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J. Biol. Chem., Vol. 277, Issue 20, 17415-17427, May 17, 2002

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A Tale of Two Controversies

DEFINING BOTH THE ROLE OF PEROXIDASES IN NITROTYROSINE FORMATION IN VIVO USING EOSINOPHIL PEROXIDASE AND MYELOPEROXIDASE-DEFICIENT MICE, AND THE NATURE OF PEROXIDASE-GENERATED REACTIVE NITROGEN SPECIES^*

Marie-Luise Brennan^§, Weijia Wu, Xiaoming Fu, Zhongzhu Shen, Wei Song, Heather Frost^¶, Caryn Vadseth^¶, Laura Narine, Elizabeth Lenkiewicz, Michael T. Borchers, Aldons J. Lusis^**, James J. Lee, Nancy A. Lee, Husam M. Abu-Soud, Harry Ischiropoulos^¶, and Stanley L. Hazen^§

From the  Department of Cell Biology and the ^§ Department of Cardiovascular Medicine and Center for Cardiovascular Diagnostics and Prevention, Cleveland Clinic Foundation, Cleveland, Ohio 44195, the ^¶ Stokes Research Institute, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania 19104, the  Departments of Biochemistry and Molecular Biology, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259, and the ^** Departments of Medicine, Human Genetics, Microbiology, Immunology, and Molecular Genetics, UCLA, Los Angeles, California 90095

Received for publication, December 27, 2001, and in revised form, February 22, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Nitrotyrosine is widely used as a marker of post-translational modification by the nitric oxide (^·NO, nitrogen monoxide)-derived oxidant peroxynitrite (ONOO). However, since the discovery that myeloperoxidase (MPO) and eosinophil peroxidase (EPO) can generate nitrotyrosine via oxidation of nitrite (NO), several questions have arisen. First, the relative contribution of peroxidases to nitrotyrosine formation in vivo is unknown. Further, although evidence suggests that the one-electron oxidation product, nitrogen dioxide (^·NO₂), is the primary species formed, neither a direct demonstration that peroxidases form this gas nor studies designed to test for the possible concomitant formation of the two-electron oxidation product, ONOO, have been reported. Using multiple distinct models of acute inflammation with EPO- and MPO-knockout mice, we now demonstrate that leukocyte peroxidases participate in nitrotyrosine formation in vivo. In some models, MPO and EPO played a dominant role, accounting for the majority of nitrotyrosine formed. However, in other leukocyte-rich acute inflammatory models, no contribution for either MPO or EPO to nitrotyrosine formation could be demonstrated. Head-space gas analysis of helium-swept reaction mixtures provides direct evidence that leukocyte peroxidases catalytically generate ^·NO₂ formation using H₂O₂ and NO as substrates. However, formation of an additional oxidant was suggested since both enzymes promote NO-dependent hydroxylation of targets under acidic conditions, a chemical reactivity shared with ONOO but not ^·NO₂. Collectively, our results demonstrate that: 1) MPO and EPO contribute to tyrosine nitration in vivo; 2) the major reactive nitrogen species formed by leukocyte peroxidase-catalyzed oxidation of NO is the one-electron oxidation product, ^·NO₂; 3) as a minor reaction, peroxidases may also catalyze the two-electron oxidation of NO, producing a ONOO-like product. We speculate that the latter reaction generates a labile Fe-ONOO complex, which may be released following protonation under acidic conditions such as might exist at sites of inflammation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Nitric oxide (^·NO, nitrogen monoxide)-derived oxidants are believed to participate in antimicrobicidal activities as part of normal host defenses, cell signaling reactions, and a variety of homeostatic mechanisms. However, excess or inappropriate formation of NO-derived oxidants has also been linked to oxidative tissue injury and protein modification through nitration of tyrosine residues in inflammatory disorders (1-4). Evidence of a role for NO-derived oxidants in oxidative damage is primarily based upon immunohistological and mass spectrometric detection of nitrotyrosine (NO₂-Tyr) (5-7). Although originally thought to serve as a specific marker for protein oxidation by peroxynitrite (ONOO), it is now clear that a variety of reactive species may promote nitration of protein tyrosine (Tyr) residues in vitro. However, neither the identity nor the relative contribution of distinct intermediates in nitration reactions in vivo is known.

By far the most widely studied nitrating intermediate is ONOO, the reactive species formed during near diffusion-limited interaction of superoxide (O) and ^·NO (1). ONOO promotes both nitration and hydroxylation reactions of targets and may be described as possessing chemical reactivity akin to a pair of caged radicals: hydroxyl radical (^·OH) and nitrogen dioxide (^·NO₂) (1). Recent studies demonstrate that at physiological levels of carbon dioxide/bicarbonate (CO₂/HCO), ONOO predominantly exists as the more potent nitrating species, nitrosoperoxocarbonate (ONOOCO₂) (8-10). Production of ^·NO and O is a characteristic feature at sites of inflammation. Consequently, both potential adverse effects of excess ^·NO production and the detection of nitrotyrosine have been predominantly attributed to formation of ONOO and ONOOCO₂ (1-4, 8-10).

Over the past several years, substantial evidence has accrued suggesting that leukocyte peroxidases may also serve as enzymatic participants in generation of reactive nitrogen species (11-15). Neutrophils, monocytes, and eosinophils are the terminal effector cells of the innate immune system and are abundant cellular constituents at sites of inflammation. The leukocyte peroxidases myeloperoxidase (MPO)¹ and eosinophil peroxidase (EPO) are some of the most abundant proteins in these cells (16-18). They utilize hydrogen peroxide (H₂O₂) and a variety of organic and inorganic low molecular weight substrates to generate an array of reactive oxidants and diffusible radical species, including nitrating intermediates (19-22). MPO and EPO are unique in their ability to use halides and pseudohalides as co-substrates to generate hypohalous acids. At plasma levels of halides (Cl, 100 mM; Br, 100 µM), MPO uses Cl as co-substrate to form hypochlorous acid (HOCl) (23, 24) and molecular chlorine (Cl₂) (25) as chlorinating oxidants. In contrast, EPO selectively utilizes plasma levels of Br over Cl to generate hypobromous acid (26). We have shown that chlorotyrosine (Cl-Tyr) and bromotyrosine (Br-Tyr) serve as specific markers of protein oxidation by MPO- and EPO-generated chlorinating and brominating oxidants, respectively, in vitro (27-29) and in vivo (30-33).

