Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Superoxide-mediated Formation of Tyrosine Hydroperoxides and Methionine Sulfoxide in Peptides through Radical Addition and Intramolecular Oxygen Transfer

2009; Elsevier BV; Volume: 284; Issue: 22 Linguagem: Inglês

10.1074/jbc.m809396200

ISSN

1083-351X

Autores

Péter Nagy, Anthony J. Kettle, Christine C. Winterbourn,

Tópico(s)

Biochemical effects in animals

Resumo

The chemistry underlying superoxide toxicity is not fully understood. A potential mechanism for superoxide-mediated injury involves addition to tyrosyl radicals, to give peptide or protein hydroperoxides. The rate constant for the reaction of tyrosyl radicals with superoxide is higher than for dimerization, but the efficiency of superoxide addition to peptides depends on the position of the Tyr residue. We have examined the requirements for superoxide addition and structurally characterized the products for a range of tyrosyl peptides exposed to a peroxidase/O2⋅− system. These included enkephalins as examples of the numerous proteins and physiological peptides with N-terminal tyrosines. The importance of amino groups in promoting hydroperoxide formation and effect of methionine residues on the reaction were investigated. When tyrosine was N-terminal, the major products were hydroperoxides that had undergone cyclization through conjugate addition of the terminal amine. With non-N-terminal tyrosine, electron transfer from O2⋅− to the peptide radical prevailed. Peptides containing methionine revealed a novel and efficient intramolecular oxygen transfer mechanism from an initial tyrosine hydroperoxide to give a dioxygenated derivative with one oxygen on the tyrosine and the other forming methionine sulfoxide. Exogenous amines promoted hydroperoxide formation on tyrosyl peptides lacking a terminal amine, without forming an adduct. These findings, plus the high hydroperoxide yields with N-terminal tyrosine, can be explained by a mechanism in which hydrogen bonding of O2⋅− to the amine increases is oxidizing potential and alters its reactivity. If this amine effect occurred more generally, it could increase the biological reactivity of O2⋅− and have major implications. The chemistry underlying superoxide toxicity is not fully understood. A potential mechanism for superoxide-mediated injury involves addition to tyrosyl radicals, to give peptide or protein hydroperoxides. The rate constant for the reaction of tyrosyl radicals with superoxide is higher than for dimerization, but the efficiency of superoxide addition to peptides depends on the position of the Tyr residue. We have examined the requirements for superoxide addition and structurally characterized the products for a range of tyrosyl peptides exposed to a peroxidase/O2⋅− system. These included enkephalins as examples of the numerous proteins and physiological peptides with N-terminal tyrosines. The importance of amino groups in promoting hydroperoxide formation and effect of methionine residues on the reaction were investigated. When tyrosine was N-terminal, the major products were hydroperoxides that had undergone cyclization through conjugate addition of the terminal amine. With non-N-terminal tyrosine, electron transfer from O2⋅− to the peptide radical prevailed. Peptides containing methionine revealed a novel and efficient intramolecular oxygen transfer mechanism from an initial tyrosine hydroperoxide to give a dioxygenated derivative with one oxygen on the tyrosine and the other forming methionine sulfoxide. Exogenous amines promoted hydroperoxide formation on tyrosyl peptides lacking a terminal amine, without forming an adduct. These findings, plus the high hydroperoxide yields with N-terminal tyrosine, can be explained by a mechanism in which hydrogen bonding of O2⋅− to the amine increases is oxidizing potential and alters its reactivity. If this amine effect occurred more generally, it could increase the biological reactivity of O2⋅− and have major implications. Free