FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2039864897> ?p ?o ?g. }

• W2039864897 endingPage "38169" @default.
• W2039864897 startingPage "38160" @default.
• W2039864897 abstract "Myeloperoxidase uses hydrogen peroxide to oxidize numerous substrates to hypohalous acids or reactive free radicals. Here we show that neutrophils oxidize melatonin to N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) in a reaction that is catalyzed by myeloperoxidase. Production of AFMK was highly dependent on superoxide but not hydrogen peroxide. It did not require hypochlorous acid, singlet oxygen, or hydroxyl radical. Purified myeloperoxidase and a superoxide-generating system oxidized melatonin to AFMK and a dimer. The dimer would result from coupling of melatonin radicals. Oxidation of melatonin was partially inhibited by catalase or superoxide dismutase. Formation of AFMK was almost completely eliminated by superoxide dismutase but weakly inhibited by catalase. In contrast, production of melatonin dimer was enhanced by superoxide dismutase and blocked by catalase. We propose that myeloperoxidase uses superoxide to oxidize melatonin by two distinct pathways. One pathway involves the classical peroxidation mechanism in which hydrogen peroxide is used to oxidize melatonin to radicals. Superoxide adds to these radicals to form an unstable peroxide that decays to AFMK. In the other pathway, myeloperoxidase uses superoxide to insert dioxygen into melatonin to form AFMK. This novel activity expands the types of oxidative reactions myeloperoxidase can catalyze. It should be relevant to the way neutrophils use superoxide to kill bacteria and how they metabolize xenobiotics. Myeloperoxidase uses hydrogen peroxide to oxidize numerous substrates to hypohalous acids or reactive free radicals. Here we show that neutrophils oxidize melatonin to N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) in a reaction that is catalyzed by myeloperoxidase. Production of AFMK was highly dependent on superoxide but not hydrogen peroxide. It did not require hypochlorous acid, singlet oxygen, or hydroxyl radical. Purified myeloperoxidase and a superoxide-generating system oxidized melatonin to AFMK and a dimer. The dimer would result from coupling of melatonin radicals. Oxidation of melatonin was partially inhibited by catalase or superoxide dismutase. Formation of AFMK was almost completely eliminated by superoxide dismutase but weakly inhibited by catalase. In contrast, production of melatonin dimer was enhanced by superoxide dismutase and blocked by catalase. We propose that myeloperoxidase uses superoxide to oxidize melatonin by two distinct pathways. One pathway involves the classical peroxidation mechanism in which hydrogen peroxide is used to oxidize melatonin to radicals. Superoxide adds to these radicals to form an unstable peroxide that decays to AFMK. In the other pathway, myeloperoxidase uses superoxide to insert dioxygen into melatonin to form AFMK. This novel activity expands the types of oxidative reactions myeloperoxidase can catalyze. It should be relevant to the way neutrophils use superoxide to kill bacteria and how they metabolize xenobiotics. Catalysis of hypochlorous acid production is the accepted physiological function of myeloperoxidase (MPO) 2The abbreviations used are: MPO, myeloperoxidase; HPLC, high performance liquid chromatography; AFMK, N1-acetyl-N2-formyl-5-methoxykynuramine; PMA, phorbol 12-myristate 13-acetate; TMB, 3,3′,5,5′-tetramethylbenzidine; LC/MS, liquid chromatography/mass spectrometry. (1.Klebanoff S.J. Proc. Assoc. Am. Physicians. 1999; 111: 383-389Crossref PubMed Scopus (327) Google Scholar, 2.Kettle A.J. Winterbourn C.C. Redox Report. 1997; 3: 3-15Crossref PubMed Scopus (588) Google Scholar). This heme enzyme is the major protein in neutrophils and is also present in monocytes, macrophages, microglia (3.Rodrigues M.R. Rodriguez D. Russo M. Campa A. Biochem. Biophys. Res. Commun. 2002; 292: 869-873Crossref PubMed Scopus (64) Google Scholar), and neurons (4.Nagra R.M. Becher B. Tourtellotte W.W. Antel J.P. Gold D. Paladino T. Smith R.A. Nelson J.R. Reynolds W.F. J. Neuroimmunol. 1997; 78: 97-107Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 5.Green P.S. Mendez A.J. Jacob J.S. Crowley J.R. Growdon W. Hyman B.T. Heinecke J.W. J. Neurochem. 