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The presence of nitrotyrosine is suggestive of the action of peroxynitrite but not absolute proof. Peroxynitrite is difficult to trap and assay in bio...
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MARCH 1999 VOLUME 12, NUMBER 3 © Copyright 1999 by the American Chemical Society

Articles A Novel Superoxide Dismutase-Based Trap for Peroxynitrite Used To Detect Entry of Peroxynitrite into Erythrocyte Ghosts Andrew J. Macfadyen,†,‡ Christopher Reiter,‡,§,| Yingxin Zhuang,‡,§ and Joseph S. Beckman*,‡,§ Departments of Pediatrics, Anesthesiology, and Biochemistry and Molecular Biology and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, Alabama 35233 Received November 17, 1998

Peroxynitrite (ONOO-) is a relatively stable oxidant produced by activated macrophages and neutrophils. To detect peroxynitrite, a novel human superoxide dismutase (SOD) trap was developed by substituting a tyrosine near the copper in the active site. The copper can catalyze nitration of this tyrosine by peroxynitrite. The nitrated tyrosine can serve as a reporter for peroxynitrite by measuring the extent of nitration with Western blots developed with a nitrotyrosine antibody. The new SOD mutant differs from bovine SOD whose sole tyrosine is far removed from the active site. Nitration of bovine SOD was second-order with respect to SOD concentration, whereas nitration of the new mutant SODs followed first-order kinetics with respect to peroxynitrite. The tyrosine SODs were used to assess whether peroxynitrite crosses erythrocyte membranes through the band 3 anion exchange protein. Tyrosine-containing SOD entrapped within normal human erythrocyte ghosts became nitrated in proportion to peroxynitrite concentration. The band 3 anion exchange protein inhibitors, phenyl isothiocyanate (PITC) and 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS), inhibited up to 90% of the nitration. The erythrocyte membrane proteins, spectrin, band 3 anion exchange protein, and proteins 4.1 and 4.2, were also nitrated. Nitration of erythrocyte membrane proteins was also inhibited by PITC and DIDS. These data suggest that the band 3 anion exchange protein is the major route for the entry of peroxynitrite into erythrocytes. The ability of peroxynitrite to cross cell membranes can contribute to its toxicity by allowing access to intracellular target molecules.

Introduction Peroxynitrite (ONOO-)1 is a powerful oxidant that is formed by the diffusion-limited reaction between nitric oxide and superoxide at a rate that is 3 times faster than the rate at which superoxide dismutase (SOD) can scavenge superoxide (1). Peroxynitrite can oxidize sulfhydryl groups, zinc fingers, and iron-sulfur centers (2). Peroxynitrite has also been shown to be bactericidal (3,

4) and to induce apoptosis in many mammalian cells (58). Many metalloproteins containing copper, manganese, or iron can catalyze the nitration of tyrosines in proteins * Corresponding author. Phone: (205) 934-5422. Fax: (205) 9347437. E-mail: [email protected]. † Department of Pediatrics. ‡ Center for Free Radical Biology. § Department of Anesthesiology. | Department of Biochemistry and Molecular Biology.

10.1021/tx980253u CCC: $18.00 © 1999 American Chemical Society Published on Web 01/22/1999

