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• W2047985441 abstract "The existence of interhalogen compounds was proposed more than a century ago, but no biological roles have been attributed to these highly oxidizing intermediates. In this study, we determined whether the peroxidases of white blood cells can generate the interhalogen gas bromine chloride (BrCl). Myeloperoxidase, the heme enzyme secreted by activated neutrophils and monocytes, uses H2O2 and Cl− to produce HOCl, a chlorinating intermediate. In contrast, eosinophil peroxidase preferentially converts Br− to HOBr. Remarkably, both myeloperoxidase and eosinophil peroxidase were able to brominate deoxycytidine, a nucleoside, and uracil, a nucleobase, at plasma concentrations of Br− (100 μm) and Cl− (100 mm). The two enzymes used different reaction pathways, however. When HOCl brominated deoxycytidine, the reaction required Br− and was inhibited by taurine. In contrast, bromination by HOBr was independent of Br− and unaffected by taurine. Moreover, taurine inhibited 5-bromodeoxycytidine production by the myeloperoxidase-H2O2-Cl−- Br− system but not by the eosinophil peroxidase-H2O2-Cl−-Br−system, indicating that bromination by myeloperoxidase involves the initial production of HOCl. Both HOCl-Br− and the myeloperoxidase-H2O2-Cl−-Br−system generated a gas that converted cyclohexene into 1-bromo-2-chlorocyclohexane, implicating BrCl in the reaction. Moreover, human neutrophils used myeloperoxidase, H2O2, and Br− to brominate deoxycytidine by a taurine-sensitive pathway, suggesting that transhalogenation reactions may be physiologically relevant. 5-Bromouracil incorporated into nuclear DNA is a well known mutagen. Our observations therefore raise the possibility that transhalogenation reactions initiated by phagocytes provide one pathway for mutagenesis and cytotoxicity at sites of inflammation. The existence of interhalogen compounds was proposed more than a century ago, but no biological roles have been attributed to these highly oxidizing intermediates. In this study, we determined whether the peroxidases of white blood cells can generate the interhalogen gas bromine chloride (BrCl). Myeloperoxidase, the heme enzyme secreted by activated neutrophils and monocytes, uses H2O2 and Cl− to produce HOCl, a chlorinating intermediate. In contrast, eosinophil peroxidase preferentially converts Br− to HOBr. Remarkably, both myeloperoxidase and eosinophil peroxidase were able to brominate deoxycytidine, a nucleoside, and uracil, a nucleobase, at plasma concentrations of Br− (100 μm) and Cl− (100 mm). The two enzymes used different reaction pathways, however. When HOCl brominated deoxycytidine, the reaction required Br− and was inhibited by taurine. In contrast, bromination by HOBr was independent of Br− and unaffected by taurine. Moreover, taurine inhibited 5-bromodeoxycytidine production by the myeloperoxidase-H2O2-Cl−- Br− system but not by the eosinophil peroxidase-H2O2-Cl−-Br−system, indicating that bromination by myeloperoxidase involves the initial production of HOCl. Both HOCl-Br− and the myeloperoxidase-H2O2-Cl−-Br−system generated a gas that converted cyclohexene into 1-bromo-2-chlorocyclohexane, implicating BrCl in the reaction. Moreover, human neutrophils used myeloperoxidase, H2O2, and Br− to brominate deoxycytidine by a taurine-sensitive pathway, suggesting that transhalogenation reactions may be physiologically relevant. 