INTRODUCTION

Nitrogen monoxide (nitric oxide, NO)¹ is produced by a variety of cells through the activity of constitutive and inducible forms of nitric oxide synthase (1). 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 (2, 3). 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 (4, 5).

NO reacts at a near diffusion-controlled rate with superoxide (O₂) (k = 6.7 × 10⁹ M¹ s¹) (6) 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 (7, 8). At physiological pH the protonated form of ONOO, peroxynitrous acid (ONOOH) (pK_a = 6.8), is highly unstable and rapidly decomposes to nitrate (NO₃). 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. 9). ONOO/ONOOH reacts with proteins, leading to the oxidation of cysteine, methionine, and tryptophan residues, and can induce protein carbonyl formation and nonspecific fragmentation (10, 11, 12). In addition, ONOO/ONOOH can react readily with phenolic compounds to form nitrated, hydroxylated, and dimerized products (13, 14, 15, 16, 17), 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 (18), neutrophils (19), and endothelial cells (20) 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 (21), atherosclerosis (22), and rheumatoid arthritis (23), has been attributed to ONOO formation. However, in all of these cases direct proof for the production of ONOO in biological systems is lacking, even though its formation in vivo is favorably predicted (24).

Under inflammatory conditions, multiple well characterized reactive oxygen species (ROS) are produced from phagocytic cells (25). For instance, stimulated neutrophils and macrophages produce significant levels of superoxide (O₂) and hydrogen peroxide (H₂O₂) as a result of the activation of the respiratory burst oxidase (26). In the case of neutrophils, some of the H₂O₂ that is produced under these conditions is converted to the strong oxidant hypochlorous acid (HOCl) by the action of myeloperoxidase as shown in Reaction 1.  HOCl produced from activated human neutrophils has been shown to react with amines (taurine, lysine, and arginine) and tyrosine to form N-chloramines and 3-chlorotyrosine (Cl-Tyr), respectively (27, 28), where the latter has been proposed to serve as a selective marker of HOCl production in vivo (29).

In addition to ROS, macrophages (30) and neutrophils (19) can also simultaneously produce large fluxes of NO through the activation of inducible nitric oxide synthase; however, the ability of human neutrophils to produce NO is debated (31). Once formed, NO can react with several biological targets, primarily thought to involve heme-iron, hyperreactive sulfhydryls and protein radicals (32, 33, 34). NO can also react with O₂ in aqueous solution to produce nitrite (NO₂) via a complex mechanism thought to involve a variety of reactive nitrogen species (RNS) including NO₂ and dinitrogen trioxide (N₂O₃) (35). In fact, NO₂ has been used as a marker of NO production in vitro and in vivo and has been shown to reach concentrations of up to 4 µM in synovial fluid from patients with rheumatoid arthritis (36) and as high as 20 µM in human airway fluids (37). These RNS produced during an inflammatory response could theoretically react with a number of ROS to form various novel species. Indeed, the interaction of HOCl with NO or NO₂ has been proposed to form species capable of nitrosylating and nitrating organic substrates (38, 39).

The present study was undertaken to examine the potential interactions of NO-derived RNS with the inflammatory oxidant HOCl in an attempt to characterize more fully the various species that may be formed under complex physiological inflammatory conditions. Our results indicate that NO₂ reacts with HOCl to form an intermediate species, postulated to be nitryl chloride (Cl-NO₂) and/or chlorine nitrite (Cl-ONO), that is capable of nitrating, chlorinating, and dimerizing phenolic compounds including tyrosine. We propose that the formation of Cl-NO₂ and/or Cl-ONO by this reaction represents a novel mechanism of inflammation-mediated biological damage, and offers an additional or alternative mechanism of tyrosine nitration independent of ONOO formation.

EXPERIMENTAL PROCEDURES

DL-Phenylalanine, DL-tyrosine, N-acetyl-L-tyrosine (NAT), N-acetyl-L-phenylalanine (NAP), NO₂-Tyr, Cl-Tyr, 4-hydroxyphenylacetic acid (HPA), 3-nitro-4-hydroxyphenylacetic acid (NO₂-HPA), chlorophenylalanine isomers, sodium hypochlorite (NaOCl), sodium nitrite (NaNO₂), and bovine serum albumin (BSA; essentially fatty acid-free) were obtained from Sigma. 3-Chloro-4-hydroxyphenylacetic acid (Cl-HPA), 4-methoxyphenylacetic acid (MPA), and nitronium tetrafluoroborate (NO₂BF₄; 0.5 M solution in sulfolane) were from Aldrich. Nitric acid, sulfuric acid, and chlorosulfonic acid were obtained from Fisher Scientific (Pittsburgh, PA). Nitrogen monoxide (nitric oxide, NO) (3,000 ppm in O₂-free N₂) was obtained from Scott-Marrin, Inc. (Riverside, CA). Dityrosine was synthesized by oxidation of L-tyrosine with horseradish peroxidase (type I; Sigma) and H₂O₂. Peroxynitrite (ONOO) was synthesized and quantified as described previously (40). HOCl concentrations were determined spectrophotometrically at 290 nm (pH 12,  = 350 M¹ cm¹). All buffer solutions were treated with Dowex-50 chelating resin to remove transition metals prior to experiments.

