©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Chlorination of Tyrosyl Residues in Peptides by Myeloperoxidase and Human Neutrophils (*)

Neil M. Domigan (1), Timothy S. Charlton (2), Mark W. Duncan (2)(§), Christine C. Winterbourn (1), Anthony J. Kettle (1)(¶)

From the (1)Free Radical Research Group, Christchurch School of Medicine, P. O. Box 4345, Christchurch, New Zealand and the (2)Biomedical Mass Spectrometry Unit, University of New South Wales, Sydney, New South Wales 2052, Australia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Hypochlorous acid is the major strong oxidant generated by human neutrophils, and it has the potential to cause much of the tissue damage that these inflammatory cells promote. It is produced from hydrogen peroxide and chloride by the heme enzyme myeloperoxidase. To unequivocally establish that hypochlorous acid contributes to inflammation, a stable and unique marker for its reaction with biomolecules needs to be identified. In this investigation we have found that reagent hypochlorous acid reacts with tyrosyl residues in small peptides and converts them to chlorotyrosine. Purified myeloperoxidase in combination with hydrogen peroxide and chloride, as well as stimulated human neutrophils, chlorinated tyrosine in the peptide Gly-Gly-Tyr-Arg. Rather than reacting directly with the aromatic ring of tyrosine, hypochlorous acid initially reacted with an amine group of the peptide to form a chloramine. The chloramine then underwent an intramolecular reaction with the tyrosyl residue to convert it to chlorotyrosine. This indicates that tyrosyl residues in proteins that are close to amine groups will be susceptible to chlorination. Peroxidases are the only enzymes capable of chlorinating an aromatic ring. Furthermore, myeloperoxidase is the only human enzyme that produces hypochlorous acid under physiological conditions. Therefore, chlorotyrosine will be a specific marker for the production of hypochlorous acid in vivo and for the involvement of myeloperoxidase in inflammatory tissue damage.


INTRODUCTION

Neutrophils and monocytes are implicated in the tissue damage that occurs in numerous inflammatory pathologies, such as atherosclerosis, adult respiratory distress syndrome, rheumatoid arthritis, ischemia reperfusion injury, and inflammatory bowel disease(1, 2, 3) . They may also promote cancers that are associated with inflammation(4, 5) . In numerous studies it has been proposed that oxidants generated by neutrophils and monocytes cause the damage that these phagocytic cells perpetrate(6) . However, to date there is a lack of convincing evidence that oxidants cause tissue injury in vivo. This arises principally because of the difficulties in detecting short-lived oxidants. Consequently, specific markers of oxidants need to be found to either confirm or refute their role in inflammation. Ideally, these should be stable and unique reaction products of oxidatively modified biomolecules.

Stimulated phagocytes generate vast amounts of superoxide that dismutates to form hydrogen peroxide(7) . These two oxidants are relatively benign but can give rise to more reactive secondary oxidants including hypochlorous acid, hydroxyl radical, and singlet oxygen(7) . Hypochlorous acid is produced from hydrogen peroxide and chloride by the heme enzyme myeloperoxidase(8) , and it is the major strong oxidant generated by neutrophils(9, 10, 11) . It is also produced in appreciable amounts by monocytes(12) . Hypochlorous acid reacts most rapidly with thiols and thioethers, but the products are not unique to this oxidant (13, 14). Hypochlorous acid also reacts readily with amines to form chloramines(15) ; however, they are unstable and decay to form aldehydes or nitriles(16) .

For a useful marker to be derived from hypochlorous acid, a stable carbon-chlorine bond must be generated. Reagent hypochlorous acid and that derived from myeloperoxidase, hydrogen peroxide, and chloride, chlorinate unsaturated fatty acids and cholesterol to form chlorohydrins(17) . The chlorine atom of hypochlorous acid is also incorporated into proteins(18) . The amino acids that are most likely to be chlorinated are tyrosine, tryptophan, and histidine(19) . There has been extensive work on the iodination of tyrosine, mainly involving thyroid peroxidase in the production of thyroid hormones(20) . Peroxidases, including myeloperoxidase, also brominate tyrosyl residues of proteins(21) . Chlorination of tyrosine has received little attention. At nonphysiological pH, chloroperoxidase chlorinates free tyrosine (22) and tyrosyl residues in barley -amylase(23) . Hypochlorous acid reacts with free tyrosine to produce a ring chlorinated nitrile derivative(24) , but, as far as we are aware, its reaction with tyrosyl residues in peptides and proteins has not been investigated.

