34–36). Using the peroxynitrite-mediated nitration of BSA model described previously (37) the effect of plasma antioxidants was examined. Figure 2 shows that uric acid, and to a lesser extent ascorbate, inhibited the formation of nitrotyrosine in BSA by peroxynitrite in a concentration-dependent manner. The IC50 for the inhibition of tyrosine nitration by uric acid is 100 mM. This concentration falls within the physiological range for uric acid, 100– 500 mM. Tyrosine nitration was also inhibited by the presence of free sulfhydryl groups with an IC50 for cysteine of approximately 200 mM. In three different

FIG. 2. Inhibition of tyrosine nitration by uric acid and ascorbate. Fatty acid-free BSA (4 mg/ml) reacted with 2 mM chemically synthesized peroxynitrite in 100 mM phosphate buffer, 100 mM dtpa, pH 7.4, in the presence of different concentrations of uric acid and ascorbate. The yield of tyrosine nitration was determined from the absorbance at 430 nm at pH 11.5. Results represent means { SD of three different experiments for at least six different concentrations of the inhibitor.

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plasma samples the total sulfhydryl concentration was 491 { 48 mM. These results are in apparent contradiction with the data presented in Fig. 2, which indicated that bolus addition of peroxynitrite or peroxynitrite generated by SIN-1 nitrated tyrosine residues of plasma proteins in freshly prepared plasma which contains these antioxidants. Therefore, we examined potential secondary reactions of peroxynitrite that may explain this apparent contradiction. Bicarbonate/CO2 enhanced peroxynitrite-mediated protein tyrosine nitration. Addition of bicarbonate/ CO2 enhanced the peroxynitrite-mediated nitration of BSA in a concentration-dependent manner (Fig. 3). Ad-

FIG. 3. Concentration-dependent increase in nitrotyrosine yield by the addition of bicarbonate at three levels of pH. Fatty acid-free BSA (4 mg/ml) reacted with 2 mM chemically synthesized peroxynitrite in 100 mM PBS, 100 mM dtpa at pH 5.8, 7.6, and 10.0. The yield of tyrosine nitration was determined from the absorbance at 430 nm at pH 11.5. Results represent the mean { SD of three different experiments.

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TABLE I

Bicarbonate Abolishes Uric Acid and Ascorbate-Mediated Inhibition of Tyrosine Nitration Nitrotyrosine yield [mM] ONOO0 ONOO0 ONOO0 ONOO0 ONOO0 ONOO0

/ / / / /

uric acid ascorbate uric acid / bicarbonate ascorbate / bicarbonate bicarbonate

80.3 12.8 60.6 90.4 138.1 150.7

{ { { { { {

3.6 2.6 2.7 5.3 3.7 2.9

Note. Fatty acid-free BSA at 4 mg/ml was reacted with 2 mM chemically synthesized peroxynitrite in 100 mM potassium phosphate-buffered saline containing 100 mM dtpa, pH 7.4 1 0.1 (n Å 3) in the presence of either 200 mM uric acid or ascorbate. The same experiment was repeated in the presence of 25 mM bicarbonate. The yield of nitration was determined from the absorbance at 430 nm and results represent means { SD of three different experiments.