NO Experiments

NO (3,000 ppm in O₂-free N₂) was bubbled through continuously stirred 100-ml solutions of phosphate buffer (100 mM KH₂PO₄, pH 7.4) at a flow rate of 20 ml/min. Buffer solutions were purged with either air or purified N₂ for 30 min prior to NO exposure and were continuously sparged with either gas throughout the experiment to maintain an oxygenated or deoxygenated solution, respectively. At various time points, aliquots of the solution were withdrawn and purged briefly with N₂ to remove residual NO, and NO₂ production was determined spectrophotometrically using Griess reagent (1% sulfanylamide, 0.1% N-(1-naphthyl)ethylenediamine, and 2.5% H3PO₄) (41). In separate experiments, HPA (5 mM) was added to the buffer solutions prior to NO exposure. At various time points, 0.5-ml aliquots of the solution were withdrawn from the reaction mixture and immediately reacted with HOCl (1 mM). After 10 min, the reaction mixture was adjusted to pH 10-11 with 1 M NaOH. NO₂-HPA was measured spectrophotometrically at 430 nm;  = 4400 M¹ cm¹ (16).

Buffered solutions (100 mM KH₂PO₄) of HPA, NAT, NAP, or MPA (1-5 mM) were adjusted to the desired pH (5.0-8.5) with either 10% NaOH or 5% H₃PO₄ prior to experimentation. Where appropriate, NO₂ was added at the desired concentration (0.1-3.0 mM) in 5-ml sample volumes. HOCl was then added to the solutions at 25 °C as a small drop (<20 µl) while continuously vortexing. Although reactions are nearly instantaneous, incubations were allowed to proceed for 10 min before reduced GSH was added at 1 mM concentration to scavenge any unreacted HOCl. In separate experiments ONOO (in 1.2 M NaOH) or NO₂BF₄ (in sulfolane) were reacted with the various substrates in a similar manner. The products of the reactions involving HPA as substrate were analyzed directly by HPLC. Since nitrated, chlorinated, and dimerized N-acetylated derivatives of tyrosine were not available, reaction mixtures utilizing NAT as substrate were first hydrolyzed (see below) to liberate the free amino acids and their modified products prior to HPLC analysis. Reaction mixtures involving MPA were transferred to a quartz cuvette, and the absorbance spectrum was measured between 280 and 500 nm at pH 7.4. 

Reactions of BSA with NO₂/HOCl

NO₂ and HOCl (both 500 mM) were loaded into 1-ml syringes that were attached to an automated syringe pump. Teflon tubing from both syringes converged into a single tube and allowed reaction of the two components for a brief period (<1 s). The reaction effluent was allowed to drop approximately 6 cm immediately into 10-ml solutions of BSA (10 mg/ml) in 100 mM KH₂PO₄ (pH 7.4), which were continuously stirred. The volume of each drop was calibrated (33 µl), and final concentrations of oxidant exposure were calculated. In some cases, NO₂ in one of the syringes was replaced with 100 mM KH₂PO₄ (pH 7.4) so as to allow exposure of BSA to HOCl alone under the same conditions. Following addition of the NO₂/HOCl mixture or HOCl alone, the solutions were stirred for 15 min and were then quenched by the addition of excess GSH. NO₂-Tyr, Cl-Tyr, and dityrosine formation in the samples were determined by HPLC following acid hydrolysis as described below.

Caution: the reagents used and products formed in this synthesis are highly irritant and corrosive to the eyes, skin, and mucous membranes. All of the procedures involved in the synthesis of Cl-NO₂ must be performed in a fume hood to ensure proper ventilation, and appropriate eye and skin protection must be worn. At room temperature, Cl-NO₂ exists as a gas (boiling point, 15 °C) and presents a serious inhalation hazard if not handled properly. The procedure for the synthesis of Cl-NO₂ is essentially that previously described (42, 43) with slight modification. Sulfuric acid (61 g) was added dropwise to vigorously stirred nitric acid (50 g) at 0 °C in a 500-ml three-necked round bottom flask equipped with a dropping funnel. A cold finger receiving flask (150 ml) was attached to the reaction vessel by a short segment of Teflon tubing, and the flask was immersed in a cooling mixture of dry ice/acetone. After 10 min, chlorosulfonic acid (85 g) was slowly added dropwise via the attached funnel into the mixture of nitric and sulfuric acids over a 4-h period. During the entire synthesis procedure, a gentle flow of N₂ gas was delivered through the apparatus to enhance the evolution and collection of gaseous Cl-NO₂. It is important that the chlorosulfonic acid is added at a slow enough rate such that brown gas does not appear above the reaction mixture. The colorless gaseous Cl-NO₂ evolved from the reaction mixture was carried by the gentle flow of N₂ into the cold finger receiving flask, where it condensed as a pale yellow liquid. The product was purified by passing ozonized air through the liquefied gas to oxidize any nitrosyl chloride (Cl-NO) that may have been present as a contaminant. The product was carefully transferred to sealed glass vials and stored at 80 °C until used in experiments. The yield of Cl-NO₂ is typically 80-90% (approximately 50 g), and the purity has been reported to be 98-99% (42). To confirm the identity of the reaction product, the purified product was diluted in methanol, and the absorbance spectrum was immediately measured. The observed absorbance spectrum of synthetic Cl-NO₂ showed a series of characteristic absorption maxima between 300 and 400 nm, similar to that reported previously (43).