This study was undertaken to determine the products formed when hypochlorous acid reacts with tyrosine in peptides. The ability of purified myeloperoxidase and stimulated neutrophils to chlorinate tyrosyl residues was also investigated. We show that reagent hypochlorous acid and that generated by myeloperoxidase and stimulated neutrophils chlorinate tyrosyl residues to produce chlorotyrosine. We propose that chlorotyrosine should prove to be a specific marker for hypochlorous acid production during inflammation.


EXPERIMENTAL PROCEDURES

Materials

Myeloperoxidase was purified from human leukocytes as described previously(25) . Its purity index (A/A) was greater than 0.72 and its concentration was determined using = 91,000 M cm/heme(26) . Peptides, amino acids, phorbol myristate acetate, bovine liver catalase, bovine erythrocyte superoxide dismutase, 5,5`-dithiobis(2-nitrobenzoic acid), and monochlorodimedon were purchased from the Sigma. 5-Thio-2-nitrobenzoic acid was prepared from 5,5-dithiobis(2-nitrobenzoic acid) as described previously(27) . Enzymes used for peptide hydrolysis were obtained from Boehringer Mannheim (Sydney, Australia). Hydrogen peroxide solutions were prepared daily by diluting a 30% stock and calculating its concentration using = 43.6 M cm(28) . Hypochlorous acid was purchased from Reckitt and Colman (NZ) Ltd. (Auckland, NZ). Its concentration was determined by measuring the loss in absorbance at 290 nm after reacting it with monochlorodimedon ( = 19,000 M cm)(29) .

Isolation of Human Neutrophils

Neutrophils were isolated from the blood of healthy donors by Ficoll-Hypaque centrifugation, dextran sedimentation, and hypotonic lysis of red cells(30) . Cell preparations contained 95-97% neutrophils. After isolation, neutrophils were resuspended in phosphate-buffered saline (PBS)()(10.0 mM sodium phosphate buffer, pH 7.4, with 140 mM sodium chloride) containing 1.0 mM calcium chloride, 0.5 mM magnesium chloride, and 1 mg/ml of glucose. Neutrophils were also isolated from the blood of a myeloperoxidase-deficient individual, whose cells have been shown to have approximately 5% of normal peroxidase activity and produce hypochlorous acid at less than 5% of the levels generated by normal neutrophils(31) .

Oxidant Production by Neutrophils

Production of superoxide by neutrophils stimulated with phorbol myristate acetate (100 ng/ml) was determined by measuring superoxide dismutase-inhibitable cytochrome c reduction(32) . To determine hypochlorous acid production, cells were stimulated in the presence of 15 mM taurine, and the formation of taurine chloramine was measured by its ability to oxidize 2-nitro-5-thiobenzoate(27) .

Chlorination of Peptides in Cell Free Systems

Peptides were dissolved in PBS, and hypochlorous acid was added while vortexing the solution. Reacted peptides were normally left at room temperature for at least 2 hours before analysis. To follow the time course of chlorination, samples were removed at intervals, added to 200 µM dithiothreitol to scavenge any chloramines, and then subjected to amino acid analysis. Gly-Gly-Tyr-Arg was also chlorinated with myeloperoxidase, hydrogen peroxide, and chloride. Reactions were run at 20 °C and started by adding hydrogen peroxide to PBS containing myeloperoxidase and peptide. To prevent inactivation of myeloperoxidase, hydrogen peroxide was added in 50-µM aliquots at 10-min intervals. After an hour of incubation, peptides were subjected to amino acid analysis.

Chlorination of Gly-Gly-Tyr-Arg by Neutrophils

Neutrophils (2 10/ml) were suspended in PBS containing 1.0 mM calcium chloride, 0.5 mM magnesium chloride, 1 mg/ml of glucose, and 300 µM Gly-Gly-Tyr-Arg and stimulated with 100 ng/ml of phorbol myristate acetate. After 1 h at 37 °C, cells were pelleted by centrifugation, and the supernatant was removed for amino acid analysis.