radical-mediated oxidative damage occurs in numerous diseases and is thought to contribute to the aging process. The primary radical generated by the reduction of oxygen is superoxide (O2⋅−), a relatively benign radical that nevertheless must be removed by superoxide dismutases (SODs) 2The abbreviations used are: SOD, superoxide dismutase; YM, tyrosinemethionine; YM-S=O, tyrosine-methionine sulfoxide; MY, methionine-tyrosine; YG, tyrosine-glycine; GY, glycine-tyrosine; YGGFM, Met-Enk, methionine-enkephalin; YGGFL, Leu-Enk, leucine-enkephalin; YPFF, Endo2, endomorphin 2; GYGGFM, Gly-Met-Enk, glycine-methionine-enkephalin; Boc-YGGFM, tert-butoxycarbonyl-methionine-enkephalin; RFYVVM, thrombospondin-1 (1016–1021); YSFKDMGLGR, human C5a anaphylatoxin (Tyr65, Phe67)-C5a (65–74); MEVDPIGHLY, MAGE-3 antigen (167–176); HPA, p-hydroxyphenylacetic acid; HOHICA, 3a-hydroxy-6-oxo-2,3,3a,6,7,7a-hexahydro-1H-indol-2-carboxylic acid; HACHD, 4-alanyl-4-hydroxy-cyclohexadienone; XO, xanthine oxidase; HRP, horseradish peroxidase; LC/MS, liquid chromatography/electrospray mass spectrometry; ESI, electrospray ionization. for an organism to survive in an aerobic environment (1Imlay J.A. Annu. Rev. Biochem. 2008; 77: 755-776Crossref PubMed Scopus (1139) Google Scholar). A number of potentially damaging reactions of O2⋅− have been identified (1Imlay J.A. Annu. Rev. Biochem. 2008; 77: 755-776Crossref PubMed Scopus (1139) Google Scholar, 2Winterbourn C.C. Nat. Chem. Biol. 2008; 4: 278-286Crossref PubMed Scopus (1789) Google Scholar, 3Liochev S.I. Fridovich I. IUBMB Life. 1999; 48: 157-161Crossref PubMed Google Scholar, 4Beckman J.S. Koppenol W.H. Am. J. Physiol. 1996; 271: C1424-C1437Crossref PubMed Google Scholar). One of these, which has received relatively little attention, is the addition of O2⋅− to other radicals to form hydroperoxides (5Winterbourn C.C. Parsons-Mair H.N. Gebicki S. Gebicki J.M. Davies M.J. Biochem. J. 2004; 381: 241-248Crossref PubMed Scopus (94) Google Scholar, 6Winterbourn C.C. Kettle A.J. Biochem. Biophys. Res. Commun. 2003; 305: 729-736Crossref PubMed Scopus (99) Google Scholar). This reaction has been shown to occur readily with tyrosine and Tyr-containing dipeptides, resulting in the formation of tyrosine hydroperoxides (5Winterbourn C.C. Parsons-Mair H.N. Gebicki S. Gebicki J.M. Davies M.J. Biochem. J. 2004; 381: 241-248Crossref PubMed Scopus (94) Google Scholar, 6Winterbourn C.C. Kettle A.J. Biochem. Biophys. Res. Commun. 2003; 305: 729-736Crossref PubMed Scopus (99) Google Scholar, 7Jin F. Leitich J. von Sonntag C. J. Chem. Soc. Perkin Trans. II. 1993; : 1583-1588Crossref Scopus (138) Google Scholar). Hydroperoxides are potentially damaging reactive oxygen species. Formation on proteins can result in detrimental structural and functional changes (8Davies M.J. Biochim. Biophys. Acta. 2005; 1703: 93-109Crossref PubMed Scopus (1117) Google Scholar). Protein hydroperoxides are also oxidants that can injure other biomolecules. Tyrosyl radicals are generated in many physiological situations and proteins are major targets for reactive oxidants (9Dean R.T. Fu S.L. Stocker R. Davies M.J. Biochem. J. 1997; 324: 1-18Crossref PubMed Scopus (1470) Google Scholar). In proteins exposed to free radicals, regardless of the initial site of attack, the resultant radical commonly localizes to Tyr (10Bobrowski K. Wierzchowski K.L. Holcman J. Ciurak M. Int. J. Radiat. Biol. 1990; 57: 919-932Crossref PubMed Scopus (60) Google Scholar, 11Garrison W.M. Chem. Rev. 1987; 87: 381-398Crossref Scopus (645) Google Scholar, 12Prutz W.A. Siebert F. Butler J. Land E.J. Menez A. Montenaygarestier T. Biochim. Biophys. Acta. 1982; 705: 139-149Crossref Scopus (83) Google Scholar, 13Zhang H. Zielonka J. Sikora A. Joseph J. Xu Y. Kalyanaraman B. Arch. Biochem. Biophys. 