Cl-NO₂ (10 ml) was placed in a 50-ml sparging flask immersed in a cooling dry ice/acetone bath. A stream of N₂ gas was allowed to flow through a glass tube fitted with a fritted glass fitting, which was submerged into the undiluted Cl-NO₂, and was bubbled through the liquid at a flow rate of 75 ml/min. Gaseous Cl-NO₂ evolved into the headspace exited through a glass tube connected to the top of the flask into a glass reaction vessel containing a solution of the analyte to be exposed. Cl-NO₂ in N₂ gas was allowed to bubble through the 100 mM phosphate-buffered solutions (25 ml, pH 7.4) of NAT (5 mM), NAP (5 mM), MPA (1 mM), or BSA (10 mg/ml) for various periods of time (0-120 s). Aliquots (500 µl) of the solutions were sampled at various time points and subjected to acid hydrolysis, and the levels of the modified amino acids were determined by HPLC as described below.

Spectral Characterization of NO₂/HOCl Reaction Intermediates

To characterize the intermediate(s) produced by reaction of NO₂ and HOCl we have utilized a continuous flow reaction with photodiode array (PDA) spectrophotometric detection. NO₂ and HOCl (both at 25 mM in 50 mM KH₂PO₄, pH 6.0) were independently pumped into a mixing junction at a flow rate of 0.3 ml/min. Upon mixing, the reaction effluent was immediately directed into the flow cell of a Waters 996 PDA detector, and the absorbance spectrum of the reaction products was continuously monitored over the range 300-400 nm. In some cases the reaction medium was supplemented with 25% methanol (HPLC grade) in order to compare the absorbance spectra with that of authentic Cl-NO₂ in methanol, as described above.

All reaction mixtures were analyzed by HPLC, using a 5-µm Spherisorb ODS-2 reverse-phase C-18 column. Samples containing HPA were analyzed directly following experiments without sample preparation by isocratic elution from the column with a mobile phase consisting of 100 mM KH₂PO₄ (pH 3.5)/methanol (70/30, v/v) and UV detection at 274 nm. Samples including NAT, NAP, or BSA were first hydrolyzed in 6 M HCl at 110 °C in sealed glass vials for 4 and 24 h, respectively, to obtain the free amino acids and their modified products. The hydrolysates were then dried using a Centrivap (Labconco) and resuspended in the appropriate mobile phase. Tyrosine, NO₂-Tyr, and Cl-Tyr were analyzed by isocratic elution from the column with 50 mM KH₂PO₄ (pH 3.0)/methanol (92/8, v/v) and subsequent UV detection at 274 nm (15). Dityrosine was detected simultaneously by on-line fluorescence detection using a Waters 470 scanning fluorescence detector (excitation, 284 nm; emission, 410 nm) (15). Phenylalanine (Phe) and ortho-, meta-, and para-chlorophenylalanine (o-, m-, and p-Cl-Phe) were separated on the same column cited above, utilizing a mobile phase consisting of 50 mM KH₂PO₄ (pH 3.0)/methanol (85/15, v/v) with UV detection at 220 nm. Peaks were identified and quantitated using authentic external standards. Peak identity was determined by adding to the sample the authentic compound to establish a match in the HPLC retention time. Peak identity was confirmed using a Waters 996 PDA detector. A spectral match between the authentic chemical and the sample analyte of greater than 90% constituted positive identification.

RESULTS

Interactions of NO, NO₂, and HOCl

Aliquots of HPA solutions purged with NO were reacted with HOCl at various time points to determine if a species is formed under these conditions that is capable of nitrating this model phenolic compound. NO₂-HPA was immediately formed upon HOCl addition, and the yield increased in a manner dependent on the length of time the solution had been purged with NO. The extent of NO₂-HPA formation was significantly lower under deoxygenated (N₂-sparged) conditions (Fig. 1A), suggesting that autoxidation of NO is involved in the reaction with HOCl. NO rapidly autoxidizes to NO₂ when bubbled through air-saturated solutions, whereas formation of NO₂ is significantly diminished under deoxygenated conditions (Fig. 1B). As shown in Fig. 1, A and B, the formation of NO₂-HPA parallels NO₂ production, suggesting that a nitrating species is formed by reaction of HOCl with NO₂ or an intermediate species formed during NO autoxidation.

Oxygen dependence of HPA nitration by the interaction of NO and HOCl.

Modification of HPA and Tyrosine by NO₂/HOCl Reaction

A series of experiments were designed using HPA as a substrate to determine whether NO₂ reacts with HOCl to produce a nitrating species. Indeed, the addition of HOCl to solutions containing NO₂ and HPA resulted in the immediate (<1 s) formation of a persistent yellow color indicative of phenolic nitration. The presence of NO₂-HPA was confirmed by subsequent HPLC analysis and detection by PDA. Nitration of HPA by the reaction of NO₂ and HOCl in solution was found to be pH-dependent (Fig. 2). Maximal formation of NO₂-HPA from this reaction occurred at neutral pH, whereas its formation decreased at increasingly acidic or basic pH values and is independent of ionic strength (10-200 mM phosphate) (data not shown). The pH profile of NO₂-HPA formation by the reaction of NO₂ and HOCl (Fig. 2) indicates that the reaction involves HOCl and not ClO, because of the rapid decrease in nitration at pH > 7.5 (the pK_a of HOCl). Since the pK_a of NO₂ is approximately 3.4, it is NO₂, and not HNO₂, that is the reacting species at all of the pH levels we have studied (pH 5.0-8.5). The decreasing yield of NO₂-HPA at low pH is similar to that determined for the reaction of tyrosine with NO₂ (15, 44) or ONOO (15, 16) and may well be due to the lower reactivity of the phenol relative to the phenolate species.