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(9, 10). Carbon dioxide also reacts rapidly with peroxynitrite and enhances the nitration of many proteins (1114). Modification of tyrosine with a nitro group can alter protein function and increase the level of protein degradation (15-20). Nitrotyrosine has been demonstrated by immunostaining in a variety of disease states, such as ARDS and atherosclerosis (21-23). Peroxynitrite is produced by activated macrophages and neutrophils (24, 25). A subpopulation of human neutrophils can induce nitric oxide synthase after ingesting opsonized bacteria and then become immunoreactive for nitrotyrosine (25). Although other nitrogen species such as nitric oxide, nitrogen dioxide, or nitrite may produce nitrotyrosine (26), endogenous thiols, ascorbate, and alternative targets in tissues suppress tyrosine nitration by nitrogen dioxide (27, 28). The presence of nitrotyrosine is suggestive of the action of peroxynitrite but not absolute proof. Peroxynitrite is difficult to trap and assay in biological systems. As an approach for circumventing this problem, we have developed several mutant human Cu, Zn SOD with tyrosines introduced in the rim of the active site surrounding the copper atom. Human SOD has no tyrosine and does not cross react with nitrotyrosine antibodies after peroxynitrite treatment. However, the copper in Cu, Zn SOD reacts with peroxynitrite and catalyzes tyrosine nitration on other proteins without inactivating Cu, Zn SOD (26, 29, 30). We reasoned that nitration of a tyrosine introduced by site-directed mutagenesis into the active site of Cu, Zn SOD could be a useful probe for assaying for peroxynitrite. Although the human and bovine Cu, Zn SODs are closely related, the bovine SOD contains a single tyrosine at position 108, which is far removed from the active site. We have previously shown that the copper in bovine SOD is required to catalyze nitration by peroxynitrite (26). Furthermore, one dimer of SOD catalyzes the nitration of tyrosine 108 on a separate dimer of SOD. The kinetics for nitration were second-order with respect to SOD concentration, and a small amount of copper-containing SOD catalyzed the nitration of tyrosines on a larger amount of copper deficient SODs. Addition of other proteins competitively interfered with the nitration of SOD. In brain homogenates, bovine SOD effectively nitrated several other brain proteins such as neurofilament L (31). However, nitration of the bovine SOD itself could not be detected in peroxynitrite-treated homogenates. Consequently, nitration of bovine SOD is not an effective trap for peroxynitrite in biological systems. In this study, we have utilized the tyrosine-modified human SODs to show that peroxynitrite anion may traverse erythrocyte membranes through the band 3 anion exchange protein. Two different mechanisms have recently been shown to allow for the passage of peroxynitrite across membranes. Peroxynitrite can rapidly diffuse across liposome membranes (32). Peroxynitrous acid (ONOOH) would be expected to cross membranes, since it should be about as hydrophobic as hydrogen peroxide and hydrogen peroxide is known to readily cross membranes. However, Marla et al. (32) also found that the rate of crossing liposome membranes was indepen1 Abbreviations: ONOO-, peroxynitrite; DIDS, 4,4′-diisothiocyanatostilbene-2,2′-disulfonate; PITC, phenyl isothiocyanate; SOD, superoxide dismutase; ONOOH, peroxynitrous acid; ECL, chemiluminescent; E133Y, mutant with glutamate at position 133; G141Y, mutant with glycine at position 141; SITS, 4-acetamido 4′-isothiocyanatostilbene2,2′-disulfonate; IPTG, isopropyl β-D-thioglucoside; DTT, dithiothreitol.

Macfadyen et al.

dent of pH and suggested that liposome membranes pose little barrier to peroxynitrite anion. In contrast, two groups (33, 34) report that a large fraction of peroxynitrite anion traverses erythrocyte membranes through anion channels. The band 3 anion exchange protein of the erythrocyte is the major route of anions of many sizes and shapes, including bicarbonate, into the red cell. Peroxynitrite is about the same size as bicarbonate and could potentially pass through the band 3 anion exchanger. Previously, superoxide anion has been shown to cross the red cell membrane (35). Band 3 anion exchangers are inhibited by the stilbenes, 4-acetamido 4′-isothiocyanatostilbene-2,2′-disulfonate (SITS), 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS), and phenyl isothiocyanate (PITC), which bind to a lysine residue (36-38). Previously, the inhibitors SITS and DIDS were used to demonstrate that superoxide anion crosses the red cell membrane via anion channels (39). The efflux of superoxide anion detected in the extracellular space of cat brains treated with arachidonic acid or bradykinin is blocked by DIDS and by phenylglyoxal, another inhibitor of the band 3 anion channel (40, 41). Consequently, anion channels may be major routes for the passage of negatively charged oxidants across cell membranes. One potential limitation in the previous studies (33, 34) was the use of an indirect assay for peroxynitrite based upon the oxidation of oxyhemoglobin, which can also be oxidized by hydrogen peroxide and nitrite, which are common contaminants in solutions of peroxynitrite. Control experiments with decomposed peroxynitrite suggested that these contaminants were not primarily responsible for the oxidation of hemoglobin. We have also investigated the passage of peroxynitrite across erythrocyte ghost membranes using the tyrosine SODs and found that even a substantially larger fraction of peroxynitrite passage can be blocked with amine-binding anion channel inhibitors than previously reported (33, 34).