Because of the high levels of halides in biological matrices, halogenating oxidants were thought to account for the major reactive species formed by MPO and EPO. However, recent in vitro studies with purified enzymes and isolated leukocytes have shown that MPO (11, 12, 14, 28) and EPO (13, 33) are capable of promoting aromatic nitration reactions through the use of nitrite (NO) as substrate, even in the presence of plasma levels of halides (28, 33). Peroxidases are capable of catalyzing both one- and two-electron oxidation reactions of substrates (19-22,34). Consequently, both one- and two-electron oxidation products of NO have been suggested to serve as potential diffusible oxidants formed by MPO and EPO (11, 35). The formal oxidation states of nitrogen in NO and its potential one- and two-electron oxidation products include: nitrite (NO; +3), nitrogen dioxide (^·NO₂; +4), nitronium cation (NO₂⁺; +5), and peroxynitrite (ONOO; +5). Although the reactive nitrogen species formed following peroxidase-catalyzed oxidation of NO have not been directly established, rapid kinetics studies support formation of the one-electron oxidation product, ^·NO₂ (36). It has been proposed that peroxidases might also generate the two-electron oxidation product of NO, ONOO, in reactions analogous to the addition of OH to halides forming hypohalous acids (35). However, direct evidence to support formation of ONOO by peroxidases has not yet been reported.

The relative contributions of peroxidases to formation of ^·NO-derived oxidants in vivo are unknown. Similarly, few studies have characterized the nature of the nitrating intermediate(s) formed following peroxidase-catalyzed oxidation of NO. Herein we have utilized recently developed MPO knockout (KO) (31) and EPO-KO (32) mice to provide evidence for the contributions of leukocyte peroxidases to protein NO₂-Tyr formation in vivo using multiple distinct models of inflammation. We also provide direct evidence that ^·NO₂ gas is a physiological oxidant formed as a preferred product during MPO- and EPO-catalyzed oxidation of NO. Finally, we provide several lines of evidence suggesting that MPO and EPO can also utilize NO as substrate to form a ONOO-like oxidant, which may be liberated from a labile Fe-ONOO complex at acid pH.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Solvents H₃PO₄, NaH₂PO₄, and Na₂HPO₄ were purchased from Fisher Chemical Co. Chelex 100 resin (200-400 mesh, sodium form) was purchased from BioRad. Methane sulfonic acid and bromide were from Fluka Chemical Co. (Ronkonkoma, NY). L-[¹³C₆]tyrosine, [¹³C₉¹⁵N₁]tyrosine, and L-[²H₄]tyrosine were purchased from Cambridge Isotopes, Inc. (Andover, MA). Deoxy[1',2'-³H]guanosine, 5'-triphosphate was obtained from Amersham Biosciences, Inc. All other reagents were purchased from Sigma unless otherwise indicated. All gasses were of the highest quality available (Praxair, Cleveland, OH).

Animals-- The animal studies described were all performed using approved protocols from the Animal Research Committees of the Cleveland Clinic Foundation and Mayo Clinic Foundation. 129/SvJ age- and sex-matched EPO-KO and wild-type (WT) mice were used for the allergen lung challenge and helminth antigen-induced peritonitis studies. Age- and sex-matched C57BL/6J MPO-KO and WT mice (>96% genetic homogeneity) were used for candidiasis and thioglycollate/zymosan-induced peritonitis studies.

Aeroallergen Lung Challenge Model-- EPO-KO and WT mice were sensitized or sham treated by intraperitoneal injection with either normal saline as control or 20 µg of ovalbumin (grade IV, Sigma) and 2.25 mg of Imject® Alum (Pierce) on days 0 and 14. On days 24, 25, and 26, animals were challenged with 20-min inhalations of a 1% ovalbumin or normal saline aerosol. On day 28, bronchoalveolar lavage fluid was obtained by normal saline lavage, and differentials were performed on cytospin preparations of isolated cells. Lung tissue was harvested and immediately placed in antioxidant buffer consisting of phosphate-buffered saline (PBS) supplemented with 100 µM butylated hydroxytoluene (BHT) and 100 µM diethylenetriaminepentaacetic acid (DTPA) overlaid with argon and stored at -80 °C until analysis.

Helminth Protein-induced Peritonitis Model-- EPO-KO and WT mice were sensitized by subcutaneous injection with 250 µg of whole protein extract from the helminth Mesocestoides corti and 8 × 10⁹ heat-killed pertussis organisms (Michigan Department of Public Health, Lansing, MI). Animals were challenged on day 21 by intraperitoneal injection with 200 µg of M. corti. At 72 h, elicited peritoneal leukocytes (typically >2 × 10⁶ cells, 25% eosinophils) were activated by intraperitoneal injection with zymosan (250 mg/kg of body weight). Peritoneal lavage was performed 4 h later with PBS containing 100 µM BHT and 100 µM DTPA, and centrifugation at 1000 rpm for 10 min at 4 °C was used to separate recovered peritoneal cells and lavage fluid. Cell pellets were resuspended in PBS containing 100 µM BHT and 100 µM DTPA, and differentials were performed on cytospin preparations of isolated cells. Both cells and cell-free lavage fluid were overlaid with argon, immediately frozen, and stored at 80 °C until analysis. Superoxide production was measured as the superoxide dismutase-inhibitable reduction of cytochrome c (37, 38) using eosinophils isolated from EPO-KO and WT mice on an interleukin-5 transgenic background (to induce eosinophilia) as described (32), and neutrophils were isolated from MPO-KO and WT mice as described (31).

Candidiasis Model-- Candida albicans was cultured in Sabouraud dextrose broth, collected by centrifugation, washed in ice-cold water, and resuspended in sterile normal saline. Inocula were prepared and injected intraperitoneally into MPO-KO and WT mice. Peritoneal lavage was performed 24 h later with PBS containing 100 µM BHT and 100 µM DTPA, and centrifugation at 1000 rpm for 10 min at 4 °C was used to separate recovered peritoneal cells and lavage fluid. Cell pellets were resuspended in PBS containing 100 µM BHT and 100 µM DTPA, and differentials were performed on cytospin preparations of isolated cells. Both cells and cell-free lavage fluid were overlaid with argon, immediately frozen, and stored at 80 °C until analysis.

Zymosan-induced Peritonitis Model-- MPO-KO and WT animals were injected intraperitoneally with 1 ml of 4% thioglycollate broth. Twenty hours after recruitment, mice were injected with zymosan (250 mg/kg). Peritoneal lavage was performed 4 h later with PBS containing 100 µM BHT and 100 µM DTPA, and centrifugation at 1000 rpm for 10 min at 4 °C was used to separate recovered peritoneal cells and lavage fluid. Cell pellets were resuspended in PBS containing 100 µM BHT and 100 µM DTPA, and differentials were performed on cytospin preparations of isolated cells. Both cells and cell-free lavage fluid were overlaid with argon, immediately frozen, and stored at 80 °C until analysis.

Cell Counts and Differentials-- Cells were visually counted using a hemacytometer. Wright's Giemsa stain was used to stain samples, and the cell type was distinguished microscopically based on staining morphology.