2008; 10.1016/j.abb.2008.11.018Google Scholar). Tyrosyl radicals are also produced from tyrosyl peptides through the action of peroxidases such as myeloperoxidase, and are generated during the catalytic cycle of enzymes such as ribonucleotide reductase and cyclooxygenase (14Stubbe J.A. van der Donk W.A. Chem. Rev. 1998; 98: 705-762Crossref PubMed Scopus (1368) Google Scholar). Tyrosyl radicals undergo a variety of subsequent reactions. They readily dimerize to form dityrosine, which has been well documented as a product of oxidative injury (15Beal M.F. Free Radic. Biol. Med. 2002; 32: 797-803Crossref PubMed Scopus (700) Google Scholar, 16Heinecke J.W. Li W. Francis G.A. Goldstein J.A. J. Clin. Investig. 1993; 91: 2866-2872Crossref PubMed Scopus (302) Google Scholar). Another oxidative biomarker, nitrotyrosine, is also formed via tyrosyl radicals (4Beckman J.S. Koppenol W.H. Am. J. Physiol. 1996; 271: C1424-C1437Crossref PubMed Google Scholar, 15Beal M.F. Free Radic. Biol. Med. 2002; 32: 797-803Crossref PubMed Scopus (700) Google Scholar, 17Souza J.M. Peluffo G. Radi R. Free Radic. Biol. Med. 2008; 45: 357-366Crossref PubMed Scopus (325) Google Scholar). However, one of their most favored reactions is with O2⋅− (5Winterbourn C.C. Parsons-Mair H.N. Gebicki S. Gebicki J.M. Davies M.J. Biochem. J. 2004; 381: 241-248Crossref PubMed Scopus (94) Google Scholar, 7Jin F. Leitich J. von Sonntag C. J. Chem. Soc. Perkin Trans. II. 1993; : 1583-1588Crossref Scopus (138) Google Scholar, 18d'Allesandro N. Bianchi G. Fang X. Jin F. Schuchmann H.-P. von Sonntag C. J. Chem. Soc. Perkin Trans. II. 2000; : 1862-1867Crossref Scopus (48) Google Scholar, 19Jonsson M. Lind T. Reitberger T.E. Eriksen T.E. Merenyi G. J. Phys. Chem. 1993; 97: 8229-8233Crossref Scopus (110) Google Scholar). The reaction has a rate constant several times higher than that for dimerization (7Jin F. Leitich J. von Sonntag C. J. Chem. Soc. Perkin Trans. II. 1993; : 1583-1588Crossref Scopus (138) Google Scholar, 20Mozziconacci O. Mirkowski J. Rusconi F. Pernot P. Bobrowski K. Houee-Levin C. Free Radic. Biol. Med. 2007; 43: 229-240Crossref PubMed Scopus (27) Google Scholar) and is favored over dityrosine formation in situations where both tyrosyl and O2⋅− radicals are generated (7Jin F. Leitich J. von Sonntag C. J. Chem. Soc. Perkin Trans. II. 1993; : 1583-1588Crossref Scopus (138) Google Scholar, 20Mozziconacci O. Mirkowski J. Rusconi F. Pernot P. Bobrowski K. Houee-Levin C. Free Radic. Biol. Med. 2007; 43: 229-240Crossref PubMed Scopus (27) Google Scholar). The reaction of O2⋅− with phenoxyl radicals results in either repair of the parent phenol (reaction 2, Fig. 1b) or addition to form a hydroperoxide (reaction 3). With tyrosine, most of the O2⋅− reacts by addition (7Jin F. Leitich J. von Sonntag C. J. Chem. Soc. Perkin Trans. II. 1993; : 1583-1588Crossref Scopus (138) Google Scholar, 20Mozziconacci O. Mirkowski J. Rusconi F. Pernot P. Bobrowski K. Houee-Levin C. Free Radic. Biol. Med. 2007; 43: 229-240Crossref PubMed Scopus (27) Google Scholar). The structure of tyrosine hydroperoxide has not been determined directly but inferred from NMR studies of the corresponding monoxide derivative formed by slow decomposition (7Jin F. Leitich J. von Sonntag C. J. Chem. Soc. Perkin Trans. II. 1993; : 1583-1588Crossref Scopus (138) Google Scholar). These were shown to be bicyclic compounds formed by conjugate addition of the amino group to the phenol ring (HOHICA, designated I and named in full in Fig. 1b, proposed to arise from reactions 5 and 6). Hydroperoxide formation has been observed with peptides but only when tyrosine is N-terminal or the reaction is promoted by amino compounds (5Winterbourn C.C. Parsons-Mair H.N. Gebicki S. Gebicki J.M. Davies M.J. Biochem. J. 2004; 381: 241-248Crossref PubMed Scopus (94) Google Scholar). The amine effect has implications for hydroperoxide formation on proteins, but the mechanism is not understood. It has also been postulated that the repair mechanism involves singlet oxygen release from an intermediate (reaction 4) rather than electron transfer (reaction 2) (18d'Allesandro N. Bianchi G. Fang X. Jin F. Schuchmann H.