2004; 90: 724-733Crossref PubMed Scopus (282) Google Scholar). It reacts with hydrogen peroxide to form the redox intermediate compound I, which oxidizes chloride to hypochlorous acid with coincident regeneration of the native enzyme (Reactions 1 and 2). HOCl indicates hypochlorous acid. compound I + CI-+ H→ MPO + HOCIeq 1 compound I + CI-+ H→ MPO + HOCIeq 2 Myeloperoxidase also promotes the oxidation of numerous substrates (RH) to free radical intermediates via the classical peroxidase cycle involving compound I and compound II (Reactions 3 and 4). compound I + RH → compound II + R⋅+H+eq 3 compound II + RH → MPO + R⋅+H-eq 4 Klebanoff (6.Klebanoff S.J. J. Bacteriol. 1968; 95: 2131-2138Crossref PubMed Google Scholar) was the first to show that myeloperoxidase has potent antimicrobial activity because of its ability to generate hypochlorous acid. He proposed that myeloperoxidase produces hypochlorous acid inside phagosomes where it reacts with and kills ingested bacteria. However, during microbial killing myeloperoxidase functions in the presence of a high flux of superoxide (7.Hampton M.B. Kettle A.J. Winterbourn C.C. Blood. 1998; 92: 3007-3017Crossref PubMed Google Scholar). Superoxide reacts rapidly with native enzyme to produce oxymyeloperoxidase or compound III (Reaction 5; k5 = 2 × 106 M–1 s–1 (8.Kettle A.J. Sangster D.F. Gebicki J.M. Winterbourn C.C. Biochim. Biophys. Acta. 1988; 956: 58-62Crossref PubMed Scopus (50) Google Scholar)), which is the dominant form of myeloperoxidase in stimulated neutrophils (9.Winterbourn C.C. Garcia R. Segal A.W. Biochem. J. 1985; 228: 583-592Crossref PubMed Scopus (125) Google Scholar). Reactions of superoxide with myeloperoxidase are likely to be important in host defense because it has been demonstrated that superoxide enhances myeloperoxidase-dependent killing of Staphylococcus aureus by isolated human neutrophils (10.Hampton M.B. Kettle A.J. Winterbourn C.C. Infect. Immun. 1996; 64: 3512-3517Crossref PubMed Google Scholar). The nature of this interaction has yet to be revealed. MPO +O2-→ compound IIIeq 5 Compound III reacts sluggishly with potential substrates (11.Yokota K. Yamazaki I. Biochem. Biophys. Res. Commun. 1965; 18: 48-53Crossref PubMed Scopus (49) Google Scholar), but it is reduced by ascorbate (12.Marquez L.A. Dunford H.B. J. Biol. Chem. 1990; 265: 6074-6078Abstract Full Text PDF PubMed Google Scholar) and acetaminophen (13.Marquez L.A. Dunford H.B. Arch. Biochem. Biophys. 1993; 305: 414-420Crossref PubMed Scopus (23) Google Scholar). It is conceivable that compound III potentiates oxygen, like the analogous intermediates of related enzymes such as cytochromes P450 (14.Newcomb M. Hollenberg P.F. Coon M.J. Arch. Biochem. Biophys. 2003; 409: 72-79Crossref PubMed Scopus (149) Google Scholar) and nitric-oxide synthase (15.Knowles R.G. Moncada S. Biochem. J. 1994; 298: 249-258Crossref PubMed Scopus (2507) Google Scholar). Indeed, reactions of compound III have been invoked to explain the hydroxylation of phenol (16.Subrahmanyam V.V. Kolachana P. Smith M. Free Radic. Res. Commun. 1991; 15: 285-295Crossref PubMed Scopus (33) Google Scholar) and salicylate (17.Kettle A.J. Winterbourn C.C. J. Biol. Chem. 1994; 269: 17146-17151Abstract Full Text PDF PubMed Google Scholar) by myeloperoxidase and superoxide. Superoxide also reduces compound II of myeloperoxidase (Reaction 6). This reaction prevents accumulation of compound II and the associated inhibition of hypochlorous acid production (18.Kettle A.J. Winterbourn C.C. Biochem. J. 1988; 252: 529-536Crossref PubMed Scopus (152) Google Scholar, 19.Kettle A.J. Winterbourn C.C. Biochem. J. 1989; 263: 823-828Crossref PubMed Scopus (62) Google Scholar). Similarly, reduction of compound II by superoxide boosts the catalytic activity of myeloperoxidase (20.Kettle A.J. Winterbourn C.C. Biochemistry. 2001; 40: 10204-10212Crossref PubMed Scopus (80) Google Scholar). compound II +O2- + 2H+→MPO +O2 +H2Oeq 6 We have found that neutrophils and macrophages oxidize melatonin to a chemiluminescent product, presumably N1-acetyl-N2-formyl-5-methoxykynuramide (AFMK) (21.de Oliveira S.S. Ximenes V.F. Catalani L.H. Campa A. Biochem. Biophys. Res. Commun. 