Sample Processing-- Lung tissue and cell pellets were thawed and immediately homogenized in PBS containing 100 µM BHT and 100 µM DTPA using a Potter-Elvehjem tissue homogenizer with a PTFE pestle. Protein concentrations of lung tissue homogenate, cell pellet homogenates, and supernatants were determined using a Bradford-based Bio-Rad protein assay with IgG as the protein standard.

Preparation of Stable Isotope-labeled Oxidized Tyrosine Standards-- 3-Chloro[¹³C₆]tyrosine and 3-chloro[¹³C₉¹⁵N]tyrosine standards were prepared and isolated following exposure of either L-[¹³C₆]tyrosine or L-[¹³C₉¹⁵N]tyrosine to HOCl (1:1, mol/mol) in 20 mM phosphoric acid as described (33). 3-Bromo[¹³C₆]tyrosine and 3-bromo[¹³C₉¹⁵N]tyrosine standards were similarly synthesized by exposure of either L-[¹³C₆]tyrosine or L-[¹³C₉¹⁵N]tyrosine to hypobromous acid (1:1, mol/mol) and isolation by preparative reverse phase HPLC (33). 3-Nitro[¹³C₆]tyrosine and 3-nitro[¹³C₉¹⁵N₁]tyrosine were synthesized by reaction of either L-[¹³C₆]tyrosine or L-[¹³C₉¹⁵N₁]tyrosine to ONOO (1:1, mol/mol) and isolated as described (33).

Protein Hydrolysis-- Protein was delipidated and desalted using two sequential extractions with a single phase mixture of H₂O/methanol/H₂O-saturated diethyl ether (1:3:8 v/v/v). Oxidized tyrosine standards (2 pmol each) and universal labeled tyrosine (2 nmol) were added to protein pellets. For nitrotyrosine analyses, proteins were hydrolyzed by incubating the desalted protein pellet with degassed 6N HCl supplemented with 1% phenol for 24 h under argon atmosphere. For chlorotyrosine and bromotyrosine analyses, samples were hydrolyzed in degassed 4N methane sulfonic acid supplemented with 1% phenol for 24 h at 100 °C under argon atmosphere. Amino acid hydrolysates were resuspended in Chelex-treated water and applied to mini solid-phase C18 extraction columns (Supelclean LC-C18SPE mini-column, 3 ml; Supelco, Inc., Bellefone, PA) pre-equilibrated with 0.1% trifluoroacetic acid. Following sequential washes with 2 ml of 0.1% trifluoroacetic acid, oxidized tyrosines and tyrosine were eluted with 2 ml of 30% methanol in 0.1% trifluoroacetic acid, dried under vacuum, and then analyzed by mass spectrometry as described below.

Mass Spectrometry (MS)-- For nitrotyrosine analyses, tandem mass spectrometry was performed using electrospray ionization and detection with an ion trap mass spectrometer (LCQ Deca, ThermoFinigann, San Jose, CA) interfaced with a Thermo SP4000 high performance liquid chromatograph (HPLC). Samples were suspended in equilibration solvent (H₂O with 0.1% formic acid) and injected onto an Ultrasphere C18 column (Phenominex, 5 µm, 2.0 × 150 mm). L-Tyrosine and its oxidation products were eluted at a flow rate of 200 µl/min using a linear gradient generated against 0.1% formic acid in methanol, pH 2.5, as the second mobile phase. Analytes were monitored in positive ion mode with full scan product ion MS/MS at unit resolution. Response was optimized with a spray voltage setting of 5 kV and a spray current of 80 µA. The heated capillary voltage was set at 10 V and the temperature to 350 °C. Nitrogen was used both as sheath and auxiliary gas, at a flow rate of 70 and 30 arbitrary units, respectively. The precursor ion isolation width was 1.0 and 3.0 for nitrotyrosine and tyrosine, respectively. The analyte abundance was evaluated by measuring the chromatographic peak areas of selected product ions extracted from the full scan total ion chromatogram, according to the corresponding ion trap product ion spectra. The ions monitored for each analyte were: 3-nitro[¹²C₆]tyrosine (mass-to-charge-ratio (m/z) 227181, and 210); 3-nitro[¹³C₆]tyrosine (m/z 233187, and 216); 3-nitro[¹³C₉¹⁵N₁]tyrosine (m/z 237190, and 219); [¹²C₆]tyrosine (m/z 182136, and 165); and [¹³C₉¹⁵N₁]tyrosine (m/z 192145, and 174). The maximum ion injection time was 200 ms; a scan rate was used that permitted a minimum sampling rate of at least 9 points/chromatographic peak. Tyrosine and nitrotyrosine were base line-resolved under the HPLC conditions employed, permitting programming of the LCQ Deca for analysis over a span of 0 to 7 min for detection of tyrosine isotopomers and from 7 min on for detection of 3-nitrotyrosine isotopomers.

For 3-chlorotyrosine and 3-bromotyrosine analyses, gas chromatography/mass spectrometry (GC/MS) was performed after derivatization of amino acids to their n-propyl/heptafluorylbutyryl or n-propyl/pentafluorylpropionyl derivatives using a Finnigan Voyager GC/MS in the negative ion chemical ionization mode, as previously described (30).

For all analyses, results were normalized to the content of the precursor amino acid L-tyrosine, which was monitored within the same injection of each oxidized amino acid. Both 3-chlorotyrosine and 3-bromotyrosine were routinely detected at 1 fmol on-column with a signal to noise ratio of >10:1, and 3-nitrotyrosine was routinely detected at 10 fmol on-column with a signal to noise ratio of >10:1. When presented normalized to the level of the precursor amino acid tyrosine, all tyrosine oxidation products were detectable at <1 µmol/mol tyrosine under the conditions employed. Intrapreparative formation of 3-bromo[¹³C₉¹⁵N]tyrosine, 3-chloro[¹³C₉¹⁵N]tyrosine, or 3-nitro[¹³C₉¹⁵N]tyrosine was routinely monitored for all analyses and was usually negligible under the sample preparation conditions employed (i.e. 5% of the level of the natural abundance product observed). On the rare occasion where intrapreparative nitration or halogenation exceeded 5% of the level of the natural abundance analyte monitored, repeat sample preparation and mass spectrometric analyses were performed.

Oxidation of Targets by ONOO or by Peroxidase-H₂O₂-NO System-- Purified human MPO and EPO free from cross-contamination were isolated and quantified as described (33). ONOO (NO-free) was quantified spectrophotometrically (₃₀₂ = 1670 M¹cm¹) (39). The concentration of reagent H₂O₂ was also determined spectrophotometrically (₂₄₀ = 39.4 M¹cm¹) (40). NO₂-G (8-nitroguanine) and NO₂-dG (8-nitrodeoxyguanosine) were prepared as standards by reaction of appropriate precursors with ONOO (1:1, mol:mol), isolation by preparative HPLC, and structure and purity confirmation by NMR and LC/ESI/MS prior to use. All of the solutions and buffers were freshly prepared using Chelex-treated water. Once prepared, the solutions and buffers were bubbled with argon for 15 min immediately prior to use. Unless otherwise specified, reactions were initiated by the addition of oxidant (ONOO or H₂O₂, 1 mM final concentration) in 150 mM phosphate buffer containing 115 µM DTPA. Target concentrations were (in mM): 2-dG, 2.5; salicylate, 5; tyrosine, 1.5; phenylalanine, 5. Where indicated, peroxidase reactions included the following at 37 °C for 60 min: EPO, 57 nM; MPO, 57 nM; H₂O₂, 1 mM final added in 4 × 250 µM increments each 10 min; and NO, 1 mM.