Reaction of Chloramines with Gly-Gly-Tyr-Arg

Chloramines of taurine, glycine and lysine were formed by adding 100 µM hypochlorous acid to an equivalent concentration of the amines in PBS. After 5 min, Gly-Gly-Tyr-Arg was added to each chloramine, and the mixture was incubated at room temperature for 60 min. Residual chloramines and amino acid composition were then determined.

Amino Acid Analysis

Peptides were hydrolyzed under vacuum for 12 h at 100 °C in the presence of rapidly boiling hydrochloric acid with 1% phenol and then subjected to amino acid analysis by the method outlined by Cohen and Strydom(33) . Concentrations of tyrosine and chlorotyrosine in hydrolyzed peptides were calculated by comparing their peak heights in HPLC chromatograms with those obtained for a series of standards of known concentration. Free tyrosine and chlorotyrosine were analyzed by reverse phase HPLC with diode array detection. Analysis was performed using a 220 4.6 mm Spheri 5 octadecylsilane 5-µm column and an isocratic mobile phase of water/methanol/acetic acid (60:40:1).

Analysis of Chloramine Formation

Chloramines were determined by adding 2-nitro-5-thiobenzoate and measuring the loss in absorbance at 412 nm ( = 14,100 M cm)(27) . To confirm that oxidation of 2-nitro-5-thiobenzoate was due the chloramine of Gly-Gly-Tyr-Arg and not unreacted hypochlorous acid, monochlorodimedon, which reacts rapidly with hypochlorous acid but not with chloramines(34) , was added to hypochlorous acid 1 min after the peptide. There was no loss of monochlorodimedon, indicating that all the hypochlorous acid had already reacted (data not shown).

Mass Spectrometry

MALDI-tof Mass Spectrometry

Molecular weights of the untreated peptide and the peptide treated with reagent hypochlorous acid were determined by MALDI-tof mass spectrometry on a Lasermat (Finnigan MAT, San Jose, CA). The matrix for the thermolytic digest was 2,5-dihydroxybenzoic acid (10 mg/ml), and the matrix for the chymotryptic A4 digest was -cyano-4-cinnamic acid (5 mg/ml). Both matrices were prepared in acetonitrile/water/trifluoroacetic acid (35:65:0.1) (v/v). A two-point internal calibration using a matrix ion and the [M+H] ion for metenkephalin was used to improve mass accuracy for the thermolysin digests. All spectra are the averages of five laser shots/slide. Samples were prepared by adding the matrix solution (0.5 µl) and a solution of the peptide sample (0.1 µl) to stainless steel Lasermat targets. Samples were dried at room temperature and then placed in the instrument.

Enzyme Digests

For the thermolysin digest, the untreated peptide (4.4 nmol) and the peptide treated (2.1 nmol) with reagent hypochlorous acid were dried, reconstituted in 10 mM Tris-HCl buffer, pH 8.0, containing 10 mM calcium chloride, and incubated with thermolysin (0.022 nmol) in a final volume of 4.5 µl at 37 °C for 30 min. Samples were stored at 4 °C for 2 days before analysis. For the chymotrypsin A4 digest, the untreated (8.8 nmol) and treated (4.2 nmol) peptides were reconstituted in 10 mM Tris-HCl buffer, pH 8.2, and incubated with chymotrypsin (0.04 nmol) in a final volume of 3.5 µl at 37 °C for 14 h. For both digests, control samples that contained the appropriate enzyme and buffer but no peptide were also analyzed.

Electrospray Ionization Mass Spectrometry

Electrospray mass spectrometry was performed on a TSQ 7000 mass spectrometer (Finnigan MAT). Untreated peptide (4.4 nmol) and peptide treated with reagent hypochlorous acid (2.1 nmol) were introduced by injection into a fixed loop (5 µl). The mobile phase was methanol/water/acetic acid (50:50:1) (v/v) pumped by a Series II 1090 liquid chromatograph (Hewlett-Packard Co., Palo Alto, CA) at 200 µl/min. Mass spectra were recorded for the treated and untreated peptides, and mass spectrometry/mass spectrometry product ion spectra of [M+H] (m/z 50-500) and [M+2H] (m/z 50-280) were also recorded. The collision gas was methane (4 millitorr), and the collision energy was -100 eV. All mass spectra were averaged across the peak, and the background was subtracted.