dition of bicarbonate at neutral, acidic, or alkaline pH significantly increased the yield of nitration compared to the spontaneous nitration by peroxynitrite. The concentration of bicarbonate required to attain maximal nitration increased as the pH increased, 2.5 mM at pH 5.8, 5 mM at pH 7.6, and greater than 50 mM at pH 10.0. In addition, the maximal quantity of nitrotyrosine formed was greater at pH 7.6 than at pH 5.8. Moreover, addition of 25 mM bicarbonate overcame the inhibition in tyrosine nitration by 200 mM uric acid and/or ascorbate (Table I). However, both uric acid and ascorbate were still inhibitory over the reaction of peroxynitrite and bicarbonate/CO2 alone. Potential mechanism for the effect of bicarbonate/ CO2 . The results shown in Fig. 3 indicate that the concentration of bicarbonate required to reach maximal nitration is lowest at lower pH and that the maximal yield of nitrotyrosine is highest above pH 6.8. These data suggested that the species involved are CO2 and peroxynitrite anion because most of the bicarbonate at acidic pH will be as dissolved CO2 and peroxynitrite anion is the prevalent species at alkaline pH. This is confirmed by Fig. 4 which shows that the yield of nitrotyrosine is dependent upon the pH with a halfmaximal nitration point that approximates to the pKa of peroxynitrite, 6.8, when the concentration of CO2 is kept constant. Figure 5 shows that CO2 acts catalytically in mediating the nitration of tyrosine residues. Stepwise addition of peroxynitrite resulted in a linear increase in nitrotyrosine yield even at concentrations of peroxynitrite that exceed the quantity of CO2 by more than 20-fold. It should be noted that the measurements of absorbance made in Fig. 5 were carried out at the pH of the reaction and therefore cannot be converted to nitrotyrosine yield because absorbance is only maximal at alkaline pH.

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FIG. 4. pH profile for the nitration of tyrosine residues by peroxynitrite at constant CO2 concentration. Fatty acid-free BSA (4 mg/ml) reacted with 2 mM chemically synthesized peroxynitrite in 100 mM PBS, 100 mM dtpa at various levels of pH. Bicarbonate was added prior to peroxynitrite addition in sufficient quantity to give a final CO2 concentration of 0.06 mM. The yield of tyrosine nitration was determined from the absorbance at 430 nm at pH 11.5. Results represent the mean { SD of three different experiments.

However, CO2 can be seen to be catalytic at all three pH values. It is unlikely that CO2 catalyzed the formation of nitrotyrosine through a radical mechanism based on three lines of evidence. First, as shown in Fig. 2, addition of 200 mM ascorbate had only a negligible effect on CO2-catalyzed nitration. If nitrogen dioxide was the nitrating species, as it would be in a bicarbonate radical-mediated reaction, ascorbate should have had a significant inhibitory effect (38). Second, Fig. 5 demon-

FIG. 5. Catalytic action of CO2-mediated nitration by peroxynitrite. Sequential additions of 0.1 mM (open symbols) and 0.2 mM (closed symbols) peroxynitrite were made to 4 mg/ml fatty acid-free BSA, 0.06 mM CO2 in 100 mM PBS, 100 mM dtpa at pH 6 (circles), pH 7.5 (squares), pH 9 (diamonds). pH was controlled to within {0.1 pH unit by addition of 3 N HCl post-peroxynitrite addition.

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strated that CO2 was not consumed during the peroxynitrite-mediated nitration. Therefore, an intermediate formed by the reaction of peroxynitrite with CO2 is likely to be responsible for the enhanced protein tyrosine nitration. This finding is consistent with the conclusions reported previously (31). Third, nitrotyrosine but not dityrosine is formed at pH 8.0–8.5 in the presence of CO2 . The yield of dityrosine, in the absence of added bicarbonate, increases from nondetectable at pH 7.4 to 95 { 21 mM at pH 8.25. DISCUSSION