2000; 279: 657-662Crossref PubMed Scopus (55) Google Scholar, 22.Rodrigues M.R. Rodriguez D. Henrique C.L. Russo M. Campa A. J. Pineal Res. 2003; 34: 69-74Crossref PubMed Scopus (23) Google Scholar, 23.Silva S.O. Rodrigues M.R. Carvalho S.R. Catalani L.H. Campa A. Ximenes V.F. J. Pineal Res. 2004; 37: 171-175Crossref PubMed Scopus (97) Google Scholar). The reaction required superoxide and was blocked by azide, which is an inhibitor of heme enzymes. Catalase, which scavenges hydrogen peroxide, had little effect on formation of the chemiluminescent product. Collectively, these results suggested that myeloperoxidase uses superoxide to oxidize an organic substrate without the need for hydrogen peroxide. In this investigation our main objectives were to identify the chemiluminescent product and to establish whether it is formed by the classical peroxidase cycle (Reactions 1, 3, and 4) or by a novel mechanism that may be relevant to the physiological function of myeloperoxidase. Melatonin, 5-hydroxytryptamine hydrochloride (serotonin), superoxide dismutase, bovine liver catalase, xanthine oxidase, phorbol 12-myristate 13-acetate (PMA), dl-methionine, taurine, 3,3′,5,5′-tetramethylbenzidine (TMB), acetaldehyde, diethylenetriaminepentaacetic acid, and cytochrome c were purchased from Sigma. Dimethyl sulfoxide (Me2SO) and mannitol were purchased from Merck. All the reagents used for buffers were of analytical grade. Myeloperoxidase was purified from human neutrophils as described previously. Its concentration was determined using its absorption at 430 nm(ϵ430 nm = 89,000 m–1 cm–1 per heme) (24.Kettle A.J. Methods Enzymol. 1999; 300: 111-120Crossref PubMed Scopus (28) Google Scholar). Its purity index (A430/A280) was at least 0.75. Hydrogen peroxide was prepared by diluting a 30% stock solution (Merck) and calculating its concentration using its absorption at 240 nm (ϵ240 nm = 43.6 m–1 cm–1) (25.Beers R.J. Sizer I.W. J. Biol. Chem. 1952; 195: 133-140Abstract Full Text PDF PubMed Google Scholar). Hypochlorous acid was prepared by diluting a concentrated commercial chlorine bleach solution and calculating its concentration using its absorption at 292 nm (ϵ292 nm = 350 m–1 cm–1) (24.Kettle A.J. Methods Enzymol. 1999; 300: 111-120Crossref PubMed Scopus (28) Google Scholar). Melatonin stock solutions were prepared by dissolving it in water containing 10% dimethylformamide. The final concentration of dimethylformamide in the assays was 1% or less. These solutions were prepared daily. PMA was dissolved in Me2SO so that its final concentration in neutrophil reactions was 1% v/v. Isolation of Human Neutrophils—Neutrophils were isolated from the blood of healthy donors by Ficoll-Paque centrifugation, dextran sedimentation, and hypotonic lysis of red cells (24.Kettle A.J. Methods Enzymol. 1999; 300: 111-120Crossref PubMed Scopus (28) Google Scholar). After isolation, neutrophils were resuspended in 10 mm phosphate buffer containing 10 mm potassium chloride and 140 mm sodium chloride (PBS), plus 1 mm calcium chloride, 0.5 mm magnesium chloride, and 1 mg/ml glucose. This complete buffer is referred to as Buffer A. Determination of Hypochlorous Acid Production by Neutrophils and Purified Myeloperoxidase—Neutrophils (2 × 106 cells/ml) were preincubated at 37 °C in Buffer A with 5 mm taurine and varying concentrations of melatonin for 10 min. When used, superoxide dismutase was present at 20 μg/ml. Cells were stimulated by the addition of 100 ng/ml PMA. After 30 min the reactions were stopped by the addition of 20 μg/ml catalase. The neutrophils were then pelleted by centrifugation, and the supernatant put on ice. Formation of hypochlorous acid was measured by assaying accumulated taurine chloramine (see below). Purified myeloperoxidase (10 nm) was incubated in PBS with 5 mm taurine, and reactions were started by adding 50 μm H2O2. After 5 min, the reactions were stopped by adding catalase (20 μg/ml), and accumulated taurine chloramine was measured. When used, serotonin was present at 5 μm. Taurine chloramine was assayed by adding 200 μl of neutrophil supernatant or the myeloperoxidase reaction system to 50 μl of a solution of containing 10 mm TMB and 100 μm potassium iodide in 50% dimethylformamide and 400 mm acetic acid. Under these conditions taurine chloramine oxidizes TMB to a blue product with an absorbance maximum at 655 nm. 3Dypbukt, J. M., Bishop, C., Brooks, W. M., Thong, B., Eriksson, H., and Kettle, A. J. (2005)Free Radic. Biol. Med., in press. A standard curve was