Reaction of NO₂ (1 mM) and HOCl (1 mM) converted approximately 4% of HPA to NO₂-HPA, similar to the reported yields of NO₂-HPA obtained from reaction of ONOO (1 mM) with this substrate (16). The reaction of NO₂ alone with HPA did not yield NO₂-HPA at any of the pH values studied herein. In the absence of NO₂, HOCl directly converted HPA into Cl-HPA at nearly 40% yield. Increasing the concentration of NO₂ (0.1-3.0 mM) in the reaction mixture decreased the yield of Cl-HPA and resulted in a concomitant increase in the formation of NO₂-HPA (Fig. 3), indicating that NO₂ competed with HPA for reaction with HOCl. When initial NO₂ and HOCl concentrations were equal (1 mM), the extent of chlorination was approximately 4-fold higher than nitration. Addition of NO₂ in excess over HOCl (up to 3-fold) did not significantly increase the yield of NO₂-HPA, suggesting that the nitrating species is produced by a 1:1 reaction of HOCl with NO₂. Similarly, excess NO₂ did not significantly decrease formation of Cl-HPA, suggesting that the product formed by this reaction is an efficient chlorinating agent as well as a nitrating species.

We also used NAT as a substrate for the reaction of NO₂ with HOCl to simulate tyrosine residues in proteins. As shown in Table I, qualitatively and quantitatively similar results were obtained. However, whereas HOCl alone caused very small amounts of dityrosine to be produced (<0.8 µM), the combined addition of NO₂ and HOCl induced a 15-fold increase in dityrosine formation. Since dityrosine is formed by the combination of two tyrosyl radicals, this result indicated that the reaction between NO₂ and HOCl produces a species that is capable of carrying out a one-electron oxidation of tyrosine to form the tyrosyl radical. Reactions of NAT with ONOO and NO₂BF₄ were also studied in order to compare the nitration mechanisms with that of NO₂/HOCl. Although the reaction of ONOO (1 mM) with NAT led to NO₂-Tyr levels that were 2-fold higher than that achieved by NO₂/HOCl, the levels of dityrosine were nearly identical. The NO⁺₂ species (NO₂BF₄) reacted with NAT to form NO₂-Tyr but in lower yields than with either ONOO or NO₂/HOCl treatments. The reaction of NO₂BF₄ with NAT also yielded relatively high levels of dityrosine, suggestive of tyrosyl radical intermediates in its mechanism of tyrosine nitration.

Table I. Comparative modification of NAT by reactions of HOCl, HOCl/NO2, ONOO and NO+2 N-Acetyltyrosine (5 mM) was dissolved in phosphate buffer (pH 7.4, 100 mM) and reacted with the various reagents (all at 1 mM) as described under ``Experimental Procedures.'' The yields of NO2-Tyr, Cl-Tyr, and dityrosine were determined following acid hydrolysis of the N-acetylated derivatives. Values are the mean ± S.D. of four separate experiments. Reaction NO2-Tyr Cl-Tyr Dityrosine µM µM µM N-Acetyltyrosine +NO2 NDa ND ND +HOCl ND 652  ± 42 0.8  ± 0.2 +HOCl/NO2 40  ± 4 312  ± 24 14.3  ± 2.3 +ONOO 86  ± 7 ND 13.2  ± 3.1 +NO2BF4 9  ± 2 ND 5.9  ± 0.7 a  ND, not detected.

Modification of Tyrosine in BSA by NO₂/HOCl

Reaction of HOCl with solutions of BSA containing NO₂ resulted in NO₂-Tyr formation, but more slowly than with pure HPA or NAT as substrate. However, when NO₂ and HOCl were allowed to react just before the addition to BSA (using a dual syringe pump), rapid formation of NO₂-Tyr was observed in a dose-dependent manner (Fig. 4A). A small amount of dityrosine (approximately 1 µM) could also be detected in BSA treated in this manner. The species produced by the reaction of NO₂ and HOCl also reacted with BSA to produce relatively high levels of Cl-Tyr (Fig. 4B). However, when NO₂ was omitted from one of the syringes (replaced by phosphate buffer), significantly higher levels of Cl-Tyr were detected in BSA, again suggesting that NO₂ was reacting with HOCl. No detectable levels of these modified tyrosine products were found in acid hydrolysates of control (nonexposed) solutions of BSA.

Characterization of NO₂/HOCl Reaction Product(s)

To determine the identity of the product(s) formed by reaction of NO₂ with HOCl we utilized a continuous flow dual pump PDA system. The spectrum of NO₂ at pH 6.0 (50 mM KH₂PO₄) under continuous flow through the PDA detector shows a single absorbance maximum at approximately 370 nm (not shown). The addition of HOCl to the continuous flow apparatus via a separate pump leads to the degradation of the NO₂ absorption and the concomitant formation of a series of maxima observed between 320 and 420 nm (Fig. 5A). Johnson and Margerum (45) have studied the reaction of NO₂ with HOCl and have suggested that nitryl chloride (Cl-NO₂) is a product. Authentic Cl-NO₂ was synthesized as described under ``Experimental Procedures,'' and the absorbance spectrum of this species (in methanol, a polar solvent for which Cl-NO₂ is more stable as compared with aqueous conditions) is shown in Fig. 5C for comparison and shows a series of absorption maxima between 320 and 400 nm, characteristic of that for Cl-NO₂ reported previously (43). The addition of methanol to the reaction mixture of NO₂ and HOCl caused a hypsochromic shift (20 nm) in the absorption spectrum (Fig. 5B) without affecting the characteristic series of maxima observed in the absence of methanol. Although the absorbance spectra of authentic Cl-NO₂ and that determined for the product of the reaction between NO₂ and HOCl show much similarity, the slight differences may be due to the different conditions for which the spectra were obtained (100% methanol for Cl-NO₂, and 25% methanol for NO₂/HOCl reaction) and to interference of unreacted NO₂ in the spectrum of the NO₂/HOCl reaction product. Hence, we can conclude that the product formed by this reaction shows characteristics similar to those of Cl-NO₂.