5-Bromouracil incorporated into nuclear DNA is a well known mutagen. Our observations therefore raise the possibility that transhalogenation reactions initiated by phagocytes provide one pathway for mutagenesis and cytotoxicity at sites of inflammation. diethylenetriaminepentaacetic acid gas chromatography mass spectrometry high pressure liquid chromatography Reactive oxidants generated by phagocytic white blood cells are critical to host defense because they kill invading pathogens (1Dunford H.B. Heme Peroxidases. John Wiley & Sons, Inc., New York1999Google Scholar, 2Klebanoff S.J. Clark R.A. The Neutrophil: Function and Clinical Disorders. North Holland Biochemical Press, Amsterdam1978Google Scholar, 3Hurst J.K. Barrette Jr., W.C. CRC Crit. Rev. Biochem. Mol. Biol. 1989; 24: 271-328Crossref PubMed Scopus (220) Google Scholar, 4Tobler A. Koeffler H.P. Harris J.R. Blood Cell Biochemistry: Lymphocytes and Granulocytes. Plenum Press, New York1991: 255-288Google Scholar, 5Kettle A.J. Winterbourn C.C. Redox Rep. 1997; 3: 3-15Crossref PubMed Scopus (592) Google Scholar). However, they are also potentially dangerous because they may damage tissues at sites of inflammation. The heme enzyme myeloperoxidase, synthesized and secreted by neutrophils and monocytic cells, is an important source of oxidants. It uses H2O2generated by the phagocyte NADPH oxidase to produce potent cytotoxins. At plasma halide concentrations, its major initial product is HOCl (6Harrison J.E. Schultz J. J. Biol. Chem. 1976; 251: 1371-1374Abstract Full Text PDF PubMed Google Scholar,7Foote C.S. Goyne T.E. Lehrer R.I. Nature. 1981; 301: 715-716Crossref Scopus (228) Google Scholar). Cl−+H2O2+H+→HOCl+H2OEquation 1 HOCl can oxidize sulfhydryl groups (8Carr A.C. Winterbourn C.C. Biochem. J. 1997; 327: 275-281Crossref PubMed Scopus (93) Google Scholar), halogenate and oxygenate unsaturated lipids (9Winterbourn C.C. van den Berg J.J.M. Roitman E. Kuypers F.A. Arch. Biochem. Biophys. 1992; 296: 547-555Crossref PubMed Scopus (235) Google Scholar, 10Heinecke J.W. Li W. Mueller D.M. Bohrer A. Turk J. Biochemistry. 1994; 33: 10127-10136Crossref PubMed Scopus (140) Google Scholar), and halogenate aromatic compounds (11Domigan N.M. Charlton T.S. Duncan M.W. Winterbourn C.C. Kettle A.J. J. Biol. Chem. 1995; 270: 16542-16548Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 12Hazen S.L. Hsu F.F. Mueller D.M. Crowley J.R. Heinecke J.W. J. Clin. Invest. 1996; 98: 1283-1289Crossref PubMed Scopus (233) Google Scholar, 13Hazen S.L. Heinecke J.W. J. Clin. Invest. 1997; 99: 2075-2081Crossref PubMed Scopus (755) Google Scholar). Myeloperoxidase-derived chlorinating agents also generate secondary oxidants such as monochloramines, dichloramines (14Marquez L.A. Dunford H.B. J. Biol. Chem. 1994; 69: 7950-7956Abstract Full Text PDF Google Scholar, 15Test S.T. Lampert M.B. Ossanna P.J. Thoene J.G. Weiss S.J. J. Clin. Invest. 1984; 74: 1341-1349Crossref PubMed Scopus (129) Google Scholar, 16Thomas E.L. Grisham M.B. Jefferson M.M. Methods Enzymol. 1986; 132: 569-585Crossref PubMed Scopus (339) Google Scholar), and amino acid-derived aldehydes (17Stetmaszynska T. Zgliczynski J.M. Eur. J. Biochem. 1978; 92: 301-308Crossref PubMed Scopus (85) Google Scholar, 18Hazen S.L. d'Avignon A. Anderson M.M. Hsu F.F. Heinecke J.W. J. Biol. Chem. 1998; 273: 4997-5005Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 19Anderson M.M. Requena J.R. Crowley J.R. Thorpe S.R. Heinecke J.W. J. Clin. Invest. 1999; 104: 103-113Crossref PubMed Scopus (325) Google Scholar). We previously demonstrated that HOCl generated by myeloperoxidase is in equilibrium with molecular chlorine (Cl2) through a reaction that requires chloride (Cl−) and H+(12Hazen S.L. Hsu F.F. Mueller D.M. Crowley J.R. Heinecke J.W. J. Clin. Invest. 1996; 98: 1283-1289Crossref PubMed Scopus (233) Google Scholar, 20Henderson J.P. Byun J. Heinecke J.W. J. Biol. Chem. 1999; 274: 33440-33448Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). HOCl+H++Cl−⇄Cl2+H2OEquation 2 Cl2 generated by this pathway has been implicated in the production of 3-chlorotyrosine and 5-chlorodeoxycytidine by activated neutrophils (12Hazen S.L. Hsu F.F. Mueller D.M. Crowley J.R. Heinecke J.W. J. Clin. Invest. 