generated by adding reagent hypochlorous acid to PBS containing taurine. The absorbance measurements were made in Spectra Max 190 plate reader (Molecular Devices). Measurements of Superoxide Production by Stimulated Neutrophils—Superoxide production was measured as a superoxide dismutase-inhibitable reduction of ferricytochrome c (26.Fridovich I. Greenwald R.A. Handbook of Methods for Oxygen Radical Research. CRC Press, Inc., Boca Raton, FL1985: 213-215Google Scholar). Neutrophils (2 × 106 cells/ml) were preincubated for 10 min at 37 °C in Buffer A containing 20 μg/ml catalase and 100 μm ferricytochrome c. Cells were stimulated by adding 100 ng/ml PMA, and the change in absorbance at 550 nm was measured over the 2nd min of the reaction after an initial lag. Preparation of AFMK Standard—AFMK was prepared by ozone oxidation of melatonin as reported previously (27.Sakiyama F. Masuda N. Nakayama T. Katsuragi Y. Chem. Lett. 1978; 8: 893-986Crossref Google Scholar) and purified by HPLC. The identity of AFMK was confirmed by mass spectrometry ([M + 1]+ m/z = 265) using a Quattro II electrospray ionization-mass spectrometer (Micromass, Manchester, UK) and by fluorescence spectrophotometry (λex = 340 nm; λem = 460 nm) using a Varian F-4500 spectrophotometer (21.de Oliveira S.S. Ximenes V.F. Catalani L.H. Campa A. Biochem. Biophys. Res. Commun. 2000; 279: 657-662Crossref PubMed Scopus (55) Google Scholar). Determination of AFMK Production by Activated Neutrophils—Neutrophils (2 × 106 cells/ml) were incubated at 37 °C in Buffer A with 50 μm melatonin. Cells were stimulated with 50 ng/ml PMA, and subsequently reactions were stopped by adding 20 μg/ml catalase. Neutrophils were then pelleted by centrifugation and put on ice. Loss of melatonin and formation of AFMK were determined by HPLC after comparing peak areas with standard curves for the purified standards. The HPLC system used was a Shimatzu LC-10A coupled to SPD-10A UV-visible detector and RF535 fluorescence detector. Samples were separated isocratically on a Luna C18 reversed phase column (250 × 4.6 mm, 5 μm) using 1 mm KH2PO4, pH 4.0, acetonitrile (3:1) as the mobile phase with a flow rate of 1 ml/min. Absorbances were monitored at 254 nm, and fluorescence was recorded using excitation and emission wavelengths of 340 and 460 nm, respectively. Oxidation of Melatonin by Isolated Myeloperoxidase—Melatonin was incubated in 50 mm phosphate buffer, pH 7.4, with 10 mm acetaldehyde and varying concentrations of myeloperoxidase and xanthine oxidase. Reactions were started by the addition of xanthine oxidase, and formation of AFMK was measured by HPLC as described above. Oxidation of melatonin was monitored by either following UV absorbance changes or increases in fluorescence (λex = 340; λex = 460). Absorbance changes were recorded on an Agilent 8453 diode array spectrophotometer, and fluorescence was measured using a Hitachi F-4500 spectrophotometer. Detection of Melatonin Oxidation Products by LC/MS—The oxidation products of melatonin were separated on a Luna C18 (2.Kettle A.J. Winterbourn C.C. Redox Report. 1997; 3: 3-15Crossref PubMed Scopus (588) Google Scholar) column (particle size 5 μm, 150 × 2.6 mm) (Phenomenex, Torrance, CA) using a Surveyor HPLC pump (Thermo Corp., San Jose, CA). The column was maintained at 30 °C. The products were eluted at a flow rate of 0.2 ml/min using a linear gradient of two solvents: solvent A (5 mm ammonium acetate, pH 4.0, 25% acetonitrile) and solvent B (100% acetonitrile). The gradient was as follows: 0–10 min, 0% solvent B; 10–20 min, increased solvent B to 100%; 20–22 min, maintained solvent B at 100%; 22–25 min, decreased solvent B to 0%. The injection volume was 20 μl. The HPLC was coupled to an ion-trap mass spectrometer (ThermoFinnigan LCQ Deca XP Plus, Thermo Corp.) equipped with an electrospray ionization source. The mass spectrometer was operated with positive ionization using full scan mode (scan range 100–1000 m/z) and selected ion monitoring. Spray voltage was set at 5.0 kV, and the capillary temperature at 275 °C, and the sheath gas flow was at 26 units (instrument units). Spectral Observations of Myeloperoxidase during Oxidation of Melatonin—The absorption spectrum of myeloperoxidase was monitored during oxidation of melatonin using a Beckman DU 7500 diode array spectrophotometer. Spectra