We have examined the possibility that nitrotyrosine may be a specific marker for peroxynitrite-mediated oxidative stress under pathophysiological conditions. Although peroxynitrite can modify several amino acid residues, these modifications are not specific for peroxynitrite (37). Nitrotyrosine was found to be specifically derived from the reaction of peroxynitrite with proteins. There are a number of reactive species that can nitrate proteins; however, our data indicate that these reactions are unlikely to contribute significantly to in vivo nitrotyrosine formation. We found that when nitric oxide was generated at pathophysiological concentrations (17 mM min01) in the presence of proteins and under aerobic conditions, nitrotyrosine was not detected, although nitric oxide decomposition followed typical second-order kinetics, indicating that nitrogen dioxide was produced. Although nitrogen dioxide can nitrate tyrosine residues in vitro (38), previous work indicated that nitrogen dioxide did not contribute significantly to protein tyrosine nitration in vivo (11, 13– 15). Recently, Eiserich et al. showed that the tyrosyl radical reacts with •NO at a nearly diffusion limited rate (39). The product formed in this reaction was presumably nitrosotyrosine, which can be oxidized to nitrotyrosine. However, data in Fig. 1 showed that H2O2/ HRP, which is known to oxidize tyrosine to tyrosyl radical, when followed by addition of •NO and allowed to further oxidize did not result in detectable amounts of nitrotyrosine. Under acid conditions nitrite will react with tyrosine to form nitrosotyrosine which can be further oxidized to nitrotyrosine by nitric acid (40). Acidification of nitrite with dilute hydrochloric acid results in the formation of nitrotyrosine in BSA and this process may be responsible for the formation of nitrotyrosine in proteins at gastric pH (40). To achieve significant yield of nitrotyrosine the pH must be below 4, a condition that can only be achieved in specialized cellular compartments such as lysosomes and phagocytes. Moreover, this is a kinetically slow process (41, 42) requiring exposure of proteins for extended time, usually greater than 2 h to achieve sufficient nitrotyrosine formation. Recent data indicated that addition of 100 mM •NO to a tyrosine containing peptide formed nitro-

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tyrosine only after 7 h of exposure at pH 3.0 (43). Based on the overall third-order rate constant for the reaction of •NO with O2 , most of the •NO will be converted to NO2 and other oxides within the first hour of exposure. Therefore, the formation of nitrotyrosine reflects the slow reaction with nitrous acid formed by the acidification of nitrite in the reaction mixture. It is interesting to note that the reaction of •NO with the same peptide at pH 7.8 (closer to physiological pH) did not form nitrotyrosine but rather a peptide dimer (43). In similar experiments we observed that exposure of tyrosine to • NO at concentrations of greater than 100 mM resulted in the formation of a diazotyrosine compound at physiological pH. The reaction of 3-nitrosotyrosine with •NO yields 3-diazonium nitrate which couples to another tyrosine to form diazotyrosine (H. Ischiropoulos unpublished data). Similar results have been reported for the reaction of N-acetyltyrosine with nitrous acid (42) and for the reaction of nitrite with p-cresol where the diazonium formed was coupled to b-naphthol (44). Finally, although nitric oxide will react with H2O2 in the presence of ferrous iron or HRP to oxidize small organic molecules (45), presumably by forming reactive intermediates like peroxynitrous acid, this pathway did not result in the formation of detectable nitrotyrosine at physiological pH. In the plasma or cellular milieu there are several reactions which compete with protein nitration by peroxynitrite. It is well known that peroxynitrite will react with thiols, ascorbate, uric acid, and a-tocopherol (8, 27, 28, 34–36). Uric acid and cysteine have been shown to inhibit peroxynitrite-mediated oxidation of dihydrorhodamine 123 and luminol (30, 34). We detected nitration of plasma proteins in experiments with peroxynitrite or when nitric oxide and superoxide were generated simultaneously (Fig. 1), despite the presence of plasma antioxidants. We also found that bicarbonate increased the yield of peroxynitrite-mediated nitration at acidic, neutral, and alkaline pHs. This may account for the ability of peroxynitrite to nitrate protein tyrosine residues in the presence of low-molecular-weight antioxidants. The reaction of peroxynitrite with bicarbonate was reported previously to enhance the peroxynitrite-mediated light-emitting oxidation of luminol (40). Recently it has been shown by stopped-flow experimentation that CO2 reacts with the peroxynitrite anion with a rate constant of 2.9 1 104 M01 s01 (31). It was suggested that this reaction resulted in a ONO2CO20 adduct. Data presented in Fig. 3 indicated a higher yield of nitrotyrosine, at any given concentration of bicarbonate, at pH 5.8 than at pH 10.0 confirming the involvement of CO2 in the nitration reaction. Heterolytic cleavage of this adduct would yield NO2/ and CO320 . However, heterolytic cleavage may not be required for the nitration of tyrosine because the adduct itself may be the nitrating species. This seems