Reverse Phase HPLC Quantification of Nitration and Hydroxylation Oxidation Products Generated in Model Systems in Vitro-- All analytes were quantified using external calibration curves constructed with authentic commercial or synthetic standards. The identity of each analyte was confirmed initially by studies employing HPLC with on-line photodiode array and LC/MS (and MS/MS analysis, as needed, for isomer conformation). Subsequent routine quantification of nitration products of free tyrosine, 2-dG, phenylalanine, and 3-(4-hydroxyphenyl)propanoic acid (HPA) were routinely performed on a Beckman Gold HPLC system equipped with photodiode array detector. Product identity was confirmed by retention time, UV-visible spectra of peaks, and tandem mass spectra of isolated material in peaks compared with authentic standards. Hydroxylated products were similarly quantified using either HPLC with on-line photodiode array detection or coulometric array detection as described below.

Nitrotyrosine separations were performed on a C₁₈ column (Beckman Ultrasphere, 5 µm, 4.6 × 250 mm) equilibrated with solvent A (0.1% trifluoroacetic acid, pH 2.5). L-Tyrosine and its oxidation products were eluted at a flow rate of 1 ml/min with a linear gradient generated with solvent B (0.1% trifluoroacetic acid in methanol, pH 2.5) as follows: 0% solvent B for 5 min, 0-100% solvent B over a span of 30 min, 100% solvent B for 10 min. Nitration products of HPA were separated and quantified similarly as described previously (33). Nitration products of phenylalanine were quantified on a C₁₈ column (Beckman Ultrasphere, 5 µm, 4.6 × 250 mm) equilibrated with solvent A (0.1% trifluoroacetic acid, pH 2.5). 2-Nitrophenylalanine and 4-nitrophenylalanine were eluted at a flow rate of 1 ml/min with a linear gradient comprising solvent B (0.1% trifluoroacetic acid in methanol, pH 2.5). Nitration products of 2-dG (both NO₂-G and NO₂-dG were generated) were eluted at 1 ml/min flow rate with a gradient comprising 20 mM ammonium acetate. Hydroxylation and nitration products of salicylic acid were separated on a C₁₈ column (Beckman Ultrasphere, 5 µm, 4.6 × 250 mm) equilibrated with a mobile phase containing 30 mM sodium citrate and 27 mM sodium acetate, which was adjusted to pH 4.7 with 5 M H₂SO₄. 2,3- and 2,5-dihydroxybenzoic acid (2,3-DHB and 2,5-DHB) elute within 20 min, and nitrobenzoate was eluted using a linear gradient generated over a span of 30 min with a mobile phase of 100% acetonitrile.

Hydroxylation products of 2-dG (8-hydroxydeoxyguanosine, 8-OHdG), phenylalanine (ortho-, meta- and para-tyrosine), and tyrosine (3,4-dihydroxyphenylalanine (DOPA)) were routinely quantified by reverse phase HPLC with electrochemical (coulometric) detection on a CoulArray (ESA, Cambridge, MA) equipped with variable wavelength UV detector and electrochemical cells (six channels) arranged in series and set to increasing specified potentials (in mV): channel 1, 50; channel 2, 150; channel 3, 300; channel 4, 480; channel 5, 650; channel 6, 800. Chromatographic separations of these oxidation products were performed on a Progel TSK ODS-80 column (5 µm, 4.6 × 250 mm) equilibrated with mobile phase comprising lithium phosphate, 3 mg/liter lithium dodecyl sulfate, pH 3.2. Products were eluted at a flow rate of 1 ml/min with a nonlinear gradient generated with mobile phase comprising 50% methanol, 15 mM lithium phosphate, and 3 mg/liter lithium dodecyl sulfate, pH 3.2. Peak identity was established by demonstrating the appropriate retention time, redox potential, and ratio of integrated currents in adjacent channels and by the method of standard additions for each analyte.

The deaminated analog of tyrosine, HPA, was used in studies examining the capacity of purified peroxidases to catalyze ONOO-mediated nitration of targets. HPA and its nitrated product, 3-(4-hydroxy-3-nitrophenyl)propanoic acid (NO₂-HPA), were quantified by reverse phase HPLC with electrochemical (coulometric) detection as recently described (33). Peak identity was established by demonstrating the appropriate retention time, redox potential, and ratio of integrated currents in adjacent channels and by the method of standard additions. Authentic standards of NO₂-HPA were prepared by reaction of HPA with a molar equivalent of ONOO, and following HPLC isolation, its structure was confirmed by LC/ESI/MS/MS (33).

On-line Detection of Nitrogen Dioxide Generation by Leukocyte Peroxidases-- Purified EPO or MPO were individually added into media containing H₂O₂ and NO. The formation of ^·NO₂ was then followed in real time by analysis of the head-space gas in the reaction mixtures. The gas phase was swept by a stream of helium into a 750 °C-heated converter consisting of a quartz tube coated with gold alloy and filled with quartz wool where ^·NO₂ was reduced to ^·NO. ^·NO was then detected by chemiluminescence after reaction with ozone using a ^·NO detector (Sievers Instrument model 280, Boulder, CO). For a typical measurement, a base line was established first prior to initiation of the reaction by diverting gas flow around the gold-plated tube using the bypass mode. Upon initiation of the reaction, gas flow was then diverted through the heated gold-coated quartz tube. Specificity of the detection system for ^·NO₂ formation, and not ^·NO itself, within reactions mixtures was routinely shown by diverting gas flow to bypass the gold catalyst, resulting in complete and rapid decline of signal. Specificity of the electrode for ^·NO₂ was further confirmed in separate studies using authentic gasses.