Since Cl-NO₂ is proposed to be formed by the reaction of NO₂ with HOCl (45) and the absorption spectrum of the product formed by this reaction suggested the potential formation of Cl-NO₂ in our studies, NAT and BSA were exposed to synthetic Cl-NO₂ in order to compare its reactivity to the species produced by the NO₂/HOCl reaction. Exposure of a solution of NAT (5 mM) to a stream of gaseous Cl-NO₂ led to the formation of Cl-Tyr, NO₂-Tyr, and dityrosine to an extent dependent on the duration of exposure (Fig. 6). Formation of Cl-Tyr and dityrosine reached maximum levels at 40 and 50 s respectively, after which the products were decomposed upon further exposure to Cl-NO₂. The loss of Cl-Tyr is likely due to the formation of dichloro-Tyr, as has been shown for the chlorination of tyrosine by Cl₂ gas (46). In contrast, the formation of NO₂-Tyr continued to increase over the entire exposure period, suggesting that NO₂-Tyr is stable under Cl-NO₂ reaction conditions. The ratio of Cl-Tyr to NO₂-Tyr formed by Cl-NO₂ averaged 5:1 during the early segment of exposure (20-40 s), and is similar to the ratio of 4:1 obtained by reaction of NO₂/HOCl with NAT.

To determine whether the reactions studied with NAT as substrate are relevant to reactions with intact proteins, gaseous Cl-NO₂ was bubbled through solutions of BSA (10 mg/ml). The time-dependent formation of NO₂-Tyr, Cl-Tyr, and dityrosine in BSA exposed to Cl-NO₂ is summarized in Table II. The profile of modified tyrosines was qualitatively similar to that observed for the reaction of NAT with Cl-NO₂, except that the yields of the products were lower, especially at the early time points. Whereas the rapid consumption of NAT by Cl-NO₂ was initiated immediately, the initial loss of tyrosine residues in BSA exposed to Cl-NO₂ was less dramatic, potentially because of competitive reactions with other targets in BSA. The slower initial rate of tyrosine loss in BSA paralleled the oxidation of free sulfhydryl groups in BSA. However, a much more rapid loss of tyrosine ensued following complete depletion of free sulfhydryl groups in BSA (data not shown). The data suggest that reaction of Cl-NO₂ with other amino acid residues (i.e., cysteine, methionine, and lysine) and/or the nonspecific oxidation of the peptide backbone are also important.

Table II. Modification of tyrosine residues in BSA exposed to synthetic Cl-NO2 Solutions of BSA (10 mg/ml) were exposed to Cl-NO2 as described under ``Experimental Procedures.'' At various time points, aliquots (250 µl) of the BSA solution were taken and subjected to acid hydrolysis to liberate free amino acids and their modified products. The products were identified and quantitated using HPLC. Values represent the mean ± S.D. of three separate experiments. Exposure Time NO2-Tyr Cl-Tyr Dityrosine s µM µM µM 0 ND ND ND 30 3  ± 0.2 35  ± 4 0.4  ± 0.1 60 118  ± 14 101  ± 8 2.9  ± 0.3 90 508  ± 35 44  ± 9 2.6  ± 0.2 120 883  ± 28 10  ± 1 1.3  ± 0.1 a  ND, not detected.

Since dityrosine is a significant product of the reaction between tyrosine and NO₂/HOCl, it is likely that radical species are involved in the reaction mechanisms. To test this hypothesis, we utilized the O-methylated derivative of HPA, MPA, a substrate that cannot form phenolic radicals. Fig. 7 compares the absorbance spectra of the products formed when NO₂/HOCl or NO⁺₂ was reacted with MPA at pH 7.4. NO⁺₂, derived from the nitryl salt NO₂BF₄, appears capable of nitrating MPA (pH 7.4) by electrophilic aromatic substitution giving rise to a strong absorbance maximum at 380 nm (Fig. 7, spectrum C). However, the reaction of NO₂/HOCl with MPA failed to give rise to an absorbance in this region (Fig. 7, spectrum B), suggesting that formation of a phenolic radical is an obligatory step in the nitration mechanism involved in this reaction pathway. Reaction of synthetic Cl-NO₂ or HOCl with MPA produced a product(s) with a spectrum nearly identical to that obtained for the NO₂/HOCl reaction (data not shown) and suggested that Cl-NO₂ was also incapable of nitrating MPA, implicating radical intermediates in the nitration mechanism of this species. The increased absorbance between 290 and 340 nm observed in both of these cases could be due to chlorination of MPA, suggesting that intermediate phenoxyl radical formation is not a compulsory step in aromatic chlorination.