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plausible because the rate of hydration of the nitronium ion is considerably greater than its reaction with tyrosine. We have previously reported the low efficiency of tetrafluoroborate nitronium salts to nitrate aromatics in aqueous solutions (11). In addition bicarbonate lowers the Escherichia coli-killing ability of peroxynitrite (29) presumably by forming an adduct that will rapidly hydrolyze in the solution before encountering its targets. The formation of a reactive intermediate may be favored because bicarbonate/CO2 has been found to lower the activation energy for the decomposition of peroxynitrite. The accompanying paper by Denicola et al. (32) reported a value of 12 kcalrmol01 for the activation energy of peroxynitrite decomposition in the presence of bicarbonate. This value compares favorably with the value of 13.9 kcalrmol01 obtained by our stopped-flow experiments at physiological pH using 50 mM bicarbonate and the value 12.2 kcalrmol01 of reported previously for the Fe3/ –EDTA-catalyzed nitration of aromatics (12). Overall, based on the observations presented in this paper and in conjunction with the proposal of this adduct, we suggest the following mechanism for the CO2catalyzed nitration of tyrosine by peroxynitrite. Peroxynitrite anion reacts with CO2 to form a reactive intermediate. This intermediate is a strong nitrating agent which attacks the ortho position of tyrosine to form nitrotyrosine. Protonation of the carbonate generated from the intermediate leads to the regeneration of CO2 . The mechanism of CO2-catalyzed nitration can be expressed in the following half reactions: ONOO0 / CO2 r ONOOC(O)O0 ONOOC(O)O0 / Y-protein r NO2 0 Y-protein / CO320 / H/ CO320 / 2H/ r CO2 / H2O. These reactions can be summarized to give an overall reaction equation: ONOO0 / Y-protein / CO2 / H/ r NO2 0 Y-protein / CO2 / H2O. The catalysis of nitration by CO2 may be important under pathologic conditions known to increase the levels of both bicarbonate/CO2 and ONOO0, such as inflammation and ischemia–reperfusion injury. As such, nitrotyrosine has been detected in human and animal conditions associated with activation of inflammatory cells, acute lung injury, and ischemia–reperfusion (13– 15, 19–25). Moreover, the work of Denicola et al. showed that the concentration of bicarbonate/CO2 and the rate of reaction with peroxynitrite in the extracellu-

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lar environment can outcompete with other reactive pathways such as sulfhydryls for peroxynitrite (32). This observation is consistent with the data showing ONOO0-mediated protein tyrosine nitration in plasma, which contains sulfhydryls and other potential scavengers of peroxynitrite. Overall, the data indicate that the reaction of CO2 with peroxynitrite produces a strong nitrating agent and redirects the reactivity of peroxynitrite toward protein tyrosine nitration. Therefore, this reaction may be a major pathway for peroxynitritemediated protein nitrotyrosine formation in vivo. ACKNOWLEDGMENTS We are grateful to Drs. J. S. Beckman and Y. Z. Ye for providing the anti-nitrotyrosine antibody, Drs. John P. Crow (University of Alabama, at Birmingham), Willem Koppenol (ETH, Zurich), and Rafael Radi and Ana Denicola (University of the Republic, Montevideo) for helpful discussions, and Mrs. June Nelson for expert technical assistance. This work was supported by grants from the Pennsylvania Lung and Southeastern Pennsylvania Heart Associations (H.I.), from the National Institutes of Health ES-05211 (S.R.T.), and the Council for Tobacco Research (S.R.T.). Dr. Ischiropoulos is a Parker B. Francis Fellow in Pulmonary Research and Dr. Gow is supported by a National Research Service Award from the National Institute of Health, Heart, Lung, and Blood Division (Grant HL07748). Note added in Proof. During review of this manuscript a paper was published which also provided evidence for the role of CO2 in peroxynitrite-mediated nitration. Uppu, R. M., Squadrito, G. L., and Pryor, W. A. (1996) Arch. Biochem. Biophys. 327, 335–343.