1996; 98: 1283-1289Crossref PubMed Scopus (233) Google Scholar, 20Henderson J.P. Byun J. Heinecke J.W. J. Biol. Chem. 1999; 274: 33440-33448Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Elevated levels of protein-bound 3-chlorotyrosine and myeloperoxidase are found in human atherosclerotic tissue, strongly suggesting that oxidative reactions involving HOCl damage proteins in this chronic inflammatory disorder (13Hazen S.L. Heinecke J.W. J. Clin. Invest. 1997; 99: 2075-2081Crossref PubMed Scopus (755) Google Scholar, 21Daugherty A. Dunn J.L. Rateri D.L. Heinecke J.W. J. Clin. Invest. 1994; 94: 437-444Crossref PubMed Scopus (1134) Google Scholar). Chronic inflammation also increases the risk of cancer, raising the possibility that reactive intermediates generated by neutrophils, monocytes, and macrophages might damage nucleic acids and compromise the integrity of the genome (22Weitzman S.A. Gordon L.I. Blood. 1990; 76: 655-663Crossref PubMed Google Scholar, 23Ohshima H. Bartsch H. Mutat. Res. 1994; 305: 253-264Crossref PubMed Scopus (971) Google Scholar, 24Wiseman H. Halliwell B. Biochem. J. 1996; 313: 17-29Crossref PubMed Scopus (1974) Google Scholar). Genetic epidemiological studies have revealed that a polymorphism in the myeloperoxidase promoter region alters the risk for various cancers (25Schabath M.B. Spitz M.R. Zhang X. Delclos G.L. Wu X. Carcinogenesis. 2000; 21: 1163-1166Crossref PubMed Google Scholar, 26Piedrafita F.J. Molander R.B. Vansant G. Orlova E.A. Pfahl M. Reynolds W.F. J. Biol. Chem. 1996; 271: 14412-14420Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 27Reynolds W.F. Chang E. Douer D. Ball E.D. Kanda V. Blood. 1997; 90: 2730-2737Crossref PubMed Google Scholar, 28London S.J. Lehman T.A. Taylor J.A. Cancer Res. 1997; 57: 5001-5003PubMed Google Scholar, 29Cascorbi I. Henning S. Brockmoller J. Gephart J. Meisel C. Muller J.M. Loddenkemper R. Roots I. Cancer Res. 2000; 60: 644-649PubMed Google Scholar, 30Le Marchand L. Seifried A. Lum A. Wilkens L.R. Cancer Epidemiol. Biomarkers Prev. 2000; 9: 181-184PubMed Google Scholar). These results suggest that myeloperoxidase may play an important role in carcinogenesis, perhaps by generating mutagenic oxidants during the inflammatory response. A structurally related heme protein, eosinophil peroxidase, is released by activated eosinophils, which help kill invading parasites. This peroxidase contributes to the characteristic staining of eosinophils. At plasma concentrations of halide (100 mmCl−, 20–100 μm bromide, <1 μm iodide; Refs. 31Holzbecher J. Ryan D.E. Clin. Biochem. 1980; 13: 277-278Crossref PubMed Scopus (50) Google Scholar and 32Ramsey P.G. Martin T. Chi E. Klebanoff S.J. J. Immunol. 1982; 128: 415-420PubMed Google Scholar), eosinophil peroxidase preferentially oxidizes bromide (Br−) to produce the potent brominating agent HOBr (33Weiss S.J. Test S.T. Eckmann C.M. Ross D. Regiani S. Science. 1986; 234: 200-203Crossref PubMed Scopus (210) Google Scholar, 34Mayeno A.N. Curran A.J. Roberts R.L. Foote C.S. J. Biol. Chem. 1989; 264: 5660-5668Abstract Full Text PDF PubMed Google Scholar, 35Thomas E.L. Bozeman P.M. Jefferson M.M. King C.C. J. Biol. Chem. 1995; 270: 2906-2913Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Br−+H2O2+H+→HOBr+H2OEquation 3 Like HOCl, HOBr oxidizes biomolecules at sites of eosinophilic inflammation (36Wu W. Samoszuk M.K. Comhair S.A.A. Thomassen M.J. Farver C.F. Dweik R.A. Kavuru M.S. Erzurum S.C. Hazen S.L. J. Clin. Invest. 2000; 105: 1455-1463Crossref PubMed Scopus (269) Google Scholar). DNA may be one important target because eosinophil peroxidase brominates deoxycytidine in vitro. 