RESULTS

Reaction of Hypochlorous Acid with Peptides Containing Tyrosine

When the peptide Gly-Gly-Tyr-Arg was reacted with equimolar reagent hypochlorous acid, there was a loss in its tyrosine content, whereas the amounts of glycine and arginine present were unaffected (Fig. 1, A and B). Concomitant with the loss in tyrosine, there was production of a novel compound that coeluted with 3-chlorotyrosine on HPLC analysis (Fig. 1B). Addition of hypochlorous acid to three other peptides caused a similar loss in tyrosine and formation of chlorotyrosine (). The yield of chlorotyrosine ranged from 30 to 55% of the hypochlorous acid added, and 50-75% of the reacted tyrosine was accounted for as chlorotyrosine. When hypochlorous acid was added to free tyrosine, there was no detectable formation of chlorotyrosine, although 60% of the amino acid had reacted (). Addition of hypochlorous acid to Gly-Gly-Tyr-Arg up to a concentration of 150 µM resulted in increasing formation of chlorotyrosine (Fig. 2). The yield of chlorotyrosine was approximately 60%. At greater than 150 µM hypochlorous acid, there was a progressive decline in the concentration of chlorotyrosine detected.


Figure 1: Amino acid analysis of hydrolyzed Gly-Gly-Tyr-Arg before and after reaction with hypochlorous acid. A, the untreated peptide Gly-Gly-Tyr-Arg (100 µM). B, peptide treated with 50 µM hypochlorous acid in PBS. Cl Tyr, chlorotyrosine.




Figure 2: Reaction of Gly-Gly-Tyr-Arg with reagent hypochlorous acid or myeloperoxidase/hydrogen peroxide/chloride. Gly-Gly-Tyr-Arg (100 µM) was either reacted with varying concentrations of hypochlorous acid in PBS () or incubated in PBS at 20 °C with 20 nM myeloperoxidase, and chlorination was started by adding varying concentrations of hydrogen peroxide (). After 2 h the peptide was hydrolyzed, and the production of chlorotyrosine was determined by amino acid analysis. Data are representative of two experiments.



Gly-Gly-Tyr-Arg was also chlorinated with purified myeloperoxidase, hydrogen peroxide, and chloride. There was no chlorination in the absence of enzyme, hydrogen peroxide, or chloride. Production of chlorotyrosine increased with increasing concentrations of hydrogen peroxide up to a maximum of 200 µM (Fig. 2). With 100 µM hydrogen peroxide, about 45% of the hydrogen peroxide was accounted for by the formation of chlorotyrosine. Above 200 µM hydrogen peroxide, the production of chlorotyrosine declined.

Analysis of Chlorinated Gly-Gly-Tyr-Arg by Mass Spectrometry

Direct MALDI-tof analysis of the untreated and treated Gly-Gly-Tyr-Arg confirmed the presence of one chlorine atom in the treated sample (). To locate the site of substitution, fragments of the peptide were generated by the addition of thermolysin, which should cleave the Gly-Tyr bond, and the masses were determined by MALDI-tof. Ion abundances for the intact peptides decreased over time, and new species appeared (Fig. 3). For the untreated peptide m/z 338.8 corresponds to [YR+H], and for the treated peptide m/z 373.3 corresponds to [YR-Cl+H] (Fig. 3). The mass difference of 34.5 Da is accounted for by the substitution of a proton for a chlorine atom. All other ions in the spectra were derived from either the matrix, the buffer, or the internal calibrant, metenkephalin (Fig. 3, m, b, and me, respectively).


Figure 3: MALDI-tof mass spectra of thermolysin digests of untreated Gly-Gly-Tyr-Arg (A) or peptide treated with equimolar hypochlorous acid (B). For details see ``Experimental Procedures.'' b, buffer; m, matrix; me, metenkephalin.