Experimental Procedures Preparation of Erythrocyte Ghosts. All reagents were purchased from Sigma Chemical Co. (St. Louis, MO). Blood from healthy human volunteers was freshly drawn, and ghosts were prepared by the method described by Steck (42) as modified by Lynch and Fridovich (35). The unsealed ghosts were incubated overnight at 4 °C with 2.5 or 5 mg/mL recombinant tyrosinecontaining human Cu, Zn SOD, resealed, washed, and then treated with 25, 50, or 100 µM DIDS or with 2 mM PITC for 60 min at 37 °C in the dark (33). The ghosts were washed three times to remove excess DIDS or PITC and immediately exposed to 1 mM peroxynitrite that had been previously treated with manganese dioxide to remove hydrogen peroxide. The reaction buffer was 100 mM sodium phosphate with 0.1 mM EDTA at pH 7.4. The final pH was unchanged by the addition of the peroxynitrite. Total protein content was determined by the BCA protein assay (Pierce, Rockford, IL). Approximately 50 µg of protein was loaded onto a 10% polyacrylamide gel (Biorad Minigel system) as previously described (43). After the gel was transferred to a nitrocellulose membrane, protein was visualized with Ponceau S, which can be washed off without interfering with subsequent antibody binding for Western blots. The membrane was scanned on a PS Silverscan III instrument using NIH Image 1.61, a public domain program (http://rsb.info.nih.gov/nihimage). After the solution was washed with water to remove the Ponceau S stain, the membrane was blocked for 30 min with nonfat dried milk dissolved in phosphate-buffered saline and 0.5% Tween-20, and the mixture was incubated in the same buffer overnight at 4 °C with the 1A6 monoclonal antibody against nitrotyrosine (Upstate Biologicals, Inc., Lake Placid, NY)

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Figure 1. (A) Nitration of E133Y SOD compared to that of bovine SOD. The concentrations of E133Y SOD were (b) 0.031, (9) 0.063, ([) 0.125, (2) 0.25, and (1) 0.5 mg/mL. The initial peroxynitrite concentration was 200 µM. Nitration followed first-order kinetics except at the lowest concentrations where the E133Y SOD was fully nitrated before peroxynitrite had fully decayed. Bovine SOD (0) at 2 mg/mL showed no detectable nitration. All reactions were conducted at 37 °C with final concentrations of 50 mM sodium phosphate (pH 7.5) and 50 µM DTPA. Spectra were recorded with an Applied Photophysics stopped-flow spectrometer. Peroxynitrite diluted in water was used in one syringe, and buffer and SOD were used in the other syringe at twice the final concentration. The initial absorbances were subtracted due to the small absorbance of SOD at 430 nm. Equivalent results were obtained with the tyrosine SOD mutation G141Y. (B) The yield of nitrotyrosine in E133Y under the same experimental conditions after treatment with 200 µM peroxynitrite. at 1:2000 (43). The membrane was then washed three times with phosphate-buffered saline and 0.5% Tween-20 for 10 min and incubated with goat anti-mouse HRP-conjugated antibody (Boehringer Mannheim Corp., Indianapolis, IN) at 1:2000 for 1 h. The membrane was washed five times for 5 min each, developed with enhanced chemiluminescent (ECL) staining, and exposed to Hyperfilm-ECL (Amersham Life Science, Inc., Arlington Heights, IL). After automated processing, the film was scanned using NIH Image 1.61. To verify the specificity of the nitrotyrosine antibody, separate blocking experiments were conducted with 10 mM nitrotyrosine dissolved in the incubation buffer during incubation with the primary antibody. Tyrosine-Containing Human Superoxide Dismutase. Point mutations in the human SOD gene were created using a two-cycle, polymerase chain reaction-based mutagenesis protocol (31, 44). Mutagenic primers were used to substitute tyrosine for either glutamate at position 133 (E133Y) or glycine at position 141 (G141Y). Mutations were confirmed by complete sequencing of the mutant SOD gene. NcoI and BamHI restriction sites were introduced into the mutant SOD gene, which were used for subcloning into a pET-3d vector system (Novagen, Madison, WI). The plasmid containing the SOD insert was then expressed in Escherichia coli strain BL21(DE3)plysS in Luria broth. SOD expression was induced by adding 0.3 mM isopropyl β-D-thioglucoside (IPTG), 0.1 mM ZnCl2, and 0.05 mM CuCl2, and cells were harvested after 3 h at 23 °C. The broth was centrifuged at 4000g for 10 min at 4 °C, and the pellet was resuspended in 20 mM Tris-HCl (pH 7.8) and frozen overnight. The solution was thawed, then sonicated, and treated with 0.05% polyethyleneimide to remove DNA, followed by centrifugation at 18000g for 30 min. The supernatant was loaded onto a DEAE-Sephacel column (Pharmacia) and eluted with a gradient of 0 to 200 mM NaCl in 20 mM Tris (pH 8.0). A 10% molar excess of copper sulfate and zinc sulfate was added to the fractions possessing SOD activity and the mixture incubated overnight at 4 °C. The purified SOD was concentrated to ∼30 mg/mL by ultrafiltration and stored at -80 °C until it was used. The supernatants of the ghost washings and small aliquots were tested for SOD activity by the cytochrome c method of McCord and Fridovich (45). Samples containing ghosts were first