Statistical Analysis-- Data were analyzed using an unpaired Student's t test. The threshold for significance was set at p  0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Controversy 1: Do Peroxidases Generate Nitrotyrosine in Vivo?-- To examine the potential role of leukocyte peroxidases in NO₂-Tyr formation in vivo, it was fundamentally important to ascertain that EPO and MPO were catalytically active within the model to be examined. This was accomplished by stable isotope dilution GC/MS quantification of protein Br-Tyr and Cl-Tyr content, respectively. Specificity of protein-bound Br-Tyr and Cl-Tyr as "molecular fingerprints" for EPO- and MPO-catalyzed oxidation, respectively, also had to be established by demonstrating that there was no significant formation of the biomarker in the correspondingly treated KO mouse of homogeneous genetic background. It was also critically important to evaluate the role of leukocyte peroxidases in NO₂-Tyr formation in vivo by utilizing multiple distinct models of inflammation with both EPO-KO and MPO-KO mice and to sample distinct protein compartments (e.g. tissue versus cell associated versus lavage supernatants) where practical. Models were also selected where comparable leukocyte recruitment between base line and challenged WT and KO mice were observed. Finally, we felt that it was important initially to characterize leukocytes from MPO-KO and EPO-KO mice versus their respective WT controls for O formation. Neutrophils harvested from WT versus MPO-KO mice demonstrated comparable levels of O production in response to phorbol ester stimulation (p = 0.85; data not shown). Interestingly, superoxide production was consistently enhanced ~1.6-fold in eosinophils isolated from EPO-KO mice compared with WT mice of homogeneous genetic background (26 ± 4.8 versus 41.5 ± 7.3 nmol/10⁶ cells over 1 h for WT versus EPO-KO; p < 0.001).

Eosinophil Peroxidase Generates Nitrotyrosine, a Marker of Protein Oxidation by ^·NO-derived Oxidants in Vivo-- In recent studies we demonstrated that EPO is the major pathway for generating protein-bound Br-Tyr in vivo using an aeroallergen challenge model in WT and EPO-KO mice (32). In this widely used mouse model of asthma (41, 42), animals are pre-sensitized to antigen (ovalbumin) and then exposed to nebulized antigen, eliciting a robust eosinophil-rich leukocyte infiltration in lung and airways. Leukocyte recruitment (at base line, 24 h, and 72 h) to the lungs and airways of WT and EPO-KO mice in this model has been reported as similar (32), and this similarity was reconfirmed by demonstrating comparable total cell counts and differentials in bronchoalveolar lavage recovered from WT and EPO-KO mice (p > 0.60 for all WT versus EPO-KO comparisons; data not shown). GC/MS analyses revealed significant levels of protein-bound Br-Tyr in lung and airways tissues of allergen-challenged WT mice (72 h post-challenge) but no detectable Br-Tyr in EPO-KO mice (32). These results confirm that EPO is catalytically active in this model and that Br-Tyr is a specific marker for protein oxidation by EPO.

To examine the role of EPO in NO₂-Tyr formation in this mouse model of asthma, stable isotope dilution LC/ESI/MS/MS quantification of protein-bound NO₂-Tyr was performed on lungs harvested 72 h after allergen challenge. Basal levels of NO₂-Tyr from WT and EPO-KO lungs were comparable following challenge with diluent (normal saline) (Fig. 1). Following aerosolized ovalbumin (aeroallergen) exposure, the content (expressed as a product/precursor ratio, mol/mol) of protein-bound NO₂-Tyr increased 2.3-fold in WT lungs, but did not significantly change in EPO-KO mice. This finding corresponded to a 70% reduction (p < 0.01) in the NO₂-Tyr formed over basal levels in lung proteins in the EPO-KO versus WT mice (i.e. only a 13 µmol/mol increase in KO compared with a 46 µmol/mol increase in WT) in response to the aeroallergen challenge (Fig. 1). When protein-bound NO₂-Tyr levels were expressed instead in units proportional to the total amount present/mg of lung protein, i.e. ([µmol NO₂-Tyr/mol Tyr] × [mg protein/ml lung homogenate]), comparable results were observed (data not shown). Collectively, these results demonstrate that EPO plays a dominant role in protein-bound NO₂-Tyr formation following aeroallergen challenge in the ovalbumin mouse model of asthma. They also demonstrate that EPO does not significantly contribute to the production of basal levels of NO₂-Tyr in proteins from lungs of mice.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   3-Nitrotyrosine formation in proteins recovered from lung tissue after aeroallergen challenge. EPO-KO and WT animals were sensitized with ovalbumin (Ova) or sham sensitized with normal saline on days 0 and 14 and then challenged later on days 24, 25, and 26 with aerosolized ovalbumin or normal saline as indicated. Lungs were harvested on day 28, and protein levels of bromotyrosine (BrY) (from Ref. 32) and nitrotyrosine were determined by stable isotope dilution GC/MS and LC/ESI/MS/MS, respectively. Results are normalized to the protein content of tyrosine (Y), the precursor amino acid. Lines indicate mean levels within each group. Values represent mean ± S.D.

The Contribution of Eosinophil Peroxidase to Nitrotyrosine Formation in Vivo Is Model-specific and Does Not Always Parallel the Extent of Bromotyrosine Formation-- The mouse ovalbumin challenge model of asthma does not elicit a significant degree of eosinophil degranulation (42), and only modest levels of bromotyrosine are observed in bronchoalveolar lavage proteins recovered from mice as compared with those noted in bronchoalveolar proteins recovered from human asthmatics following allergen challenge (30) or severe asthma exacerbation (33). We therefore sought to develop an alternative inflammatory model where EPO-mediated protein oxidation was more robust, as monitored by GC/MS formation of protein-bound Br-Tyr. We have previously reported that sensitization of mice with intraperitoneal injection of helminth protein from M. corti produces a dramatic eosinophil-rich infiltration of leukocytes to the peritoneum 72 h after rechallenge of both WT and EPO-KO mice (32). Using this model, we noted comparable recruitment of leukocytes between the mice, as monitored by total cell count and differentials (p > 0.70 for all WT versus EPO-KO comparisons; data not shown). Interestingly, despite robust recruitment of eosinophils to the peritoneum (typically >2 × 10⁶ cells; ~25-30% eosinophils), only modest levels of Br-Tyr were formed in WT mice (Fig. 2A). Subsequent intraperitoneal injection with yeast cell wall (zymosan), a potent trigger of leukocyte activation and degranulation, produced a dramatic rise in Br-Tyr formation in WT mice. Again, no detectable protein-bound Br-Tyr was formed in any proteins recovered from EPO-KO mice (Fig. 2A).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Protein modification by brominating and nitrating oxidants in helminth-induced peritonitis model. GC/MS analysis of bromotyrosine content (A) and LC/ESI/MS/MS analysis of nitrotyrosine content (B) in extracellular lavage proteins from an acute eosinophilic peritonitis model. EPO-KO and WT mice were sensitized by subcutaneous injection with whole protein extract from the helminth M. corti. Animals were challenged on day 21 by intraperitoneal injection with either normal saline (NS) or M. corti to elicit an eosinophil-rich peritoneal infiltrate. Levels of bromotyrosine and nitrotyrosine were measured in peritoneal lavage recovered 72 h later. Where indicated, for some animals, peritoneal lavage was recovered following M. corti challenge (72 h) and subsequent to intraperitoneal injection of zymosan (4 h). The content of bromotyrosine and nitrotyrosine in soluble proteins (lavage supernatant) was subsequently measured by GC/MS and LC/ESI/MS/MS, respectively. Results are normalized to the protein content of tyrosine, the precursor amino acid. Lines indicate mean levels within each group. Values represent mean ± S.D.