REFERENCES 1. Huie, R. E., and Padjama, S. (1993) Free Radical Res. Commun. 18, 195–199. 2. Goldstein, S., and Czapski, G. (1995) Free Radical Biol. Med. 19, 505–510. 3. Ischiropoulos, H. Zhu, L., and Beckman, J. S. (1992) Arch. Biochem. Biophys. 298, 446–451. 4. Carreras, M. C., Pargament, G. R., Catz, S. D., Poderoso, J. J., and Boveris, A. (1994) FEBS Lett. 341, 65–68. 5. Rodensa, J., Mitjavila, M. T., and Carbonell, T. (1995) Free Radical Biol. Med. 18, 869–875. 6. Kooy, N. W., and Royall, J. A. (1994) Arch. Biochem. Biophys. 310, 352–359. 7. Radi, R., Beckman, J. S., Bush, K. M., and Freeman, B. A. (1991) J. Biol. Chem. 266, 4244–4250. 8. Radi, R., Beckman, J. S., Bush, K. M., and Freeman, B. A. (1991) Arch. Biochem. Biophys. 288, 481–487. 9. Beckman, J. S., Beckman, T. W., Chen, J., Marshall, P. A., and Freeman, B. A. (1990) Proc. Natl. Acad. Sci. USA 87, 1620– 1624. 10. Crow, J. P., Spruell, C., Chen, J., Gunn, C., Ischiropoulos, H., Zhu, L., Tsai, J-H. M., Smith, C. D., Radi, R., Koppenol, W. H., Freeman, B. A., Matalon, S., and Beckman, J. S. (1994) Free Radical Biol. Med. 16, 331–338. 11. Ischiropoulos, H., Zhu, L., Chen, J., Tsai, J-H. M., Martin, J. C., Smith, C. D., and Beckman, J. S. (1992) Arch. Biochem. Biophys. 298, 431–437. 12. Beckman, J. S., Ischiropoulos, H., Zhu, L., van der Woerd, M.,

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48

GOW ET AL. Smith, C. D., Harrison, J., Martin, J. C., and Tsai, J-H. M. (1992) Arch. Biochem. Biophys. 298, 438–445.

13. Beckman, J. S., Ye, Y. Z., Anderson, P. G., Chen, J., Accavitti, M. A., Tarpey, M. M., and White, C. R. (1994) Biol. Chem. HoppeSeyler 375, 81–88. 14. Haddad, I. Y., Pataki, G., Hu, P., Beckman, J. S., and Matalon, S. (1994) J. Clin. Invest. 94, 2407–2413. 15. Kooy, N. W., Royall, J. A., Ye, Y. Z., Kelly, D. R., and Beckman, J. S. (1995) Am. J. Respir. Crit. Care Med. 151, 1250–1254. 16. Schulz, J. B., Matthews, R. T., Muqit, M. K., Browne, S. E., and Beal, M. F. (1995) J. Neurochem. 64, 936–939. 17. Schulz, J. B., Matthews, Jenkins, B. G., Ferrante, R. J., Siwek, D., Henshaw, D. R., Cipolloni, P. B., Mecocci, P., Kowall, N. W., Rosen, B. R., and Beal, M. F. (1995) J. Neurosci. 15, 8419–8429. 18. Basarga, O., Michaels, F. H., Zheng, Y. M., Borboski, L. E., Spitsin, S. V., Fu, Z. F., Tawadros, R., and Koprowski, H. (1995) Proc. Natl. Acad. Sci. USA 92, 12041–12045. 19. Szabo, C., Salzman, A. L., and Ischiropoulos, H. (1995) FEBS Lett. 363, 235–238. 20. Wizemann, T. M., Gardner, C. R., Laskin, J. D., Quinones, S., Durham, K. D., Golle, N. L., Ohnishi, S. T., and Laskin, D. L. (1994) J. Leukocyte Biol. 56, 759–768. 21. Ischiropoulos, H., Al-Mehdi, A., and Fisher, A. B. (1995) Am. J. Physiol. 269, L158–L164. 22. Kaur, H., and Halliwell, B. (1994) FEBS Lett. 350, 9–12. 23. Miller, M. J. S., Thompson, J. H., Zhang, X-J., Saodowska-Krowicka, H., Kakkis, J. L., Munshi, U. K., Sandoval, M., Rossi, J. L., Eloby-Childress, S., Beckman, J. S., Ye, Y. Z., Rodi, C. P., Manning, P. T., Currie, M. G., and Clark, D. A. (1995) Gastroenterology 109, 1475–1483. 24. Salman-Tabcheh, S., Guerin, M-C., and Torreilles, J. (1995) Free Radical Biol. Med. 19, 695–698. 25. Giorgio, S., Linares, E., de Capurro, M. L., de Bianchi, A. G., and Augusto, O. (1996) Photochem. Photobiol., in press. 26. Koppenol, W. H., Moreno, J. J., Pryor, W. A., Ischiropoulos, H., and Beckman, J. S. (1992) Chem. Res. Toxicol. 5, 834–842.