lysed with 0.5% Triton X-100 in 100 mM Tris buffer (pH 8.0). The activities of the tyrosine-containing SOD E133Y and G141Y mutants were approximately 4000 units/mg protein, which was only modestly lower than the wild-type specific activity of 5200 units/mg (46).

Results Stopped flow spectrometry showed that the extent of nitration of two tyrosine-containing mutants, E133Y and G141Y, increased by a first-order reaction with respect to peroxynitrite (Figure 1A). The yields of nitration as a function of E133Y SOD concentration are shown in Figure 1B. In contrast, bovine SOD, where the sole tyrosine at position 108 is far removed from the active site, was not measurably nitrated under these conditions. We previously observed that nitration of bovine SOD requires substantially higher concentrations of bovine SOD (6 mg/mL) and peroxynitrite (1 mM), which were necessary because the reaction was second-order with respect to SOD concentration (26). Bovine SOD does not nitrate itself when added to brain homogenates treated with peroxynitrite, but does enhance the nitration of other brain proteins (31). Peroxynitrite readily nitrated tyrosine-containing SOD sealed inside of red cell ghosts (Figure 2). When the sealed ghosts were incubated with DIDS or PITC under conditions which covalently modify the band 3 anion channel protein followed by extensive washing of the excess reagent before exposure to peroxynitrite, the extent of nitration of all proteins was substantially decreased (Figure 2A). The level of inhibition of nitration was greater with larger amounts of DIDS, with 56% inhibition of nitration occurring with 100 µM DIDS (Figure 2). PITC (2 mM) has been shown to almost fully inhibit the band 3 anion exchanger (31), and its presence inhibited nitration by 94% (Figure 3). Erythrocyte membrane proteins were also nitrated by peroxynitrite (Figure

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Figure 2. (A) Western blot of erythrocyte ghosts for nitrotyrosine using a monoclonal antibody and developed with enhanced chemiluminescense. Ghosts were incubated with 25 µM DIDS, 50 µM DIDS, 100 µM DIDS, or 2 mM PITC prior to exposure to 1 mM peroxynitrite. In the -ONOO lane, SOD-containing ghosts were not exposed to either inhibitors or peroxynitrite. (B) Coomassie blue staining pattern for protein of the same samples.

Figure 3. Nitration of mutant SOD E133Y sealed in erythrocyte ghosts treated with either 25 µM DIDS, 50 µM DIDS, 100 µM DIDS, or 2 mM PITC prior to exposure to 1 mM peroxynitrite. Western blotting was performed, and the integrated density of the staining signal under each condition was obtained, compared to the strength of the signal without inhibitors present and expressed as a percentage ( SEM (n ) 6). In the DIDS Control lane, 100 µM DIDS was added to the ghosts after they were exposed to 1 mM peroxynitrite (n ) 3).

Figure 4. Nitration of mutant SOD E133Y not entrapped in ghosts at 0.25 mg/mL (7.8 µM) in a buffer of 100 mM sodium phosphate and 0.1 mM EDTA at pH 7.4 and incubated with 25 µM DIDS, 50 µM DIDS, 100 µM DIDS, or 2 mM PITC prior to exposure to 1 mM peroxynitrite. Western blotting was performed, and the integrated density of the staining signal under each condition was obtained, compared to the strength of the signal without inhibitors present and expressed as a percentage ( SEM (n ) 6).