-P. von Sonntag C. J. Chem. Soc. Perkin Trans. II. 2000; : 1862-1867Crossref Scopus (48) Google Scholar), but this has not been studied experimentally. The objectives of this investigation were to determine the structures of the hydroperoxide and any other superoxide addition products, and to understand the mechanism of formation, using a range of synthetic and physiological tyrosyl peptides. These include the opioids Leu- and Met-Enkephalin (Leu-Enk, YGGFL; and Met-Enk, YGGFM, respectively) and Endomorphin 2 (Endo2, YPFF). The opioids have a free N-terminal Tyr that is essential for activity and are potential physiological targets for inactivation by O2⋅− addition. We also investigated whether the presence of a Met residue (as in Met-Enk) influences Tyr-hydroperoxide formation on the peptide and whether O2⋅− addition results in the formation of methionine sulfoxide. If so, this could be a physiological mechanism for production of methionine sulfoxide, which is one of the most prevalent products of oxidative stress (21Stadtman E.R. Levine R.L. Amino Acids. 2003; 25: 207-218Crossref PubMed Scopus (1413) Google Scholar, 22Moskovitz J. Flescher E. Berlett B.S. Azare J. Poston J.M. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14071-14075Crossref PubMed Scopus (241) Google Scholar). Peptides were exposed to a xanthine oxidase (XO) system to generate O2⋅− and hydrogen peroxide (H2O2) plus horseradish peroxidase (HRP) to catalyze the reaction of H2O2 with the peptide to give the tyrosyl radical (Fig. 1a). Products were analyzed using a general hydroperoxide assay (Fe2+/xylenol orange or FOX assay) and by liquid chromatography/electrospray mass spectrometry (LC/MS). We have obtained structural information on the hydroperoxides, identified a mechanism of rapid intramolecular oxidation of Met residues via a hydroperoxide intermediate, and provide an explanation for why amino groups facilitate the addition of O2⋅− to the tyrosyl radical. Reagents—Water was purified by running through a Milli-Q system (Millipore) so that its resistivity was greater than 18 mΩ-cm. All reagents and enzymes were purchased from Sigma, unless otherwise indicated. HOCl solutions were prepared from commercial bleach (Janola) and were standardized spectrophotometrically (using ϵ292 nm = 350 m–1 cm–1). The peptides: YGGFM, Boc-YGGFM, GYGGFM, MEVDPIGHLY, RFYVVM, and YSFKDMGLGR were purchased from Bachem (Bubendorf) and Tyr-Met (YM) and Met-Tyr (MY) were custom synthesized by Genscript (NJ). All peptides were >98% pure. Deuterium oxide (99.9%) was obtained from Cambridge Isotope Laboratories. Anthracene-9,10-diyldiethyl sulfate (EAS) was a generous gift of Prof. Paolo Di Mascio. The Tyr-para-hydroperoxide derivative of Gly-Tyr-Gly (Gly-Tyr-Gly-OOH) was a generous gift of Prof. Michael Davies. It was generated by the reaction of Gly-Tyr-Gly with singlet oxygen, where singlet oxygen was generated in situ by irradiation in the presence of Rose-Bengal (23Wright A. Bubb W.A. Hawkins C.L. Davies M.J. Photochem. Photobiol. 2002; 76: 35-46Crossref PubMed Scopus (186) Google Scholar). The concentration of Gly-Tyr-Gly-OOH was 500 μm (as measured by the FOX assay) in the presence of 2.5 mm Gly-Tyr-Gly. YM-sulfoxide (YM-S=O) was prepared by oxidation of YM by 1 m equivalent HOCl. No other components were detected by LC/MS indicating ∼100% conversion. The concentrations of stock solutions of H2O2 were determined iodometrically. Concentrations in solutions prepared from the stock were confirmed spectrophotometrically (ϵ(H2O2)240 nm = 43.6 m–1 cm–1). Stock solutions of XO were prepared by dilution of an ammonium sulfate suspension with 50 mm phosphate buffer, pH 7.4, and spinning through a G-25 Sephadex column to remove the ammonium sulfate. The activity of XO was measured by the cytochrome c assay and by quantifying H2O2 formation using the FOX assay. Enzyme and acetaldehyde stock solutions were prepared fresh daily and stored on ice. Peroxidase-mediated Oxidation of Tyrosine-containing Peptides in the Presence of Superoxide—Reaction