Based upon the aforementioned results, we anticipated that this model would prove particularly effective in illustrating EPO-dependent oxidation events in vivo, such as NO₂-Tyr formation. Remarkably, subsequent LC/ESI/MS/MS analyses of protein NO₂-Tyr levels failed to demonstrate any significant differences between WT and EPO-KO mice (Fig. 2B). Notably, despite 7- and 50-fold increases in Br-Tyr formation over basal levels following eosinophil recruitment with M. corti. and subsequent intraperitoneal zymosan challenge in WT mice, respectively, NO₂-Tyr levels in proteins remained unchanged (Fig. 2B). An examination of total NO₂-Tyr recovered within peritoneal lavage proteins showed similar results, with no significant differences between all WT versus EPO-KO treatments or between any of the various groups within a given mouse (p > 0.27 for all comparisons made between WT versus EPO-KO and within WT and EPO-KO groups for various treatments; data not shown). Collectively, these results demonstrate that EPO-dependent formation of protein NO₂-Tyr formation in vivo is model-dependent. They also suggest that peroxidase-dependent formation of NO₂-Tyr does not always parallel the extent of EPO-mediated Br-Tyr formation.

Myeloperoxidase Generates Nitrotyrosine in Vivo in a Site-specific Fashion-- To assess the contribution of MPO-catalyzed nitration reactions in vivo, we initially selected a candidiasis model. We had previously demonstrated that this model manifests similar leukocyte total cell counts and differentials in pleural fluid recovered from WT and MPO-KO animals (31), and the present studies confirmed these prior observations (p > 0.38 for all comparisons made; data not shown). To establish that MPO is catalytically active and promotes protein oxidation in this model, the content of protein-bound Cl-Tyr was determined using GC/MS analysis (Fig. 3A). Base-line levels of Cl-Tyr in proteins recovered within peritoneal fluids were below the detection limit in WT and MPO-KO mice. Twenty-four hours after intraperitoneal challenge with C. albicans, a significant increase in Cl-Tyr was observed in both extracellular (i.e. peritoneal lavage supernatant) and cell-associated proteins (i.e. peritoneal lavage cell pellet) recovered from WT, but not MPO-KO mice (Fig. 3A). These results confirm both the specificity of protein-bound Cl-Tyr as a marker of MPO, and that MPO catalyzes protein oxidation in this model.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Chlorotyrosine and nitrotyrosine formation in candidiasis model. MPO-KO and WT mice were either injected intraperitoneally with normal saline or infected with an intraperitoneal inoculum of C. albicans. Peritoneal lavage was harvested 24 h later and the content of protein bound chlorotyrosine (A) and nitrotyrosine (B) determined by GC/MS and LC/ESI/MS/MS, respectively, in both extracellular (peritoneal lavage supernatant) and cellular pellet proteins. Lines indicate mean levels within each group. Values represent mean ± S.D.

LC/ESI/MS/MS quantification of protein-bound NO₂-Tyr levels in the candidiasis model was then performed. Extracellular proteins recovered in peritoneal lavage supernatant demonstrated a significant ~35% increase in NO₂-Tyr content (expressed as the conventional product/precursor ratio, NO₂-Tyr/Tyr, mol/mol) 24 h after intraperitoneal challenge with C. albicans in WT mice (Fig. 3B). In contrast, following challenge, the NO₂-Tyr content of extracellular peritoneal lavage proteins in MPO-KO mice did not increase but rather decreased modestly, suggesting that the observed increase in protein NO₂-Tyr content in WT mice following C. albicans exposure was produced by MPO (Fig. 3B). We hypothesized that the modest decline in NO₂-Tyr content per protein observed resulted from increases in the concentration of extracellular proteins present in peritoneal fluid that exceeded potential increases in NO₂-Tyr formed in the candidiasis model. We therefore also examined estimates of total NO₂-Tyr formation within peritoneal proteins by multiplying the content of NO₂-Tyr within proteins (i.e. NO₂-Tyr/Tyr; mol/mol) × the recovery of protein within lavage fluid, as determined by protein concentration (note that animals used in a given experiment were similar in size and lavages were performed with identical volumes). When expressed in this manner, WT mice demonstrated a >3-fold increase in total NO₂-Tyr formation 24 h after C. albicans exposure (base line versus 24 h, 1.56 ± 1.23 versus 5.05 ± 2.23 ([µmol NO₂-Tyr/mol Tyr] × [mg protein/ml]); p < 0.002). In marked contrast, no significant change in total protein-bound NO₂-Tyr was formed 24 h after C. albicans challenge within the MPO-KO mice (baseline versus 24 h, 1.93 ± 0.94 versus 2.28 ± 0.48 ([µmol NO₂-Tyr/mol Tyr] × [mg protein/ml]); p = 0.32). Further, although comparisons between total NO₂-Tyr content in lavage proteins of WT versus MPO-KO mice at base line demonstrated no significant difference (p = 0.56), levels of total NO₂-Tyr formed 24 h after C. albicans challenge demonstrated significant ~50% reductions (i.e. 5.05 ± 2.23 versus 2.28 ± 0.48 ([µmol NO₂-Tyr/mol Tyr]) × [mg protein/ml]); p < 0.001) in MPO-KO mice compared with WT. If one looks only at the total protein-bound NO₂-Tyr generated above base-line levels in response to C. albicans challenge, this correlates to a ~90% decrease in NO₂-Tyr generation in the MPO-KO mice versus WT (p < 0.001). In marked contrast, LC/ESI/MS/MS examination of the content of NO₂-Tyr in proteins recovered in cell pellet of peritoneal lavage from WT and MPO-KO mice failed to demonstrate any significant differences (p > 0.29 for all comparisons made) either at base line or 24 h after C. albicans challenge (Fig. 3B). Expressing the data in units comparable with total NO₂-Tyr formed in cell pellet of peritoneal lavage demonstrated similar results (p > 0.45 for all comparisons made, in both WT versus MPO-KO and base line versus 24 h post-challenge within a mouse strain; data not shown).