NAP was used as a model substrate to examine in more detail the mechanisms of aromatic chlorination by the product(s) formed by reaction of NO₂ with HOCl. Exposure of NAP to HOCl, NO₂/HOCl, or Cl-NO₂ led to the formation of o-Cl-Phe, m-Cl-Phe, and p-Cl-Phe to differing extents as illustrated in Fig. 8. Treatment of NAP with HOCl alone led to the formation of o-Cl-Phe, m-Cl-Phe, and p-Cl-Phe in a ratio of 1.00/0.20/0.85. Reactions of NAP with NO₂/HOCl or synthetic Cl-NO₂ led to the formation of the o-Cl-Phe, m-Cl-Phe, and p-Cl-Phe isomers in a ratio of 1.0/0.35/0.83 or 1.0/0.40/0.83, respectively. Whereas the o-Cl-Phe:p-Cl-Phe ratios for all three treatments are nearly identical, an increase in the proportion of m-Cl-Phe formed by NO₂/HOCl and Cl-NO₂ compared with HOCl alone was observed. The ratios of o-Cl-Phe to m-Cl-Phe for HOCl, NO₂/HOCl, and Cl-NO₂ are 5.0, 2.8, and 2.5, respectively. The high proportion of o-Cl-Phe and p-Cl-Phe isomers by all of the reactions is indicative of electrophilic aromatic substitution reactions and is consistent with previous studies utilizing a variety of chlorinating agents (47). The nearly 2-fold increase in the m-Cl-Phe isomer by reactions of NAP with both NO₂/HOCl and Cl-NO₂ suggest a less selective mechanism of chlorination more typical of radical reactions. It is noteworthy that the reaction of NAP with NO₂/HOCl or Cl-NO₂ did not form detectable levels of nitrated products.

DISCUSSION

Although the mechanisms of biomolecular damage and pathology induced by individual inflammatory oxidants are in general well characterized, an understanding of the complex interactions of ROS and RNS that are likely to occur at sites of inflammation is only just beginning to emerge. The studies reported herein show that the interactions of RNS and HOCl may be important under inflammatory conditions in vivo. We have shown that NO₂, the autoxidation product of NO in biological fluids, reacts with HOCl to produce a species that can nitrate, chlorinate, and dimerize biologically relevant phenolic compounds such as tyrosine, both free and within protein. The detection of NO₂-Tyr in a variety of pathologic states (21, 22, 23) has been used to indicate the formation of ONOO in vivo. However, reaction of tyrosine with the products of the NO₂/HOCl reaction also forms NO₂-Tyr. Hence, our results suggest that NO₂-Tyr should not be regarded as a specific marker of ONOO formation, but only as a marker of RNS.

Mechanism of NO₂/HOCl Reaction

It has long been thought (48) that the reaction of NO₂ with HOCl represented a classical example of an oxygen atom transfer reaction producing NO₃. However, this type of mechanism does not easily explain the nitration and chlorination reactions observed in our studies. Our data suggest a more complex mechanism involving the formation of reactive nitrating and chlorinating intermediates. One-electron oxidation of NO₂ by HOCl, producing the reactive radical species Cl and NO₂ is one possible pathway. Since HOCl is a poor one-electron oxidant, having an estimated one-electron reduction potential (E₀) in the range of +0.17 to +0.26 V at pH 7 (38), it is unlikely that a one-electron oxidation mechanism contributes, since the E value for the NO₂/NO₂ couple is approximately +1.04 V (49). In contrast, HOCl is a strong two-electron oxidant (E₀ = +1.08 V) (38) and would favor the conversion of NO₂ to the nitryl cation (NO⁺₂) or an ``NO⁺₂-like'' species. In addition to a direct two-electron oxidation of NO₂ by HOCl, a bimolecular substitution reaction between these two reactants could be involved. In fact, contrary to the reaction mechanism previously reported (48), Johnson and Margerum (45) have suggested that HOCl reacts with NO₂ by Cl⁺ transfer, rather than O atom transfer, to yield the intermediate Cl-NO₂, which then hydrolyzes to NO₃.

The absorbance spectrum of the product of the reaction between NO₂ and HOCl (Fig. 5B) was found to be similar to that of authentic Cl-NO₂ (Fig. 5C). The spectrum of the product(s) of the NO₂/HOCl reaction is typical of alkyl nitrites (R-ONO) (50) and therefore could also indicate the formation of a Cl-O-bonded species. In fact, the transfer of Cl⁺ to the negatively charged oxygen atom in NO₂ is likely and would produce the transient intermediate species Cl-ONO. It is possible that both reactions occur (Fig. 9), the extent to which each pathway initially predominates under neutral aqueous conditions is not known. Cl-ONO can exist as both the cis- and trans-rotamers (Fig. 9), where ab initio calculations predict that the energy difference between the two rotamers is approximately 3 kcal/mol, with the cis rotamer being the more stable (51). An analogy can be drawn between Cl-ONO and HO-ONO (peroxynitrous acid), where the energy difference between cis- and trans-HO-ONO is also calculated to be approximately 3 kcal/mol (52). Once formed, Cl-ONO can readily isomerize to Cl-NO₂ (53). We propose that intermediate Cl-ONO can isomerize in aqueous solution to Cl-NO₂ by at least two mechanisms (Fig. 9): 1) intramolecular rearrangement of trans-Cl-ONO involving migration of the chlorine atom to the nitrogen atom forming Cl-NO₂, or 2) unimolecular homolysis of the Cl-O bond in Cl-ONO to form a geminate pair of solvent-caged radicals Cl and NO₂, which undergo cage return to either reform Cl-ONO or by recombination to form Cl-NO₂ (Fig. 9). Some of the solvent-caged Cl and NO₂ can escape as ``free'' radicals and could potentially explain, in part, the radical mechanisms involved in the nitration reactions we observed in the NO₂/HOCl reaction. Since Cl-NO₂ is predicted to be 10.7 and 13.8 kcal/mol lower in energy than cis- and trans-Cl-ONO (51), respectively, the isomerization of Cl-ONO to Cl-NO₂ is a favorable process that shifts the equilibrium toward Cl-NO₂. Isomerization of cis Cl-ONO to Cl-NO₂ is probably not likely, because the large size of the chlorine atom, which would presumably preclude the migration of the chlorine atom to the nitrogen atom and, hence, the trans-rotamer of Cl-ONO, is probably the species that isomerizes to Cl-NO₂, analogous to the decomposition of trans-peroxynitrous acid (trans-HO-ONO). Whereas the isomerization of trans-HO-ONO leads to nitric acid (HO-NO₂), an unreactive end product, isomerization of Cl-ONO produces another highly reactive species (Cl-NO₂). Hence, Cl-ONO and the product of isomerization, Cl-NO₂, may both be reactive oxidants with nitrating and chlorinating activity.