1J. P. Henderson and J. W. Heinecke, unpublished observation. It is noteworthy that schistosomiasis, a chronic inflammatory disease characterized by an intense eosinophilic granulomatous reaction to the eggs of the blood fluke Schistosoma, greatly increases the risk for cancer (reviewed in Refs. 37Rosin M.P. Anwar W.A. Ward A.J. Cancer Res. 1994; 54 (suppl.): 1929-1933Google Scholar, 38Ishii A. Matsuoka H. Aji T. Ohta N. Arimoto S. Wataya Y. Hayatsu H. Mutat. Res. 1994; 305: 273-281Crossref PubMed Scopus (59) Google Scholar, 39Rosin M.P. Zaki S. Ward A.J. Anwar W.A. Mutat. Res. 1994; 305: 283-292Crossref PubMed Scopus (112) Google Scholar). More than 100 years ago, inorganic chemists proposed the existence of interhalogens, which are combinations of different halogens (XX n′). Both binary (BrCl, IBr, and ICl) and ternary (ICl3) interhalogens have since been characterized. One pathway for their formation requires hypohalous acid (HOX) and halide ion (X; Refs. 40Kumar K. Margerum D.W. Inorg. Chem. 1987; 26: 2706-2711Crossref Scopus (283) Google Scholar and 41Wang T.X. Kelley M.D. Cooper J.N. Beckwith R.C. Margerum D.W. Inorg. Chem. 1994; 33: 5872-5878Crossref Scopus (169) Google Scholar). HOX+nX′+H+⇄XXn′+H2OEquation 4 HOCl reacts with Br− by this mechanism to yield molecular BrCl. Anions of interhalogens and polyhalides are also known; they include Cl 3−, Br 3−, I 3−, Br2Cl−, and BrCl 2−. Chemically, interhalogens are extremely corrosive species that attack a wide range of other compounds (42de la Mare P.B.D. Electrophilic Halogenation. Cambridge University Press, Cambridge1976Google Scholar). In the current studies, we show that myeloperoxidase generates reactive brominating species that oxidize nucleobases by a reaction involving HOCl, Br−, and formation of BrCl, an interhalogen gas. We also found that human neutrophils used myeloperoxidase, Cl−, and Br− to brominate deoxycytidine, suggesting that transhalogenation reactions may be physiologically relevant. Our observations suggest that transhalogenation reactions executed by phagocytes may represent one pathway for mutagenesis and cytotoxicity at sites of inflammation. Organic solvents, H2O2, sodium hypochlorite, and sodium phosphate were obtained from Fisher. Bis(trimethylsilyl)trifluoroacetamide and silylation grade acetonitrile were from Regis Technologies, Inc. (Morton Grove, IL). All other materials were purchased from Sigma, except where indicated. Myeloperoxidase (A 430/A 280 > 0.8) was isolated from HL-60 cells by sequential lectin affinity, ion exchange, and size exclusion chromatographies (43Heinecke J.W. Li W. Francis G.A. Goldstein J.A. J. Clin. Invest. 1993; 91: 2866-2872Crossref PubMed Scopus (302) Google Scholar, 44Rakita R.M. Michel B.R. Rosen H. Biochemistry. 1990; 29: 1075-1080Crossref PubMed Scopus (91) Google Scholar). Enzyme concentration was determined spectrophotometrically (ε430 = 178 mm−1 cm−1; Ref. 45Morita Y. Iwamoto H. Aibara S. Kobayashi T. Hasegawa E. J. Biochem. ( Tokyo ). 1986; 99: 761-770Crossref PubMed Scopus (64) Google Scholar). Porcine eosinophil peroxidase (Ahis/A 280 > 0.9) was provided by ExOxEmis (San Antonio, TX). The purity of myeloperoxidase and eosinophil peroxidase were assessed by peroxidase activity using nondenaturing polyacrylamide slab gel electrophoresis and gel system 8 (46van Dalen C.J. Whitehouse M.W. Winterbourn C.C. Kettle A.J. Biochem. J. 1997; 327: 487-492Crossref PubMed Scopus (350) Google Scholar, 47Maurer H. Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis. De Gruyter, New York1971Google Scholar). Glycerol (25% w/v) and cetyltrimethylammonium bromide (0.05% w/v) were included in all buffers. Riboflavin (0.024 mg/ml) was used as the polymerization catalyst, and the stacking gel was omitted. Peroxidase activity was visualized by incubating the gel in 400 μm tetramethylbenzidine, 10 mm sodium citrate, pH 5, 10 mm EDTA, 5 mm NaBr, and 200 μm H2O2. Chloride-free NaOCl was prepared by a modification of previously described methods (16Thomas E.L. Grisham M.B. Jefferson M.M. Methods Enzymol. 1986; 132: 569-585Crossref PubMed Scopus (339) Google Scholar). Reagent NaOCl (100 ml) mixed with ethyl acetate (100 ml) was protonated by dropwise addition of concentrated phosphoric acid (final pH ≤ 6) with intermittent shaking. The organic phase containing HOCl was washed twice with water, and HOCl was re-extracted into the aqueous phase by dropwise addition of NaOH (final pH ≥ 9). Residual ethyl acetate in the aqueous solution of chloride-free NaOCl was removed by bubbling with nitrogen gas. The concentration of NaOCl was determined spectrophotometrically (ε292 = 350 m−1cm−1; Ref. 48Morris J.C. J. Phys. Chem. 1966; 70: 3798-3805Crossref Scopus (816) Google Scholar). Taurine monochloramine was prepared by addition of HOCl to taurine (1:100; mol/mol). Taurine monochloramine concentration was determined spectrophotometrically (ε252= 429 m−1 cm−1; Ref. 16Thomas E.L. Grisham M.B. Jefferson M.M. Methods Enzymol. 1986; 132: 569-585Crossref PubMed Scopus (339) Google Scholar) Bromide-free HOBr was prepared as described (49Derbyshire, D. H., and Waters, W. A. (1950) J. Chem. Soc., 564–573.Google Scholar). Briefly, silver nitrate solution was added to ∼80 mmbromine water in a molar ratio of 1.5:1. The precipitate was removed by centrifugation, and 30 ml of the supernatant was distilled under vacuum using a foil-covered microscale distillation apparatus. The distillate was collected in a foil covered vial at 4 °C. Reagent taurine monobromamine was prepared by addition of reagent HOBr to a 100-fold excess of taurine. HOBr concentration was determined spectrophotometrically following formation of taurine monobromamine (ε288 = 430 m−1cm−1; Ref. 35Thomas E.L. Bozeman P.M. Jefferson M.M. King C.C. J. Biol. Chem. 1995; 270: 2906-2913Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). All reactions were performed in gas-tight vials and initiated by addition with a gas-tight syringe of oxidant (H2O2 or HOCl/OCl−) through a septum while vortexing the sample. Reactions were terminated by addition of l-methionine to a final concentration of 6 mm. The concentration of H2O2 was determined spectrophotometrically (ε240 = 43.6 m−1cm−1; Ref. 50Beers R.J. Sizer I.W. J. Biol. Chem. 1952; 195: 133-140Abstract Full Text PDF PubMed Google Scholar). The pH dependence of 5-bromouracil and 5-bromodeoxycytidine formation was performed using reaction mixtures containing phosphoric acid, monobasic sodium phosphate, and dibasic sodium phosphate (final concentration, 50 mm). The pH of the reaction mixture (which did not contain l-methionine) was determined at the end of the incubation. Neutrophils were prepared by density gradient centrifugation (51Heinecke J.W. Li W. Daehnke H.L.R. Goldstein J.A. J. Biol. Chem. 1993; 268: 4069-4077Abstract Full Text PDF PubMed Google Scholar) and suspended in Dulbecco's phosphate-buffered saline supplemented with 1 mg/ml dextrose, 1 mm deoxycytidine, 100 μm NaBr, and 100 μm DTPA,2 pH 5.9. Differential cell counts revealed that neutrophil preparations contained 96–100% neutrophils and 0–4% eosinophils. Cells (3 ml) were activated with 200 nm phorbol myristate acetate, incubated at 37 °C for 60 min, and maintained in suspension with intermittent inversion. The reaction was terminated by addition of methionine to 6 mm and centrifugation of the cells at 400 × g for 10 min. The supernatant was