To establish whether the site of chlorine substitution was on the tyrosine or arginine residue, the peptides were subjected to digestion with chymotrypsin A4 to remove the C-terminal arginine residue. Following digestion, the untreated and treated peptides showed the presence of a species at m/z 175. This corresponds to arginine ([R+H] calculated mass, 175.2). From these data it is apparent that both peptides contain an unmodified C-terminal arginine residue. Spectra from control slides confirmed that the m/z 175 ions were derived from the peptides. From these results it is apparent that chlorine was substituted on the tyrosyl residue.

The peptides were also examined by electrospray mass spectrometry. The [M+H] and [M+2H] ions were selected as parent ions (at Q1), and the product ion spectra (Q3) were recorded. The fragmentation pattern was entirely consistent with the presence of a single chlorine substitution on the tyrosine residue. With [M+H] ions selected at Q1, the most abundant ions observed were at m/z 136.0 for the untreated sample and m/z 170.0 for the treated sample (I). These molecular masses are probably derived from the immonium ion of the tyrosyl residue, and the mass difference of 34.0 is consistent with its chlorination(35) . Arginine, [R+H], was detected at m/z 175 in both the treated and untreated samples with similar relative abundances. No ions characteristic of chlorinated arginine were detected.

With the doubly charged [M+2H] ion at Q1, in addition to the immonium ions, two other ions consistent with the fragmentation of the tyrosyl residue were detected (I). In the untreated sample m/z 107.0 is probably derived from the p-methylphenoxy ion of tyrosine. The corresponding chlorinated ion, m/z 140.8, was detected in the treated sample. In the untreated sample the ion m/z 91.1 arising from the benzyl moiety of tyrosine was observed. The corresponding chlorinated ion, m/z 124.8, was detected in the treated sample.

The Time Course of Gly-Gly-Tyr-Arg Chlorination

Maximum formation of chlorotyrosine and loss of tyrosine occurred 2 h after adding hypochlorous acid to Gly-Gly-Tyr-Arg (Fig. 4). This was preceded by formation of peptide chloramine, which occurred in the first minute of the reaction and accounted for all the hypochlorous acid (Fig. 5). The chloramine was unstable, and its decay mirrored the loss in the tyrosine content of the tetrapeptide. Hypochlorous acid also reacted quantitatively with tyrosine and glycine to convert them to chloramines (Fig. 5). Although tyrosine chloramine was also relatively unstable, it was not converted to chlorotyrosine (). Glycine chloramine, in contrast to the other chloramines, was very stable, and only 20% of it decayed over 2 h. From these results it is apparent that hypochlorous acid initially reacts with an amine group of Gly-Gly-Tyr-Arg to form a chloramine that then chlorinates the tyrosine ring.


Figure 4: The time course for the reaction of Gly-Gly-Tyr-Arg with hypochlorous acid. Gly-Gly-Tyr-Arg (100 µM) was dissolved in PBS at 20 °C and reacted with HOCl (100 µM). The reaction was terminated at the indicated times by adding 200 µM dithiothreitol, and the peptide was then hydrolyzed for analysis of tyrosine () and chlorotyrosine (). Data are representative of two experiments.




Figure 5: The time course for the decay of chloramines. Hypochlorous acid (100 µM) was added to either 100 µM of Gly-Gly-Tyr-Arg (), tyrosine (), or glycine (). The concentration of chloramine present was determined at the indicated time points by withdrawing an aliquot and adding it to 5-thio-2-nitrobenzoic acid. Other reaction conditions were as described in Fig. 4. Data are representative of two experiments.



Chloramines of taurine, glycine, and lysine were unable to chlorinate the tyrosyl residue in Gly-Gly-Tyr-Arg. They lost 0, 6, and 15%, respectively, of their reactivity toward 2-nitro-5-thiobenzoate when incubated alone for 1 h. For each chloramine, only about 20% of its reactivity was lost after an hour when it was added to the peptide, and there was no detectable formation of chlorotyrosine (results not shown). For chloramine determinations, duplicate experiments agreed within 2 µM. From these results we conclude that the intermolecular reaction of chloramines with tyrosyl residues is not favored.