were recorded every 30 s and were the average of 10 scans. Reactions were started by adding either hydrogen peroxide (50 μm) or xanthine oxidase and acetaldehyde (10 mm) to the enzyme and melatonin. Reactions were carried out in 50 mm phosphate buffer, pH 7.4. Oxidation of Melatonin by Stimulated Human Neutrophils—Initially we sought to identify the chemiluminescent product formed when neutrophils oxidize melatonin. Neutrophils were incubated with melatonin and stimulated with the phorbol ester PMA to initiate production of superoxide and the release of myeloperoxidase. After 30 min, supernatants were assayed by liquid chromatography with an electrospray positive ionization mass detector to identify products of melatonin oxidation. Melatonin eluted at 17.7 min (m/z 233). Minor and major products eluted before melatonin (Fig. 1A). The minor product that eluted at 12.4 min had an m/z of 249 mass units (Fig. 1B), indicating a likely hydroxylated form of melatonin. The major product that eluted at 14.2 min had an m/z of 265 mass units (Fig. 1C), which corresponds to the mass of AFMK. This peak also co-eluted with authentic AFMK and had the same fluorescence and ultraviolet absorption properties as AFMK (data not shown). Melatonin underwent progressive oxidation over the first 15 min of the reaction after which its rate of loss declined by ∼75%. This slower loss was maintained for the subsequent 45 min of the incubation (Fig. 2). After 1 h ∼40% of the melatonin was oxidized. The time course for AFMK formation mirrored the loss in melatonin. Over the 1-h incubation, 8 μm AFMK was formed, which accounted for about 45% of the oxidized melatonin. The nonstoichiometric yield of AFMK was not due to breakdown of the AFMK because the authentic compound was stable when added to stimulated cells (not shown). Thus, it is likely that melatonin was oxidized to additional products. A range of inhibitors was added to neutrophils to help establish the mechanism by which the cells form AFMK (TABLE ONE). Minimal AFMK was produced when cells were not stimulated or when the NADPH oxidase was inhibited with diphenyliodonium. These results demonstrate that superoxide and/or hydrogen peroxide must be produced by neutrophils before they can oxidize melatonin. Superoxide dismutase was a strong inhibitor of AFMK formation. Catalase showed weak inhibition and blocked the reaction by only 50% at a high concentration of 100 μg/ml. A combination of superoxide dismutase and catalase was no more effective than superoxide dismutase alone. Azide, an inhibitor of myeloperoxidase, decreased production of AFMK by 70%.TABLE ONEInhibitor profile for oxidation of melatonin to AFMK by stimulated human neutrophilsReaction systemAFMK% controlComplete system100-PMA2.5 ± 0.6+Diphenyliodonium0.3+SOD 20 μg/ml13 ± 4+Catalase (20 μg/ml)68 ± 6+Catalase (100 μg/ml)49 ± 2+SOD (20 μg/ml) + catalase (20 μg/ml)13 ± 4+Azide 100 μm30 ± 4+Methionine 1 mm103 ± 2+Me2SO 1 mm105 ± 9+Mannitol108 ± 4 Open table in a new tab Various strong oxidants have been proposed to be produced by neutrophils, including hypochlorous acid, singlet oxygen, hydroxyl radical, and ozone (28.Klebanoff S.J. J. Leukocyte Biol. 2005; 77: 598-625Crossref PubMed Scopus (1730) Google Scholar). All of these oxidants are capable of oxidizing melatonin. Therefore, we added methionine to stimulated neutrophil to scavenge hypochlorous acid, singlet oxygen, and ozone. Me2SO and mannitol were added to intercept the hydroxyl radical. None of these scavengers blocked formation of AFMK. We also determined the effects of superoxide dismutase and catalase on the loss of melatonin. Superoxide dismutase (20 μg/ml) inhibited melatonin loss by 60% (7 ± 2 μm compared with 18 ± 2 μm). Catalase (20 μg/ml) was less effective, inhibiting by 39% (11 ± 2 μm compared with 18 ± 2 μm). These results point to involvement of myeloperoxidase in the oxidation of melatonin to AFMK. However, the requirement for superoxide and the nonessential requirement for hydrogen peroxide suggests an unusual mechanism of oxidation by this heme enzyme. Diffusible oxidants are unlikely to be involved because their scavengers had no effect on formation of AFMK. It is more likely that melatonin reacted directly with myeloperoxidase. Effect of Melatonin on Hypochlorous Acid Production by Neutrophils and Purified Myeloperoxidase—If