concentrated to dryness under vacuum, dissolved in 0.4 ml of HPLC solvent A, centrifuged at 14,000 × g for 10 min, and the supernatant was subjected to HPLC analysis. Uracil and deoxycytidine reaction products were analyzed by reverse-phase HPLC with a C18 column (Beckman Porasil, 5 μm resin, 4.6 × 250 mm) at a flow of 1 ml/min and UV detection at 274 and 295 nm, respectively. Uracil reactions were analyzed by injection of 100 μl of reaction mixture onto the column followed by isocratic elution with 20 mm ammonium formate. For analysis of deoxycytidine reactions, 100 μl of the reaction mixture was injected onto the column and eluted with a gradient of: 95% solvent A (0.1% trifluoroacetic acid, pH 2.5) and 5% solvent B (0.1% trifluoroacetic acid in methanol, pH 2.5) for 4 min, 5–100% solvent B over 20 min, and then 100% solvent B for 10 min. 5-Bromouracil and 5-bromodeoxycytidine yields were quantified by comparison of integrated peak areas to standard curves generated using commercially available compounds. For mass spectrometric analysis, HPLC fractions were collected and concentrated under vacuum. For NMR analysis, 10-fold concentrated reaction mixtures were fractionated on a semi-preparative C18 column (μPorasil; 5 μm resin, 10 × 250 mm; Beckman) at a flow rate of 2.5 ml/min with an isocratic gradient consisting of 90% 20 mm ammonium formate, pH 6.3, and 10% methanol. N-Chlorodeoxycytidine (retention time, 10 min) was prepared as described (20Henderson J.P. Byun J. Heinecke J.W. J. Biol. Chem. 1999; 274: 33440-33448Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) and isolated with an analytical C18 column (μPorasil; 5 μm resin, 4.6 × 250 mm; Beckman) column using 5% methanol at a flow rate of 1 ml/min. Reaction products were isolated by HPLC, solubilized in D2O, and analyzed at 25 °C with a Varian Unity-Plus 500 spectrometer (499.843 MHz for 1H) equipped with a Nalorac indirect detection probe. 1H Chemical shifts were referenced to external sodium 3-(trimethylsilyl)propionate-2,2,3,3-d 4 in D2O. Spectra were recorded from 8 transients with a 12-s preacquisition delay over a spectral width of 8000 Hz. Pyrimidine resonances of the brominated deoxycytidine (8.23 ppm; singlet, H6) and uracil (7.79 ppm; singlet, H6) reaction products were essentially identical to those of commercially available 5-bromodeoxycytidine and 5-bromouracil. When compared with substrate, the aromatic region of each product spectrum was notable for the lack of proton resonances at C-5, a downfield shift in the proton resonance at C-6, and conversion of the C-6 proton resonance from a doublet to a singlet, both of which are consistent with substitution of a bromine atom at the C-5 position. After nucleobases were dried under vacuum, residual water was removed by forming an azeotrope with 50 μl of pyridine and again drying the suspension under vacuum. DNA bases were converted to trimethylsilyl derivatives with excessbis(trimethylsilyl)trifluoroacetamide + 1% trimethylchlorosilane in acetonitrile (3:1 v/v) at 100 °C for 60 min. Aliquots (1 μl) were analyzed in the positive electron ionization mode using full mass scanning on either a Hewlett Packard 5890 Series II gas chromatograph (Santa Clarita, CA) interfaced with a Hewlett Packard 5972 Series Mass Selective Detector or a Varian Star 3400 CX gas chromatograph (Walnut Creek, CA) interfaced with a Finnegan SSQ 7000 mass spectrometer (San Jose, CA). Each gas chromatograph was equipped with a 12-m DB-1 capillary column (inner diameter, 0.2 mm; film thickness, 0.33 μm; J&W Scientific, Folsom, CA). Injector and interface temperatures were 250 and 280 °C, respectively. The initial GC oven temperature was 70 °C for 2 min, followed by a 60 °C/min increase to 180 °C and a