Chlorination of Gly-Gly-Tyr-Arg by Neutrophils

When human neutrophils were stimulated with phorbol myristate acetate in the presence of Gly-Gly-Tyr-Arg, they produced approximately 3 µM chlorotyrosine/10 cells in the first 10 min of the reaction. Under the conditions of the assay, neutrophils generate hydrogen peroxide at about 3 µM/min/10 cells(11) . Thus 10% of the hydrogen peroxide was accounted for by the production of chlorotyrosine. Formation of chlorotyrosine was enhanced by myeloperoxidase and superoxide dismutase and blocked by catalase (Fig. 6). It was fully inhibited by azide and did not occur with myeloperoxidase-deficient neutrophils, indicating that chlorination of the tyrosyl residue was catalyzed by myeloperoxidase. Enhancement of chlorination by superoxide dismutase is consistent with the ability of this enzyme to cause about a 20% increase in hypochlorous acid production by neutrophils(11) .


Figure 6: Chlorination of Gly-Gly-Tyr-Arg by stimulated human neutrophils. Neutrophils (2 10/ml) were suspended in PBS (pH 7.4) at 37 °C containing 300 µM Gly-Gly-Tyr-Arg, 0.5 mM MgCl, 1 mM CaCl, and 1 mg/ml glucose. Cells were stimulated with 100 ng/ml of phorbol myristate acetate (PMA) in the presence or absence of 20 µg/ml of superoxide dismutase (+ SOD), 100 nM myeloperoxidase (+ MPO), 1 mM azide (+ Azide), or 20 µg/ml of catalase (+ Catalase). After 1 h, cells were pelleted and the supernatants were subjected to amino acid analysis. Results are expressed as means and ranges of duplicate experiments and are representative of those obtained from the cells of three individuals. The results for the myeloperoxidase deficient neutrophils were obtained once from a single individual.




DISCUSSION

Using amino acid analysis and mass spectrometry to identify products, we have shown that reagent hypochlorous acid converts tyrosyl residues in several peptides to chlorotyrosine. It is likely that the isomer formed is 3-chlorotyrosine, because the hydroxyl group on the aromatic ring of tyrosine activates the ortho positions for substitution. The para position is also activated, but it is blocked for substitution. With each peptide, chlorotyrosine was the major product and accounted for at least 50% of the reacted tyrosine. In contrast, free tyrosine was not converted to chlorotyrosine, which is in agreement with the findings of an earlier study(24) . Conversion of tyrosyl residues to chlorotyrosine is likely to be physiological possible, because purified myeloperoxidase and that released by stimulated neutrophils catalyzed chlorination of tyrosine in Gly-Gly-Tyr-Arg. Our evidence for the requirement of myeloperoxidase in chlorination by neutrophils was that the reaction was blocked by catalase, which scavenges hydrogen peroxide, and by the heme poison, azide. In addition, neutrophils from a myeloperoxidase-deficient individual did not chlorinate the tetrapeptide. Based on our finding that several unrelated peptides were chlorinated (), formation of chlorotyrosyl residues is unlikely to be restricted to a few peptides and proteins that are uniquely susceptible to chlorination. Rather, it is likely that numerous peptides and proteins could be chlorinated by myeloperoxidase, so that chlorotyrosine may prove to be an ideal marker for inflammatory reactions of neutrophils. In support of this proposal, hypochlorous acid chlorinates tyrosine in globin.()

Hypochlorous acid initially converted an amine group on Gly-Gly-Tyr-Arg to a chloramine, which decayed considerably faster than glycine chloramine or tyrosine chloramine. As the chloramine of the tetrapeptide decayed, there was a corresponding loss of tyrosine and formation of chlorotyrosine. From these results we conclude that the chloramine on the tetrapeptide chlorinates the tyrosine ring.

Chlorination appears to involve an intramolecular rearrangement, because the chloramines of lysine, glycine, or taurine were unable to chlorinate Gly-Gly-Tyr-Arg. A similar rearrangement involving a chloramine intermediate occurs when hypochlorous acid reacts with procainamide to form 3-chloroprocainamide(36) . It is also known that aromatic chloramines rearrange in the presence of hydrochloric acid to give ring chlorinated products(37) . These Orton rearrangments are intermolecular reactions in which molecular chlorine is liberated from the chloramine and then reacts with activated aromatic rings(37) . It is unlikely that this type of rearrangement explains the chlorination of the tetrapeptide, because the reaction conditions used would not favor release of molecular chlorine. Molecular modelling of Gly-Gly-Tyr-Arg revealed that in the energy-minimized structure, the terminal amino group can come within 3 Å of the tyrosine ring. Thus, a possible mechanism could involve homolysis of the nitrogen-chlorine bond of the terminal glycine chloramine, facilitated by addition of the chlorine atom to the juxtaposed tyrosine ring. In support of this proposal, homolysis of the nitrogen-chlorine bond has been shown to occur in the decomposition of N-chloroamides and adenosine chloramine(38, 39) . It has also been suggested to occur when hypochlorous acid reacts with aminopyrine to form a chloramine, which subsequently gives rise to an aminopyrine radical(40) .