We have shown that the product(s) of the reaction between NO₂ and HOCl, authentic Cl-NO₂, or the NO⁺₂ species (NO₂BF₄) react with tyrosine to form NO₂-Tyr and dityrosine. Although none of these reactants are themselves radicals, formation of dityrosine suggests the involvement of intermediate tyrosyl radicals. The nitration of aromatic compounds by NO⁺₂ is often thought to be a classical electrophilic aromatic substitution reaction, but there is strong evidence implicating electron transfer reactions and radical intermediates in these pathways (54). This reaction mechanism involves electron transfer from the aromatic to NO⁺₂, followed by radical pair collapse, and it would explain the detection of dityrosine in our studies. Hence, we are unable to distinguish between a nitration mechanism involving NO₂ or NO⁺₂ based solely on the formation of dityrosine. However, a divergence in the characteristics of the reaction mechanisms between NO⁺₂ and the reactive nitrating species formed by the reaction of NO₂ with HOCl is evident in their reactions with MPA, the O-methylated derivative of HPA, a substrate incapable of forming phenoxyl radicals. Whereas NO⁺₂ appears capable of nitrating MPA, both the product(s) of the NO₂/HOCl reaction and synthetic Cl-NO₂ fail to do so. Similarly, the inability of NO₂/HOCl and Cl-NO₂ to nitrate phenylalanine further argues against NO⁺₂ as the species involved in tyrosine nitration.

There is evidence suggesting that the reaction of Cl-NO₂ with alkenes and aromatic compounds involves homolytic processes yielding free radical intermediates (42), probably involving both Cl and NO₂. Collis et al. (43) have found that Cl-NO₂ decomposes at room temperature by homolysis to form Cl₂ and NO₂ as shown in Reaction 3, whereby these spontaneous decomposition products may be responsible, at least in part, for the chlorinating and nitrating behavior of Cl-NO₂ in our experiments. We suggest that phenolic nitration mediated by the NO₂/HOCl reaction involves NO₂. While the nitration reactions we observed appear to be radical-mediated, chlorination of aromatic amino acids such as phenylalanine (Fig. 8) appears to be executed largely by electrophilic aromatic substitution. In general, chlorination of aromatic compounds by HOCl, tert-butyl hypochlorite, and Cl₂ has been shown to be mediated by an ionic rather than a free radical mechanism (47). The nearly 2-fold increase in the relative formation of the m-Cl-Phe isomer by reactions of phenylalanine with both NO₂/HOCl and Cl-NO₂ (Fig. 8), however, suggests the potential contribution of a less selective mechanism of chlorination, potentially involving Cl. An active chlorinating species common to HOCl and Cl-NO₂ appears to be Cl₂. In fact, the formation of Cl₂ from HOCl and Cl-NO₂ can be rationalized and would explain the similarities in their chlorinating ability. HOCl is in equilibrium with Cl₂ in aqueous solution as shown in Reaction 4. The formation of Cl₂ from Cl-NO₂ has been proposed to occur by 1) the homolysis of two molecules of Cl-NO₂ to form two Cl which combine to form Cl₂ (Reaction 3), and 2) the reaction of Cl-NO₂ with H₂O (43) as shown in Reaction 5.  Although convincing evidence suggests an electrophilic substitution mechanism for these chlorination reactions, the possibility of a mechanism involving the addition of Cl to the aromatic ring cannot be excluded for reactions involving Cl-NO₂ or NO₂/HOCl.

The mechanisms of chlorination and nitration discussed thus far have primarily involved species derived from the decomposition of either Cl-NO₂ or Cl-ONO. However, as predicted by the stoichiometry of Reactions 3 and 5, these pathways are particularly favored when Cl-NO₂ or Cl-ONO are present at high concentrations. In vivo, however, Cl-NO₂ and Cl-ONO would be expected to be produced at rates that may favor the direct reaction of either species with biological substrates that are present in relative excess. In nonpolar organic solvents Cl-NO₂ has been shown to be an efficient agent for the nitration of aromatic compounds of intermediate reactivity (55). However, an increase either in the reactivity of the aromatic substrate (from benzene to phenol) or in the polarity of the solvent causes a marked decrease in the nitrating efficiency of Cl-NO₂ and a concomitant increase in the yield of chlorinated products (56). In fact, Obermeyer et al. (57) argued against the localization of a positive charge on the ``nitryl'' group of Cl-NO₂, where the structural characteristics of Cl-NO₂ contrast those of typical stable nitryl salts (i.e., NO⁺₂BF₄). Hence, reactions involving activated aromatic substrates such as tyrosine coupled with aqueous conditions would increase aromatic chlorination by Cl-NO₂, suggesting a change from ClNO⁺₂ character to a species with considerable Cl⁺NO₂ character. Our data suggest that Cl-NO₂ has significant Cl⁺ character in aqueous solution, and it is this functionality of Cl-NO₂ that dictates its reactivity.