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27. Pryor, W. A., Jin, X., and Squadrito, G. L. (1994) Proc. Natl. Acad. Sci. USA 91, 11173–11177. 28. Bartlett, D., Church, D. F., Bounds, P. L., and Koppenol, W. H. (1995) Free Radical Biol. Med. 18, 85–92. 29. Zhu, L., Gunn, C., and Beckman, J. S. (1992) Arch. Biochem. Biophys. 298, 452–457. 30. Radi, R., Cosgrove, T. P., Beckman, J. S., and Freeman, B. A. (1993) Biochem. J. 290, 52–57. 31. Lymar, S. V., and Hurst, K. (1995) J. Am. Chem. Soc. 117, 8867– 8868. 32. Denicola, A., Trujillo, M., Freeman, B. A., and Radi, R. (1996) Arch. Biochem. Biophys. 332, 000–000. 33. Ischiropoulos, H., Duran, D., and Horwitz, J. (1995) J. Neurochem. 65, 2366–2372. 34. Kooy, N. W., Ischiropoulos, H., Beckman, J. S., and Royall, J. A. (1994) Free Radical Biol. Med. 16, 149–156. 35. Van Der Vliet, A., Smith, D., O’Neill, C. A., Kaur, H., DarleyUsmar, V., Cross, C. E., and Halliwell, B. (1994) Biochem. J. 303, 295–301. 36. Hogg, N., Joseph, J., and Kalyanaraman, B. (1994) Arch. Biochem. Biophys. 314, 153–158. 37. Ischiropoulos, H., and Al-Mehdi, A. (1995) FEBS Lett. 364, 279– 282. 38. Prutz, W. A., Monig, H., Butler, J., and Land, E. J. (1985) Arch. Biochem. Biophys. 243, 125–134. 39. Eiserich, J. P., Butler, J., Van der Vliet, A., Cross, C. E., and Halliwell, B. (1995) Biochem. J. 310, 745–749. 40. Knowles, M. E., McWeeny, D. J., Couchman, L., and Thorogood, M. (1974) Nature 247, 288–289. 41. Mirvich, S. S. (1975) Toxicol. Appl. Pharmacol. 31, 325–351. 42. Kurosky, A., and Hofmann, T. (1972) Can. J. Biochem. 50, 1282– 1296. 43. Mirza, U. A., Chait, B. T., and Lander, H. M. (1995) J. Biol. Chem. 270, 17185–17188. 44. Philpot, J. L., and Small, P. A. (1938) J. Biochem. 32, 534–539. 45. Ishiropoulos, H., Nelson, J., Duran, D., and Al-Mehdi, A. (1996) Free Radical Biol. Med. 20, 373–381.

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