final 10 °C/min ramp to 220 °C. Derivatizing agent injections were analyzed between samples to ensure that no residual analyte remained in the injection port. Cyclohexene addition products (0.2 μl) were analyzed by selected ion monitoring of ions of m/z 195–201 using an initial GC temperature of 50 °C for 2 min, followed by a 20 °C/min increase to 100 °C and a final 60 °C/min ramp to 220 °C. Aliquots from HPLC fractions were analyzed on a Waters Alliance 2670 HPLC equipped with a C18 column (Porasil, 5 μm resin, 2.1 × 150 mm; Beckman) interfaced with a Finnigan LCQ. Sample (5 μl) was injected at a flow rate of 200 μl/min. Solvent C was 1% acetic acid in 4% methanol, and solvent D was 1% acetic acid in 95% methanol. The sample was eluted from the column by a discontinuous gradient of solvent D. The gradient was: 0% solvent D for 2 min, 0–100% solvent D over 8 min, and then 100% for 10 min. Mass spectrometric analysis was carried out in full mass scanning, zoom scanning, and low energy collisionally activated dissociation modes as described (20Henderson J.P. Byun J. Heinecke J.W. J. Biol. Chem. 1999; 274: 33440-33448Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Helium was used as a damping gas and collision activation partner. The temperature of the heated capillary was 220 °C. In the full scan mode (m/z 150–300), each scan consisted of three 300-ms microscans. Full scan mass spectra consisted of 10 signal-averaged scans with subtraction of the carrier solvent background from the same number of scans. The electrospray positive ion mass spectra of authentic 5-bromodeoxycytidine and the product from myeloperoxidase, HOCl + Br−, and HOBr yielded the same major [M + H]+ ion at m/z 306. All compounds also exhibited a prominent ion at m/z 308. The relative abundances of the ions at m/z 306 and 308 reflected that of the natural isotopic abundance of79Br and 81Br, strongly suggesting that the deoxycytidine oxidation product was monobrominated. The collisionally activated dissociation tandem mass spectrum of them/z 306 ion generated a product ion atm/z 190, which is consistent with cleavage of theN-glycoside bond of 5-bromodeoxycytidine to yield79Br-substituted cytosine. The collisionally activated dissociation tandem mass spectrum of the m/z 308 ion likewise generated a product ion at m/z 192, consistent with cleavage of the brominated nucleoside to yield81Br-substituted cytosine. The electrospray ionization MS/MS spectrum of the myeloperoxidase product indicates that the bromine substitution site resides on the cytosine base of deoxycytidine. Further structural characterization of the modified nucleobase generated from deoxycytidine and uracil was achieved using GC/MS to obtain an informative electron ionization mass spectrum and GC retention time from material isolated by HPLC. This procedure yields information only about the deoxycytidine nucleobase because theN-glycoside bond of the nucleoside is hydrolyzed during the derivatization reactions. The mass spectrum of the reaction products exhibited low abundance ions consistent with the molecular ion [M+·] of the bis-trimethylsilyl derivatives (m/z 333 and 334 for brominated deoxycytidine and uracil, respectively). The major ion from each analyte was consistent with loss of CH3⋅ as expected for trimethylsilyl derivatives. The [M]⋅+ and [M-CH3⋅]+ ions exhibited prominent M+2 isotopes as expected for monobrominated compounds containing the natural 1:1 abundance of 79Br or 81Br. Ions observed for [M − Br−]+ and [M − CH3· − HBr]+ or [M − CH4 − Br−]+ fragments lacked the prominent M+2 fragments as expected for compounds that lack bromine. The positive ion mass spectra and GC retention times of the trimethylsilyl derivatives of the reaction products