mixtures consisted of acetaldehyde (1 mm unless stated otherwise), XO (typically 0.001 unit/ml), HRP (typically 140 nm), and 0.2 mm peptide in 50 mm phosphate buffer plus 50 μm diethylenetriaminepentaacetic acid (except for the FOX assay experiments). This amount of XO with 1 mm acetaldehyde corresponds to an initial rate of 2.8 μm/min O2⋅− and total production of 36 μm H2O2 over a 30-min reaction period. Reactions were started by addition of XO. They were carried out at 20–25 °C, typically for 30 min for LC/MS, 10 min for FOX and dimer analyses, and stopped by adding 20 μg/ml catalase to remove residual H2O2. When necessary the enzymes were removed from the reaction mixtures by ultracentrifugation using 10–30-kDa cutoff microconcentrators (Amicon Microcon). Samples were protected from light to avoid photochemical reactions. Dimer Quantification—Dimers were measured fluorimetrically (excitation 325 nm, emission 400 nm) with a Hitachi F-4500 fluorescence spectrofluorimeter as described in Ref. 5Winterbourn C.C. Parsons-Mair H.N. Gebicki S. Gebicki J.M. Davies M.J. Biochem. J. 2004; 381: 241-248Crossref PubMed Scopus (94) Google Scholar. Results are expressed as relative fluorescence and are related to concentrations using calibration curves obtained by generating the dimer from the relevant peptide in at least 20-fold excess and known concentrations of H2O2 in the presence of HRP. Dimer concentrations were corroborated by measuring A315 at pH 8.0 using ϵ = 5,080 m–1 cm–1. Hydroperoxide Quantification—Hydroperoxides were analyzed using a modified FOX method (5Winterbourn C.C. Parsons-Mair H.N. Gebicki S. Gebicki J.M. Davies M.J. Biochem. J. 2004; 381: 241-248Crossref PubMed Scopus (94) Google Scholar, 24Wolff S.P. Methods Enzymol. 1994; 233: 182-189Crossref Scopus (1085) Google Scholar) standardized against H2O2, under similar conditions as for dimer quantification. Liquid Chromatography (LC)-Electrospray Ionization (ESI)-Mass Spectrometry (MS)—LC-ESI-MS and LC-ESI-MS/MS analyses were performed with a Thermo Finnigan LCQ Deca XP Plus ion trap mass spectrometer (San Jose, CA) coupled to a Surveyor HPLC system and PDA detector. Positive ion mode was used for all peptides and negative ion mode for p-hydroxyphenylacetic acid (HPA) and EAS derivatives. Data were analyzed using Finnigan Xcalibur, Thermo Finnigan Qual Browser 1.3, and High Chem Mass Frontier 3.0 programs. Fragmentation patterns were analyzed using Bioworks Browser 3.1 and peptide fragment ions were assigned and discussed based on the Roepstorff-Fohlman nomenclature. Further details on chromatography conditions and detection are given under supplementary Methods. Acid Hydrolysis—Peptides were lyophilized and vapor phase hydrolyzed with 6 m HCl containing 1% (w/v) phenol, plus 50 μl of mercaptoacetic acid when products were analyzed for the recovery of 3,4-dihydroxyphenylalanine (25Gieseg S.P. Simpson J.A. Charlton T.S. Duncan M.W. Dean R.T. Biochemistry. 1993; 32: 4780-4786Crossref PubMed Scopus (173) Google Scholar). After hydrolysis, the residual HCl was evaporated and the samples were redis-solved in water and analyzed by LC-MS. Quantification of Met-Enk and Leu-Enk Products—Yields of Leu-Enk hydroperoxide and Met-Enk and Gly-Met-Enk dioxides were quantified by calibrating the LC/MS peak integral for each species. Each compound was generated using the XO/HRP system and purified by LC with the same setup as for MS analysis. For Met-Enk and Gly-Met-Enk, the product peak was collected, concentrated, and a sample reinjected to check for purity. When necessary the purification step was repeated until no contaminants were detectable. The concentrations of the enkephalin dioxides in the purified solutions were established on the basis of Phe content. An aliquot of each solution was hydrolyzed and the amount of Phe determined by LC/MS using selective ion monitoring. A standard curve was created with authentic Phe, which was identified from its retention time and fragmentation pattern. Controls