myeloperoxidase was responsible for oxidizing melatonin, then production of hypochlorous acid by neutrophils is likely to have been affected. Therefore, we measured the effect of melatonin on extracellular production of hypochlorous acid by these cells. With increasing concentration of melatonin there was progressive inhibition of hypochlorous acid production (Fig. 3A). At 50 μm melatonin, little hypochlorous acid was formed. The concentration of melatonin that inhibited hypochlorous acid production by 50% (IC50) was 18 μm. When superoxide dismutase was added to remove superoxide, the IC50 for melatonin dropped to 4 μm. To demonstrate that melatonin inhibits hypochlorous acid production by neutrophils through a direct reaction with myeloperoxidase, we determined the effect of melatonin on the chlorination activity of the isolated enzyme. Melatonin was a potent inhibitor of the initial rate of hypochlorous acid production by myeloperoxidase (Fig. 3B). Serotonin, which readily reduces compound II (29.Dunford H.B. Hsuanyu Y. Biochem. Cell Biol. 1999; 77: 449-457Crossref PubMed Scopus (40) Google Scholar), substantially decreased the ability of melatonin to inhibit the chlorination activity of the enzyme. From these results, we concluded that melatonin reacts with myeloperoxidase released by neutrophils and inhibits production of hypochlorous acid. It is likely to act by competing with chloride and reducing compound I to inactive compound II. This mechanism of inhibition has been shown to occur with tryptophan (30.Kettle A.J. Candaeis L.P. Redox Report. 2000; 5: 179-184Crossref PubMed Scopus (42) Google Scholar) and several phenolic and aromatic amines (31.Kettle A.J. Winterbourn C.C. Biochem. Pharmacol. 1991; 41: 1485-1492Crossref PubMed Scopus (164) Google Scholar). Superoxide dismutase would enhance the ability of melatonin to inhibit hypochlorous acid production by neutrophils because it would prevent superoxide from recycling compound II (Reaction 6) so that the enzyme would be trapped in this inactive form. In these assays hypochlorous acid was detected by trapping it as taurine chloramine. Therefore, it was possible that melatonin acted by scavenging hypochlorous acid, thereby preventing it from reacting with taurine in the buffer. However, at 100 μm melatonin was unable to prevent reagent hypochlorous acid from reacting with taurine (5 mm) (result not shown). Melatonin up to 100 μm had no effect on superoxide production by neutrophils (result not shown). Oxidation of Melatonin by Purified Myeloperoxidase—To confirm that myeloperoxidase was responsible for the production of AFMK by neutrophils, we determined the ability of the purified enzyme to oxidize melatonin. Xanthine oxidase and acetaldehyde were used as a source of superoxide and hydrogen peroxide. Initially, we followed oxidation of melatonin by monitoring its difference spectrum (Fig. 4, top). With the complete reaction system there were progressive increases in absorbance peaks with maxima at 242 nm and between 320 and 360 nm. There was also a progressive decline in absorbance at 290 nm. There were minimal absorbance changes in the absence of myeloperoxidase, and no change in the absence of xanthine oxidase. When superoxide dismutase was added to the reaction, oxidation of melatonin still occurred, but the absorbance changes were substantially different. There was a marked decrease in absorbance at 240 nm, smaller changes at 242 and 290 nm, a distinctive peak at 320 nm, and considerably less absorbance above 340 nm (Fig. 4, bottom). These results demonstrate that melatonin is oxidized by myeloperoxidase. Furthermore, the type of products formed in the presence of superoxide and their relative yields are different to those formed in the absence of superoxide. Reaction mixtures were then assayed by liquid chromatography with mass spectrometry to identify oxidation products of melatonin. As with neutrophils, two UV-absorbing peaks eluted before melatonin. The first was a minor peak and had an m/z of 249 mass units. The major peak had an m/z of 265 mass units and the same fluorescent and UV properties as authentic AFMK (not shown). These products correspond to a hydroxylated form of melatonin and AFMK as found with stimulated neutrophils. We also used selected ion monitoring to determine whether myeloperoxidase