2A). The most intensely stained bands were spectrin a and β, band 3, band 4.1, and band 4.2 (Figure 2A). All immunoreactivity for nitrotyrosine was eliminated when membranes were incubated with the antibody in the presence of 10 mM nitrotyrosine. No change in nitrotyrosine staining occurred when 100 µM DIDS was added to samples of ghosts that had already been reacted with peroxynitrite (Figure 3). Nitration of the tyrosinecontaining human SOD by 1 mM peroxynitrite was not affected by preincubation with either DIDS or PITC for 1 h at 37 °C in the dark (Figure 4). Consequently, these inhibitors appear to prevent a large fraction of peroxynitrite from entering erythrocytes via the band 3 anion exchanger rather than by an indirect action of scavenging peroxynitrite or interfering in the detection of modified proteins with the anti-nitrotyrosine antibodies. The ghosts were stable once sealed and retained SOD after peroxynitrite exposure (Figure 2B). The amount of protein in the supernatant after sealing decreased exponentially with each washing such that less than 3 units/ mL of SOD activity could be detected by the third wash. Treatment with DIDS or PITC and the successive washes also had no effect on ghost stability. The supernatants of ghosts exposed to peroxynitrite and centrifuged had

no detectable SOD activity, showing that peroxynitrite at the concentrations used in this study did not lyse the ghosts. Peroxynitrite caused a small amount of SOD to form a covalent dimer, which was not reduced by incubation with large amounts of dithiothreitol (DTT). Similar amounts of the dimer were formed when pure SOD alone in buffer was treated with peroxynitrite in the absence of ghosts (not shown). The SOD dimer was still immunoreactive for nitrotyrosine (Figure 2A), showing that the cross-linking was not due to dityrosine since there is only one tyrosine on each SOD and the nitrotyrosine antibody does not recognize nitrated dityrosine (43).

Discussion Peroxynitrite is a significant cytotoxic oxidant produced by activated macrophages and neutrophils (24, 25). Inflammatory cells produce large amounts of extracellular superoxide by the activation of membrane-spanning NADPH oxidases. Nitric oxide can also be produced by inflammatory cells or diffuse in from surrounding tissues. Nitric oxide will react with superoxide to form peroxynitrite in the extracellular space around inflammatory cells

Peroxynitrite Crosses Anion Channels

Figure 5. Structure of peroxynitrite anion complexed with a sodium ion. This electrically neutral species may allow peroxynitrite to traverse liposome membranes.

(21, 24). Peroxynitrite preferentially reacts with essential intracellular chemical moieties such as iron-sulfur centers, zinc thiolate fingers, and protein-bound tyrosines. Therefore, peroxynitrite would be far more damaging to target tissues if it can penetrate cellular membranes and modify intracellular molecules in a pathogen. We have shown here that peroxynitrite readily crosses erythrocyte membranes to nitrate entrapped tyrosinecontaining SOD as well as endogenous proteins in the erythrocyte membrane. Under our experimental conditions, the major route of entry for the peroxynitrite appears to be through the band 3 anion exchange proteins since covalent inhibitors of the anion channel reduced the extent of nitration of the entrapped SOD. The pKa of peroxynitrite is between 6.6 and 6.8, depending upon the ionic strength and buffering ions (47, 48). According to the Henderson-Hasselbalch equation, approximately 80% of the peroxynitrite at pH 7.4 is in the anion form. Consequently, anion channels could substantially facilitate the diffusion of peroxynitrite anion across biological membranes at physiologically relevant pHs. Two other groups have recently shown that peroxynitrite can enter erythrocytes through anion channels (33, 34). Both utilized the ability of peroxynitrite to oxidize hemoglobin as the assay and estimated that about 60% of the peroxynitrite entered erythrocytes through anion channels, on the basis of inhibition with SITS and DIDS. We observed similar inhibition with the same concentrations of DIDS, but were able to achieve 90% inhibition with 2 mM PITC. Because excess inhibitors were washed away before peroxynitrite treatment and these inhibitors did not affect nitration of SOD, their action cannot be attributed to scavenging peroxynitrite, direct inhibition of tyrosine nitration, or interference with the detection of nitrotyrosine. While a majority of the peroxynitrite appeared to be entering erythrocyte ghosts through the anion exchanger using the tyrosine-containing SODs, other evidence indicates that peroxynitrite can directly pass through lipid bilayers of liposomes. Liposome membranes only slow the diffusion of peroxynitrite about 4-fold compared to diffusion in water with relatively little of the peroxynitrite reacted with lipids during the passage through the membrane (32, 49, 50). Curiously, the rate of peroxynitrite crossing liposomes was largely pH-independent, suggesting that peroxynitrite anions might also be able to cross liposome membranes. Peroxynitrite anion can form bidentate complexes where alkaline metals like lithium and sodium can bridge the two terminal oxygens (Figure 5). We have inferred the structures of these complexes in frozen argon matrixes, using infrared spectroscopy and density functional quantum mechanical calculations (51, 52). The transient formation of these species in solution could potentially allow peroxynitrite