In a related reaction to what we report here, the tyrosyl residue in the active site of D-amino acid oxidase is chlorinated by chloramines(41) . The reaction is highly specific in that N-chloro derivatives of D-leucine, D-isoleucine, and D-norvaline are reactive, whereas hypochlorous acid, monochloramine, N-chloro-D-alanine, and N-chloro-D-valine do not chlorinate the tyrosyl residue. It was proposed that, given the structural specificity of this reaction and that N-chloro-D-leucine is a relatively unreactive chlorine donor, chlorination is likely to involve an orientated intramolecular electrophilic substitution between the bound chloramine and the tyrosyl residue at the active site. The possibility of a free radical process, as we have suggested, was not excluded.

At high levels of hypochlorous acid, formation of chlorotyrosine declined. This could result from conversion of chloramines to more unstable dichloramines(16) , further ring chlorination to dichlorotyrosine, or hypochlorous acid reacting with amide groups to promote peptide cleavage(42) .

Chloramine formation from amino groups is one of the more favored reactions of hypochlorous acid (13, 14) and has been shown to occur when neutrophils are stimulated(15) . Amines are readily regenerated by reducing agents such as thiols, ascorbate, and methionine, so that the relatively slow rearrangement to form chlorotyrosine would be in competition with these faster reactions. This is evident from the ability of dithi- othreitol to stop chlorination of tyrosyl residues when it was added to the chloramine of Gly-Gly-Tyr-Arg (Fig. 5). However, stimulated neutrophils and monocytes are likely to deplete reducing species in their immediate environment, allowing the chlorination of tyrosine to occur.

Halogenation of aromatic rings is an enzymatic activity that is unique to peroxidases(43) , and myeloperoxidase is the only human enzyme capable of producing hypochlorous acid in vivo(43) . Eosinophil peroxidase does not oxidize chloride to hypochlorous acid under physiological conditions(44, 45) . This ensures that chlorotyrosine will be a specific marker for production of hypochlorous acid by myeloperoxidase. The detection of chlorotyrosine in biological samples would establish that myeloperoxidase produces hypochlorous acid in vivo and enable assessment of the contribution that this potent oxidant makes to the tissue injury of various inflammatory pathologies.

  
Table: Reactivity of tyrosine and peptides with hypochlorous acid

Peptides and tyrosine were dissolved in PBS, and 100 µM hypochlorous was added. The data are representative of two experiments. ND, not detected.


  
Table: 0p4in Value for chymotrypsin A4 digests is the mean of seven aliquots.

  
Table: Electrospray mass spectrometry of peptide fragments

The untreated peptide Gly-Gly-Tyr-Arg (GGYR), or that treated with equimolar hypochlorous acid, was analyzed by mass spectrometry as outlined under ``Experimental Procedures.'' The measured values are the result of one or two injections of the sample. When two values were obtained, the values were averaged. All measured values were within ±0.4 of the calculated mass. [Y](imm), tyrosine immonium ion; [Y](phen), p-methylphenoxy ion; and [Y](benzyl), benzyl ion.



FOOTNOTES

*
This work was supported by the Health Research Council of New Zealand and the Canterbury Medical Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by the National Health and Medical Research Council of Australia.

To whom correspondence should be addressed.

The abbreviations used are: PBS, phosphate-buffered saline; HPLC, high pressure liquid chromatography; MALDI-tof, matrix-assisted laser desorption/ionization time of flight.

N. M. Domigan, C. C. Winterbourn, and A. J. Kettle, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. Maurice Owen for assistance with the amino acid analysis.


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