oxidizes melatonin to a dimer via the intermediate production of radicals species (Reactions 3 and 4) as it does with other organic substrates such as tyrosine (32.Heinecke J.W. Li W. Daehnke H.L. Goldstein J.A. J. Biol. Chem. 1993; 268: 4069-4077Abstract Full Text PDF PubMed Google Scholar). The expected dimer should have an m/z of 463 mass units when analyzed in the positive ion mode. Indeed, two products eluted with this m/z at about 17.5 and 18 min, indicating the formation of isomeric forms of melatonin dimer (Fig. 5A). The mass spectrum of the product eluting at 18 min gave fragments with m/z of 404.2 and 231 mass units (Fig. 5B), which are consistent with melatonin dimer (Fig. 5C). To determine how myeloperoxidase oxidizes melatonin to AFMK, we undertook inhibitor studies similar to those with neutrophils. Superoxide dismutase blocked AFMK production by greater than 90%, whereas catalase was a poor inhibitor (TABLE TWO). A combination of superoxide dismutase and catalase was no more effective than superoxide dismutase alone. Azide inhibited by 70% but methionine and Me2SO had no effect. These results are very similar to the inhibition pattern observed with neutrophils (TABLE ONE). Thus, myeloperoxidase catalyzed the oxidation of melatonin to AFMK by a reaction that was reliant on superoxide. Although myeloperoxidase used hydrogen peroxide to promote formation of AFMK, the requirement for this substrate was not as great as that for superoxide. Hypochlorous acid, singlet oxygen, and hydroxyl radical could not have been responsible for the production of AFMK because their formation was blocked in the reaction system by excluding chloride and adding methionine or Me2SO.TABLE TWOInhibitor profile for oxidation of melatonin to AFMK by purified myeloperoxidaseReaction systemAFMK% controlComplete system100-MPO15 ± 3-Xanthine0+SOD 20 μg/ml9 ± 2+Catalase 20 μg/ml85 ± 3+Catalase 100 μg/ml68 ± 2+SOD (20 μg/ml) + catalase (20 μg/ml)8 ± 1+Azide 100 μm33 ± 2+Methionine 1 mm90 ± 3+Me2SO 1 mm92 ± 2 Open table in a new tab We also undertook a separate set of experiments to determine the effect of superoxide on the formation of the oxidation products of melatonin. Superoxide could act either by being an essential reactant in the oxidation of melatonin, or it could react with myeloperoxidase to modulate the rates of formation of each of the products. To exclude its effect on the enzyme, we expressed product yields relative to the amount of melatonin oxidized (Fig. 6A). In accord with the data in TABLE TWO, AFMK production was highly reliant on superoxide. In contrast, there was less formation of dimer in the presence of superoxide. The minor hydroxylated product was unaffected by superoxide. These results suggested that superoxide was directly involved in the formation of AFMK. However, it limited formation of melatonin dimer presumably by reacting with melatonin radicals. The inability of catalase to block AFMK production suggests that myeloperoxidase uses superoxide to oxidize melatonin in a reaction that is independent of hydrogen peroxide. However, it is possible that catalase may not be able to completely prevent hydrogen peroxide from reacting with myeloperoxidase. To assess this possibility, we contrasted the effect catalase has on formation of the different products of melatonin oxidation (Fig. 6B). We reasoned that if AFMK production is solely reliant on hydrogen peroxide, then catalase should inhibit its formation to the same extent as that of melatonin dimers. Conversely, if AFMK is formed independently of hydrogen peroxide, catalase should have a greater effect on the production of dimer. In this system, myeloperoxidase oxidized 50 ± 10 μm melatonin, and this was decreased to 13 ± 4 μm in the presence of catalase (74% inhibition). Products were not formed in the absence of myeloperoxidase (Fig. 6B). We found that catalase completely blocked" @default.
• W2039864897 created "2016-06-24" @default.
• W2039864897 creator A5024538246 @default.
• W2039864897 creator A5031703106 @default.
• W2039864897 creator A5041601466 @default.
• W2039864897 creator A5061706977 @default.
• W2039864897 creator A5065707736 @default.
• W2039864897 creator A5075274024 @default.
• W2039864897 creator A5084278737 @default.
• W2039864897 date "2005-11-01" @default.
• W2039864897 modified "2023-10-16" @default.