The Contribution of Myeloperoxidase to Nitrotyrosine Formation in Vivo Is Model-specific and Does Not Always Parallel the Extent of Chlorotyrosine Formation-- The role of MPO in protein nitration in an alternative acute inflammatory model was next examined. Intraperitoneal injection of thioglycollate into WT and MPO-KO mice elicits a neutrophil-rich influx of leukocytes into the peritoneum with comparable total cell counts and differentials 20 h after challenge (p > 0.7 for all comparisons made between WT versus MPO-KO mice; data not shown). Interestingly, despite robust recruitment of leukocytes to the peritoneum (typically 14-20 × 10⁶ cells; ~70% neutrophils and ~30% monocytes), only modest levels of Cl-Tyr were formed in either soluble or cell pellet proteins recovered in peritoneal lavage from WT mice (Fig. 4A). Subsequent intraperitoneal injection with yeast cell wall (zymosan), a potent trigger of leukocyte activation and degranulation, produced a marked rise in Cl-Tyr formation in WT mice. Again, no detectable protein-bound Cl-Tyr was formed in MPO-KO mice proteins examined (Fig. 4A).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Protein modification by chlorinating and nitrating ox idants in zymosan-induced peritonitis model. MPO-KO and WT animals were injected intraperitoneally with normal saline (Bl) or thioglycollate broth (Tg). Twenty hours later, peritoneal lavages were per- formed. Where indicated, mice were injected with zymosan 20 h after injection with thioglycollate (Tg/Z), and then peritoneal lavage was performed 4 h later. A, the chlorotyrosine content in extracellular proteins and cell pellet recovered from peritoneal lavage was determined by GC/MS analysis. Results are normalized to the protein content of tyrosine, the precursor amino acid. B, LC/ESI/MS/MS analysis of nitrotyrosine (NO2Y) content in soluble lavage proteins normalized to the protein content of tyrosine (Y), the precursor amino acid. C, LC/ESI/MS/MS analysis of nitrotyrosine content in soluble lavage proteins presented in a manner proportional to the total amount of nitrotyrosine recovered within lavage: (µmol NO2-Tyr/mol Tyr) × (mg protein/ml in lavage supernatant). Lines indicate mean levels within each group. Values represent mean ± S.D.

LC/ESI/MS/MS analyses of NO₂-Tyr levels in proteins (expressed as product/precursor ratio, NO₂-Tyr/Tyr, mol/mol) recovered in the supernatant of peritoneal lavage from WT and MPO-KO mice at base line, 20 h after leukocyte recruitment with thioglycollate, and 4 h after subsequent leukocyte activation with zymosan, are shown in Fig. 4B. The same data expressed in terms proportional to total NO₂-Tyr generated, i.e. (µmol NO₂-Tyr/mol Tyr) × mg protein/ml, are illustrated in Fig. 4C. At base line, both the content of protein-bound NO₂-Tyr per protein and the total level of NO₂-Tyr formed were similar in WT and MPO-KO mice (Fig. 4, B and C). However, following both leukocyte recruitment with thioglycollate, and subsequent leukocyte activation with zymosan, protein-bound NO₂-Tyr levels in extracellular proteins recovered in peritoneal lavage supernatant were significantly decreased in the KO mice compared with their WT controls (Fig. 4, B and C). Compared with WT, MPO-KO mice demonstrated no increase in total protein-bound NO₂-Tyr in extracellular soluble proteins within peritoneal lavage following thioglycollate treatment, whereas a 50% reduction in the increase in total protein-bound NO₂-Tyr was observed in the extracellular proteins recovered in peritoneal lavage supernatant from thioglycollate-elicited/zymosan-challenged mice. LC/ESI/MS/MS examination of NO₂-Tyr levels in proteins recovered in the cell pellet of peritoneal lavage demonstrated no significant differences between WT and MPO-KO mice regardless of whether samples were obtained at base line, following recruitment of leukocytes with thioglycollate, or following subsequent activation with zymosan (Fig. 5A). Expressing the data as total NO₂-Tyr recovered in the cell pellet of the peritoneal lavage demonstrated similar results (Fig. 5B). Taken together, the results suggest that MPO plays a dominant role in the generation of NO-derived oxidants that modify extracellular proteins within these acute inflammatory models, accounting for between 50 and 90% of the total NO₂-Tyr formed depending on the model examined. In marked contrast, MPO appears to play no appreciable role in NO₂-Tyr formation within cellular proteins in either model examined.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Cellular protein modification by nitrating oxidants in zymosan-induced peritonitis model. NO2-Tyr (NO2Y) content per protein in cell pellets (expressed as NO2-Tyr/Tyr, mol/mol) (A) and NO2-Tyr recovered in cell pellets (B) presented in a manner proportional to the total amount of NO2-Tyr recovered within lavage: (µmol NO2-Tyr/mol Tyr) × (mg protein/ml in lavage supernatant). Lines indicate mean levels within each group. Values represent mean ± S.D. Y, tyrosine.

Controversy 2: What Is/Are the Nitrating Oxidant(s) Formed following Peroxidase-catalyzed Oxidation of Nitrite?-- Although significant evidence suggests that ^·NO₂ is likely to be the major oxidant formed during peroxidase-catalyzed oxidation of NO, direct detection of this species as a product of the enzymes has not yet been reported. Moreover, although EPO and MPO are capable of catalyzing two-electron oxidation reactions such as might hypothetically lead to ONOO formation (35), experiments designed to evaluate this possibility have not yet been reported. We therefore initially sought to establish whether ^·NO₂, the presumptive oxidant formed by the peroxidases, was formed. We then designed studies aimed at testing the hypothesis that ONOO might serve as an additional low abundance oxidant formed during peroxidase-catalyzed oxidation of NO. For the latter studies, we examined whether EPO or MPO could: 1) utilize NO as co-substrate to generate an oxidant that promotes hydroxylation of targets; 2) utilize NO as co-substrate to generate an oxidant that promotes nitration of targets that is enhanced by CO₂/HCO; and 3) catalyze ONOO-mediated nitration reactions, consistent with activation of ONOO through formation of a Fe-ONOO intermediate.

Direct Demonstration That Myeloperoxidase and Eosinophil Peroxidase Generate Nitrogen Dioxide as a Physiologic Oxidant in the Presence of Hydrogen Peroxide and Nitrite-- A method was developed for detecting ^·NO₂ in the gas phase above reaction mixtures following catalytic reduction to ^·NO using a heated gold-plated quartz converter and subsequent detection by chemiluminescence following reaction with ozone (see "Experimental Procedures"). Briefly, a base line was initially obtained by directing helium-swept head-space gas from a gas-tight reaction vessel directly to the chemiluminescence detector, by-passing the heated gold-plated quartz catalytic converter. Upon diversion of the gas stream to the heated catalytic converter, control studies confirmed that ^·NO₂ formation could be monitored in real time if ^·NO₂ was added within the reaction vessel. Reaction mixtures included purified human peroxidase (EPO or MPO), NO, and H₂O₂ in phosphate buffer at neutral pH and demonstrated comparable results regardless of which component was added last to initiate the reaction. Direct detection of ^·NO₂ formation by EPO and MPO is demonstrated in Fig. 6. Peroxidases were incubated individually with varying concentrations of NO (either 25 or 100 µM, Fig. 6, A and B, respectively), and reactions were initiated with a bolus addition of H₂O₂ (50 µM final). Both EPO and MPO produced ^·NO₂ in the presence of all components of the reaction mixture but not if any component (peroxidase, H₂O₂, or NO) was omitted. Regardless of the conditions employed, EPO was reproducibly more effective at generating ^·NO₂ than MPO (Fig. 6), similar to studies demonstrating EPO as a more efficient generator of free and protein-bound NO₂-Tyr formation than MPO (13).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Direct demonstration of ·NO2 production by the EPO-H2O2-NO and MPO-H2O2-NO systems. Either EPO or MPO was added to phosphate buffer containing H2O2 (50 µM) and NO (25 µM) (A) or H2O2 (50 µM) and NO (100 µM) (B). The extent of ·NO2 production was then followed in real time by gas phase analysis of helium-swept reaction mixtures following gold-catalyzed reduction to ·NO and detection by chemiluminescence, as described under "Experimental Procedures."