using internal standards of authentic Phe showed nearly 100% recovery under the applied conditions. Acid hydrolysis was >90% efficient based on Phe recovery from the parent enkephalin hydrolyzed under the same conditions. This gave the Phe content and hence the dioxide concentration in the pure sample. A known amount of pure dioxide was injected into the LC/MS to calibrate its peak integral and this calibration was used to quantify dioxide formation under experimental conditions. The instrument response was corrected by use of check standard of Leu-Enk before each set of runs. For Leu-Enk, the hydroperoxide was not stable enough to use the same procedure. Instead, the entire Leu-Enk-OOH peak from each of two chromatographic runs was collected and the combined sample was hydrolyzed and analyzed for Phe content as above. The Phe content in half the hydrolysate was related to the mean peak integral for the original samples, using the same time interval for collection and integration. This calibration was then used to quantify the hydroperoxide peak from experimental samples that were separated at the same time as the calibration sample. It has been observed (5Winterbourn C.C. Parsons-Mair H.N. Gebicki S. Gebicki J.M. Davies M.J. Biochem. J. 2004; 381: 241-248Crossref PubMed Scopus (94) Google Scholar, 26Winterbourn C.C. Pichorner H. Kettle A.J. Arch. Biochem. Biophys. 1997; 338: 15-21Crossref PubMed Scopus (65) Google Scholar, 27Pichorner H. Metodiewa D. Winterbourn C.C. Arch. Biochem. Biophys. 1995; 323: 429-437Crossref PubMed Scopus (71) Google Scholar) that radicals generated on Tyr and tyrosyl dipeptides react with O2⋅− to form hydroperoxides (5Winterbourn C.C. Parsons-Mair H.N. Gebicki S. Gebicki J.M. Davies M.J. Biochem. J. 2004; 381: 241-248Crossref PubMed Scopus (94) Google Scholar, 26Winterbourn C.C. Pichorner H. Kettle A.J. Arch. Biochem. Biophys. 1997; 338: 15-21Crossref PubMed Scopus (65) Google Scholar, 27Pichorner H. Metodiewa D. Winterbourn C.C. Arch. Biochem. Biophys. 1995; 323: 429-437Crossref PubMed Scopus (71) Google Scholar). This occurs in competition with dimerization of the radicals, and with Tyr and the XO/HRP system as in Fig. 1a, more hydroperoxide than dityrosine was formed (5Winterbourn C.C. Parsons-Mair H.N. Gebicki S. Gebicki J.M. Davies M.J. Biochem. J. 2004; 381: 241-248Crossref PubMed Scopus (94) Google Scholar). We extended these observations to the enkephalins and related peptides, and found that they were all oxidized by HRP and H2O2. Yields of dimer formation in the XO/HRP/acetaldehyde systems were 3–4-fold higher when SOD was present, indicating that in the absence of SOD, the peptide radicals reacted with O2⋅− in preference to dimerization (Fig. 2a). Using the FOX assay (24Wolff S.P. Methods Enzymol. 1994; 233: 182-189Crossref Scopus (1085) Google Scholar) to detect hydroperoxides, a positive response was obtained with Tyr-Gly (YG) but not Gly-Tyr (GY) (Fig. 2b), in agreement with previous observations (5Winterbourn C.C. Parsons-Mair H.N. Gebicki S. Gebicki J.M. Davies M.J. Biochem. J. 2004; 381: 241-248Crossref PubMed Scopus (94) Google Scholar) that hydroperoxide formation requires an N-terminal Tyr. Substantial O2⋅−-dependent hydroperoxide formation was observed with Leu-Enk and Endo2, but despite having an N-terminal Tyr, Met-Enk gave no detectable hydroperoxide by FOX analysis (Fig. 2b). It should be noted that the FOX assay was calibrated with H2O2 as no hydroperoxide standards are available, and this results in underestimation of hydroperoxide yields. The version of the assay used here gives a 6-fold lower response to tyrosine hydroperoxide than H2O2 (5Winterbourn C.C. Parsons-Mair H.N. Gebicki S. Gebicki J.M. Davies M.J. Biochem. J. 2004; 381: 241-248Crossref PubMed Scopus (94) Google Scholar). Assuming that tyrosyl peptides behave similarly, the data in Fig. 2 indicate that the enkephalins gave more hydroperoxide than dimer. For Leu-Enk, this was confirmed by direct analysis (see below). Tyr-Gly and