We propose that Cl⁺NO₂ can react directly with tyrosine via electron transfer to yield an intermediate radical pair (tyrosyl radical-Cl-NO₂) (Fig. 10). Radical pair collapse of this complex leads to the rapid formation of Cl-Tyr and NO₂ (Fig. 10, reaction A), and is the major product formed by this reaction. This electron transfer-mediated reaction mechanism is analogous to the nitration of phenolic substrates by NO⁺₂ (54). Dissociation of the radical pair complex and subsequent oxidation of NO₂ by Cl (a strongly oxidizing species, E₀ of Cl/Cl = +2.2-2.6 V (Ref. 49)) results in the formation of ``free'' tyrosyl radical and NO₂ (Fig. 10, reaction B). Tyrosyl radical and NO₂ can rapidly combine to yield NO₂-Tyr (k = 3 × 10⁹ M¹ s¹ (Ref. 44)), and dityrosine formation can be envisaged by the combination of two tyrosyl radicals (Fig. 10, reactions C and D). This proposed mechanism predicts that the yields of the various tyrosine modification products will be on the order of Cl-Tyr  NO₂-Tyr > dityrosine, consistent with the data presented herein. The proposed reaction pathway also illustrates the dependence of radical intermediates in the nitration of phenolic compounds by Cl-NO₂, as suggested by our data.

Since Cl-ONO is a potential transient intermediate in the formation of the reactive species Cl-NO₂ (Fig. 9), part of the reactivity of NO₂/HOCl may be attributed to Cl-ONO. Analogous to a proposed mechanism of ONOOH reactivity (9, 58), a vibrationally excited intermediate derived from trans-Cl-ONO (Cl-ONO^*) may be formed during its isomerization to Cl-NO₂ and contribute to nitration and chlorination of tyrosine by direct reaction. The reaction mechanisms we propose for Cl-NO₂ and Cl-ONO are analogous to those recently determined for ONOOH, whereby both direct and indirect reactions with oxidizable substrates can occur (59). A more detailed examination of the reaction kinetics and thermodynamic considerations is necessary in order to elucidate which of the proposed mechanisms predominate.

The activation and accumulation of neutrophils at sites of tissue injury, leading to the formation of HOCl and other ROS/RNS, is an essential feature of inflammation. Our data suggest that the reaction of HOCl with NO₂, derived from NO produced by other phagocytes (30), endothelial cells (2), or epithelial cells (37), may be a contributing pathway operative in tissue injury at sites of inflammation. Moreover, since Cl-NO₂ is conceivably formed in vivo and is capable of nitrating tyrosine residues, our findings may imply a role for this reaction pathway where NO₂-Tyr is detected in cases of acute inflammatory lung injury (21), atherosclerosis (22), and rheumatoid arthritis (23), a phenomena previously ascribed to ONOO formation. In fact, increased levels of NO₂ have been observed in similar cases (36, 60, 61) and further suggest the potential involvement of this pathway in vivo. Recent studies have demonstrated that myeloperoxidase, the phagocytic enzyme that catalyzes HOCl formation, is a component of sputum from cystic fibrosis patients (62), as well as other inflammatory lung diseases, and of human atherosclerotic tissue (63), underscoring the potential importance of HOCl in the pathology of each of these cases. We propose, then, that tyrosine nitration by Cl-ONO and/or Cl-NO₂, formed by the reaction of NO₂ with HOCl, represents an important and additional mechanism for inflammation-mediated tyrosine nitration in vivo, independent of ONOO formation.

Our findings indicate that NO₂ may not be an appropriate marker of NO production by neutrophils or at sites of inflammation, since it is potentially removed by reaction with simultaneously produced HOCl. Determination of NO production in tissues and fluids of patients with acute and chronic inflammation, as measured by NO₂ ``accumulation,'' is likely a gross underestimate. Moreover, NO₂ has been shown to modulate the bactericidal activity of HOCl, its mechanism proposedly mediated by direct reaction of these two species (39, 41). Our findings suggest, then, that the reaction product, Cl-NO₂, is a strongly oxidizing species that may serve as an antimicrobial agent in its own right.

An analogous reaction between hypobromous acid, a product formed by oxidation of Br catalyzed by eosinophil peroxidase (64), and NO₂ forming Br-ONO and/or Br-NO₂ can be envisioned. In fact, Reaction 6 may represent a general mechanism by which hypohalous acids (HOX) react with NO₂ to produce species capable of oxidizing biological molecules. Collectively, these reaction pathways could therefore represent an important host defense mechanism and a novel pathway for inflammation-mediated tissue injury.

We report here that NO₂ and HOCl react to form the reactive intermediates Cl-NO₂ and/or Cl-ONO, species that are capable of nitrating, chlorinating, and dimerizing phenolic compounds such as tyrosine. Our data suggest that NO₂-Tyr is not necessarily a specific marker of ONOO formation in vivo and that Cl-NO₂ and Cl-ONO may be important and previously unconsidered oxidants produced at sites of inflammation.