• W2039864897 title "Superoxide-dependent Oxidation of Melatonin by Myeloperoxidase" @default.
• W2039864897 cites W1487363849 @default.
• W2039864897 cites W1505149221 @default.
• W2039864897 cites W1547009771 @default.
• W2039864897 cites W1554781540 @default.
• W2039864897 cites W1581691059 @default.
• W2039864897 cites W1582811556 @default.
• W2039864897 cites W1612547865 @default.
• W2039864897 cites W1764308599 @default.
• W2039864897 cites W1768247331 @default.
• W2039864897 cites W1883234535 @default.
• W2039864897 cites W1973425009 @default.
• W2039864897 cites W1985127121 @default.
• W2039864897 cites W1985188262 @default.
• W2039864897 cites W1988172837 @default.
• W2039864897 cites W1989133148 @default.
• W2039864897 cites W1989415916 @default.
• W2039864897 cites W1992769946 @default.
• W2039864897 cites W1993758665 @default.
• W2039864897 cites W1994304104 @default.
• W2039864897 cites W1997746994 @default.
• W2039864897 cites W1998984444 @default.
• W2039864897 cites W2006761570 @default.
• W2039864897 cites W2007525678 @default.
• W2039864897 cites W2013697509 @default.
• W2039864897 cites W2021614013 @default.
• W2039864897 cites W2021677811 @default.
• W2039864897 cites W2023183146 @default.
• W2039864897 cites W2023851340 @default.
• W2039864897 cites W2029479550 @default.
• W2039864897 cites W2035299293 @default.
• W2039864897 cites W2036857026 @default.
• W2039864897 cites W2038851454 @default.
• W2039864897 cites W2045444107 @default.
• W2039864897 cites W2055402321 @default.
• W2039864897 cites W2059149560 @default.
• W2039864897 cites W2062196582 @default.
• W2039864897 cites W2062280245 @default.
• W2039864897 cites W2065531032 @default.
• W2039864897 cites W2067960609 @default.
• W2039864897 cites W2072346051 @default.
• W2039864897 cites W2077622710 @default.
• W2039864897 cites W2083661681 @default.
• W2039864897 cites W2084381950 @default.
• W2039864897 cites W2091407682 @default.
• W2039864897 cites W2092954618 @default.
• W2039864897 cites W2099445613 @default.
• W2039864897 cites W2104970183 @default.
• W2039864897 cites W2105633984 @default.
• W2039864897 cites W2120343652 @default.
• W2039864897 cites W2127137167 @default.
• W2039864897 cites W2153563207 @default.
• W2039864897 cites W2166677591 @default.
• W2039864897 cites W2222372567 @default.
• W2039864897 cites W2247906763 @default.
• W2039864897 cites W2323516874 @default.
• W2039864897 cites W2468858310 @default.
• W2039864897 cites W4229630303 @default.
• W2039864897 cites W4239297083 @default.
• W2039864897 doi "https://doi.org/10.1074/jbc.m506384200" @default.
• W2039864897 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16148002" @default.
• W2039864897 hasPublicationYear "2005" @default.
• W2039864897 type Work @default.
• W2039864897 sameAs 2039864897 @default.
• W2039864897 citedByCount "71" @default.
• W2039864897 countsByYear W20398648972012 @default.
• W2039864897 countsByYear W20398648972013 @default.
• W2039864897 countsByYear W20398648972014 @default.
• W2039864897 countsByYear W20398648972015 @default.
• W2039864897 countsByYear W20398648972016 @default.
• W2039864897 countsByYear W20398648972017 @default.
• W2039864897 countsByYear W20398648972018 @default.
• W2039864897 countsByYear W20398648972019 @default.
• W2039864897 countsByYear W20398648972021 @default.
• W2039864897 countsByYear W20398648972022 @default.
• W2039864897 countsByYear W20398648972023 @default.
• W2039864897 crossrefType "journal-article" @default.
• W2039864897 hasAuthorship W2039864897A5024538246 @default.
• W2039864897 hasAuthorship W2039864897A5031703106 @default.
• W2039864897 hasAuthorship W2039864897A5041601466 @default.
• W2039864897 hasAuthorship W2039864897A5061706977 @default.
• W2039864897 hasAuthorship W2039864897A5065707736 @default.
• W2039864897 hasAuthorship W2039864897A5075274024 @default.
• W2039864897 hasAuthorship W2039864897A5084278737 @default.
• W2039864897 hasBestOaLocation W20398648971 @default.
• W2039864897 hasConcept C126322002 @default.
• W2039864897 hasConcept C181199279 @default.

• next