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anion to traverse liposome membranes as an electrically neutral species. The importance of anion channels for transporting peroxynitrite across erythrocyte ghosts is difficult to reconcile with the liposome results. In part, the differences may be due to differences in lipid composition and the presence of many membrane proteins affecting membrane structure. This could reduce the concentration of hydrogen ions concentrated at the lipid-aqueous phase interface in a biological membrane compared to liposomes. Liposome membranes tend to be more curved than biological membranes due to their small radii, which could also affect the passage of smaller solutes such as peroxynitrite. We attempted to use the fluorescent traps dihydrorhodamine or dichlorofluorescein entrapped in erythrocyte ghosts as indicators of peroxynitrite crossing the membrane. These dyes are sensitive indicators for low concentrations of peroxynitrite and were immediately oxidized in erythrocyte ghosts after the addition of peroxynitrite (53). However, they leaked out of the erythrocyte ghosts rapidly enough to interfere with quantitative assessment of peroxynitrite crossing membranes. These dyes have limited utility to assess peroxynitrite in vivo because they are readily oxidized by peroxidases and by other oxidative processes present in cells. To circumvent the leakage of fluorescent dyes and the lack of specificity of other methods, we developed a novel trap for peroxynitrite by introducing a tyrosine into the active site of human Cu, Zn SOD. Mutations were systematically made at the amino acid positions forming the active site loop surrounding the catalytically active copper, and it was found that replacing glutamate 133 with tyrosine was the most effective mutation for trapping peroxynitrite in vitro. The mutant G141Y was also readily nitrated and used in many of these studies with similar results. Most of the tyrosine-containing mutants formed stable proteins that bind copper and zinc appropriately and expressed well in E. coli. Furthermore, the scavenging activity for superoxide was about 80% of that of wild-type Cu, Zn SOD. The tyrosine SOD is a relatively specific trap for peroxynitrite. It is not nitrated by millimolar concentrations of nitrite or nitric oxide (data not shown). Once nitrated, the SODs are still able to catalyze nitration on other proteins. One limitation of the tyrosine-containing SODs is their limited sensitivity for detecting peroxynitrite. The kinetics for the reaction of peroxynitrite are complex, with a maximum of 9% of peroxynitrite reacting with the SOD to yield nitrotyrosine (29). However, these SODs can be used to measure the accumulative exposure to peroxynitrite in biological systems. Although peroxynitrite is a strong oxidant, it reacts relatively slowly with most biological molecules (10). Peroxynitrite is particularly stable as an anion and can be prepared as a pure solid and stored for years without decomposition (54). Even under biologically relevant conditions, peroxynitrite is relatively unreactive with most biological molecules despite being a strong oxidant. For example, glutathione is among the better scavengers of peroxynitrite, even though peroxynitrite collides with glutathione on average more than 10 million times without reaction for each collision that results in a reaction. Peroxynitrite reacts even more slowly with other amino acids, which allows peroxynitrite to have a substantial probability of diffusing through anion chan-

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nels without reaction. We observed that the band 3 anion exchanger was nitrated, suggesting that the protein was modified by peroxynitrite. However, SOD and other ghost proteins were more susceptible to nitration than the band 3 protein, showing that substantial amounts of the peroxynitrite had entered the ghosts without directly reacting with the band 3 protein. Mallozzi et al. (54) have recently shown that nitration of band 3 disrupts phosphorylation and the association of glyceraldehyde-3phosphate dehydrogenase with the membrane. One mechanism whereby inflammatory cells might limit injury to themselves may be by not expressing anion exchange channels while the cells are producing oxidants. This would minimize the level of entry of both superoxide and peroxynitrite into the inflammatory cell. There is some evidence that inflammatory cells may not express a cation-independent chloride-bicarbonate exchange protein (55), but instead utilize a different class of sodium-coupled anion transporters. Whether these cationdependent channels can carry peroxynitrite remains to be determined. However, these channels are generally more discriminating with respect to the type of anions transported, tending to favor chloride. In contrast, anion exchange proteins can carry anions as large as phosphate and sulfate, and therefore can be sufficiently indiscriminate that they might carry peroxynitrite. Limited expression of anion exchange proteins by activated inflammatory cells may help reduce self-injury from the production of negatively charged oxidants, including superoxide and peroxynitrite.

Acknowledgment. We thank Drs. Jacinda Sampson and John Crow for their comments on the tyrosinecontaining SOD mutations. This work was supported by grants from the National Institutes of Health (Grants HL-58209 and NS-33291).

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