Eosinophil Peroxidase and Myeloperoxidase Use Nitrite as Co-substrate to Generate an Oxidant That Promotes Hydroxylation of Targets at Acid pH But Not Neutral pH-- The ability of ONOO to hydroxylate targets distinguishes it from the chemical reactivity of ^·NO₂. To explore the potential capacity of peroxidase-H₂O₂-NO systems to generate ONOO, we first developed sensitive and specific analytical methods for identifying and quantifying nitration and hydroxylation products of a panel of targets (tyrosine, 2-deoxyguanosine (2-dG), phenylalanine, and salicylic acid) as described under "Experimental Procedures." Initial studies were performed at neutral pH, and subsequent studies were performed at acidic pH. All reactions were performed in chelex-treated phosphate buffer and included a metal chelator (DTPA) to prevent trace metal ion catalyzed hydroxylation reactions. Fig. 7 illustrates how the hydroxylating versus nitrating ability of ONOO versus the oxidant generated by leukocyte peroxidase-H₂O₂-NO systems were monitored using tyrosine as a target at pH 7.0. Reactions were initiated by equimolar additions of either ONOO or H₂O₂ to peroxidase/NO mixtures. Analysis for NO₂-Tyr revealed that both EPO and MPO generated more of the nitration product than ONOO (Fig. 7B). However, no detectable DOPA, the major tyrosine hydroxylation product generated following the addition of ONOO, was produced following either EPO or MPO catalyzed oxidation of NO (Fig. 7A). Similarly, incubation of 2-dG with either the EPO-H₂O₂-NO or MPO-H₂O₂-NO system versus ONOO resulted in a roughly comparable degree of nitration (as monitored by quantification of 2-deoxy-8-nitroguanosine (NO₂-dG) and 8-nitroguanine (NO₂-G)), yet both peroxidases failed to produce any detectable hydroxylation products of 2-dG (i.e. 8-hydroxy-2-deoxyguanosine (8-OHdG)), but authentic ONOO did (Fig. 8). Using phenylalanine as a target, peroxidase-H₂O₂-NO systems failed to generate either hydroxylation (o-, m-, or p-tyrosine) or nitration products (2-nitro- and 4-nitrophenylalanine), but ONOO effectively promoted both reactions (Fig. 8). Finally, the hydroxylation and nitration product profile of salicylate following incubation with ONOO at pH 7.0 also differed significantly from that observed following incubation with peroxidase-H₂O₂-NO systems (Fig. 8). Specifically, peroxidases were much more effective than ONOO at nitration of salicylate generating nitrosalicylate. In contrast, incubation of ONOO with salicylate produced significant levels of two hydroxylation products, 2,3-DHB and 2,5-DHB, whereas peroxidase-H₂O₂-NO systems reproducibly generated only lesser amounts of the 2,5-DHB isomer (Fig. 8).

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Analysis of nitration and hydroxylation products of L-tyrosine formed by peroxynitrite, EPO-H2O2-NO or MPO-H2O2-NO. L-Tyrosine (1.5 mM) was exposed to ONOO or either the EPO-H2O2-NO or MPO-H2O2-NO systems in phosphate buffer (150 mM, pH 7.0) containing 115 µM DTPA at 37 °C for 60 min as described under "Experimental Procedures." After reaction, the contents of hydroxylation (DOPA in A) and nitration (Nitrotyrosine in B) products were determined by reverse phase HPLC with on-line coulametric array detection (A) or diode array detection (B). Reagent concentrations were: ONOO and H2O2, 1 mM (200 µM additions at 10-min intervals); eosinophil peroxidase or myeloperoxidase, 57 nM; NO, 1 mM.

View larger version (31K): [in this window] [in a new window]   Fig. 8.   Comparison of the hydroxylation and nitration products formed by ONOO, EPO-H2O2-NO, and MPO-H2O2-NO using different targets at pH 7. L-Tyrosine (1.5 mM), 2-dG (2.5 mM), phenylalanine (5 mM), or salicylate (5 mM) were individually exposed to ONOO, the EPO-H2O2-NO system, or the MPO-H2O2-NO system in phosphate buffer (150 mM, pH 7.0) containing 115 µM DTPA at 37 °C for 60 min. The content of hydroxylation and nitration products was then determined. DOPA, 8-OHdG, o-, m-, and p-tyrosine were quantified by HPLC with coulametric array detection. 2,3-DHB, nitrotyrosine (NO2-Tyr), NO2-G, NO2-dG, 2- and 4-NO2-Phe, and NO2-salicylate were determined by HPLC with diode array detection as described under "Experimental Procedures." Identification of all analytes was also confirmed by LC-ESI-MS-MS. Values represent mean ± S.D. of three independent experiments.

The preceding data strongly suggest that free ONOO is not generated by peroxidase-H₂O₂-NO systems at neutral pH. However, peroxidases often operate in acidic environments, and differences in reactions at acid versus neutral pH with peroxidases are often observed (19-22, 43). We therefore examined the hydroxylating and nitrating capacity of peroxidase-H₂O₂-NO systems versus ONOO against a similar panel of targets at pH 5. Remarkably, incubation of tyrosine with EPO-H₂O₂-NO and MPO-H₂O₂-NO systems generated significant and reproducible levels of DOPA, as observed for ONOO (Fig. 9). Levels of NO₂-Tyr formed by the EPO-H₂O₂-NO and MPO-H₂O₂-NO systems were also substantial. When 2-dG was used as target, no hydroxylation product was detected with the leukocyte peroxidase-H₂O₂-NO systems, despite the fact that nitration products were still observed (Fig. 9). Finally, exposure of salicylate to peroxidase-H₂O₂-NO systems at pH 5 again resulted in hydroxylation of the target, forming the 2,5-DHB isomer, but not 2,3-DHB (Fig. 9). Nitration was still the preferred reaction with peroxidase-H₂O₂-NO