were essentially identical to those obtained for commercially available 5-bromodeoxycytidine or 5-bromouracil. We previously demonstrated that the myeloperoxidase-H2O2-Cl− system (containing 100 mm Cl−) oxidizes deoxycytidine to 5-chlorodeoxycytidine (20Henderson J.P. Byun J. Heinecke J.W. J. Biol. Chem. 1999; 274: 33440-33448Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). When we supplemented this system with a plasma concentration of Br− (100 μm), HPLC analysis detected a peak of material that migrated with a retention time distinct from that of 5-chlorodeoxycytidine (Fig.1 A). The new oxidation product was isolated by HPLC and identified as 5-bromodeoxycytidine on the basis of its HPLC retention time, ultraviolet absorption spectrum, GC retention time and positive ion mass spectrum, electrospray ionization tandem mass spectrum, and 1H NMR spectrum (see “Methods”). Under these reaction conditions, 5-bromodeoxycytidine was also the major product when eosinophil peroxidase or lactoperoxidase oxidized deoxycytidine. It was therefore important to determine whether our myeloperoxidase preparation was contaminated by other peroxidases, even though we isolated the enzyme from HL-60 cells, a promyelocytic cell line that is not known to express other peroxidases. Myeloperoxidase was apparently pure as assessed by its heme spectrum and by denaturing polyacrylamide gel electrophoresis. Moreover, it yielded a single band of peroxidase activity that migrated with a retention time distinct from that of eosinophil peroxidase on nondenaturing gel electrophoresis. These observations indicate that the reactive intermediates that brominated deoxycytidine at plasma concentrations of Cl− and Br− resulted from the action of myeloperoxidase. To determine whether the enzyme can halogenate other pyrimidines, we incubated uracil with the myeloperoxidase-H2O2-Cl− system. In the absence of added Br−, a new peak of material (retention time, 13.7 min) was detectable in the reaction mixture by HPLC analysis (Fig. 1 B). In the presence of 100 μm Br−, we observed a second peak (retention time, 18 min). We isolated both peaks of material by HPLC and determined their structures by ultraviolet absorption spectroscopy, GC, and positive ion mass spectrometry, electrospray ionization tandem mass spectrometry, and 1H NMR spectroscopy (see “Methods”). We identified the early and late eluting materials as 5-chlorouracil and 5-bromouracil, respectively. Production of 5-bromouracil by myeloperoxidase was dependent on the concentration of Br− in the reaction mixture (Fig.2). Increasing [Br−] from 0 to 100 μm in the presence of 100 mmCl− reduced uracil chlorination but increased uracil bromination. The apparent K m values for Cl− and Br− binding by myeloperoxidase are 175 and 2 mm (46van Dalen C.J. Whitehouse M.W. Winterbourn C.C. Kettle A.J. Biochem. J. 1997; 327: 487-492Crossref PubMed Scopus (350) Google Scholar), respectively, suggesting that the enzyme did not directly oxidize Br− to generate 5-bromouracil. The relative yields of the halogenated pyrimidines also differed; under optimal conditions, the yield of 5-bromouracil was 5-fold greater than that of 5-chlorouracil. We used HPLC to investigate the reaction requirements for generation of 5-bromodeoxycytidine by the myeloperoxidase and eosinophil peroxidase-H2O2-Cl−-Br−systems (Table I). Both systems required enzyme and H2O2 and were blocked by catalase, a scavenger of H2O2. The product yield of the reaction was nearly quantitative relative to H2O2. Omitting B" @default.
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• W2047985441 title "Production of Brominating Intermediates by Myeloperoxidase" @default.
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