Peptides with N-terminal Tyr Residues—To establish the structures of the peptide hydroperoxides directly, the products of the reactions of Leu-Enk, YG, and Endo2 were analyzed by LC/MS. Each gave, in addition to the dimer, a major product with molecular mass corresponding to the native peptide + 32 (potentially the hydroperoxide) and a product at a mass of the native peptide + 16 (monoxide) (Fig. 3). In some cases two peaks of equivalent mass and fragmentation pattern were evident, presumably representing o- and p-isomers. Formation of both species was strongly inhibited by SOD. Based on peak integrals and assuming the two species had similar MS characteristics, more dioxides than monoxides were present. The monoxides are assumed to have arisen from hydrolysis of the hydroperoxides during the reaction and sample processing (as in Ref. 7Jin F. Leitich J. von Sonntag C. J. Chem. Soc. Perkin Trans. II. 1993; : 1583-1588Crossref Scopus (138) Google Scholar and shown below in Fig. 6).FIGURE 6Intermolecular reaction of Leu-Enk hydroperoxide (Leu-Enk-OOH) with (a) Met and (b) Met-Enk. Decay of Leu-Enk-OOH (10 μm in 50 mm phosphate buffer) was measured in the presence and absence of 200 μm methionine by LC/MS, or 200 μm Met-Enk by the FOX assay. Diethylenetriaminepentaacetic acid (50 μm) was present for a but not b. Leu-Enk-OOH was generated using the conditions described under "Experimental Procedures." After 30 min incubation, 20 μg/ml catalase was added and the enzymes were removed by centrifugation using an Amicon 10-kDa cutoff filter. The reaction of Leu-Enk-OOH with Met-Enk was also monitored by LC/MS, where a similar second-order rate constant was obtained (k = 0.18 m–1 s–1). Loss of the hydroperoxide was accompanied by increases in the Leu-Enk monoxide and Met-Enk sulfoxide peaks at similar rates. The rate of the reaction was also investigated at lower Met-Enk concentrations. The obtained pseudo-first ordered rate constants together with the fits of the pseudo-first order kinetic traces to a single exponential curve indicate that the overall reaction is indeed second-order (first-order for both [Leu-Enk-OOH] and [Met-Enk]).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The yield of Leu-Enk hydroperoxide was measured under the conditions of Fig. 3a with the peak integral calibrated on the basis of the Phe content of the purified product (see "Experimental Procedures"). For three independent experiments, 10.7 μm (S.D. 0.5) hydroperoxide and 4.5 μm dimer (S.D. 0.4) were formed. These accounted for the majority of the total Leu-Enk loss. This result shows that the hydroperoxide was the major product in our systems. MS/MS fragment ions were assigned for the dioxide and monoxide products. As shown for YG, the parent peptide gave the expected a1 fragment for Tyr (Fig. 4a). The monoxide (Fig. 4b) gave a1 and b1 fragment ions with the extra oxygen attached to the Tyr residue, and the dioxide (Fig. 4c) gave an a1 fragment containing both oxygens. Where there was loss of H2O from the monoxide, H2O2 was lost from the dioxide, implying that the two differ by an -OH or -OOH at the same site. Similar features were evident in the fragmentation patterns of the dioxide and monoxide products for Leu-Enk and Endo2 (not shown) and fits obtained using the Bioworks Browser (as for Met-Enk in Fig. S5) were consistent with the extra oxygens in these peptides being attached to the Tyr. YG monoxide had a very similar fragmentation pattern to the monoxide formed by exposing free Tyr to the XO/HRP system (HOHICA in Fig. S1a). Both showed the same a1 fragment with an extra oxygen and the m/z 134 peak (representing loss of water from the a1 fragment and thus a lack of aromaticity, see later). The m/z 134 peak, plus the absence of the peak representing ammonia loss (presumably reflecting lack of a free amino group), are key features that discriminate these structures from other theoretical alternatives with the same mass (II and III in Fig. 1c: data in Fig. S1, b and c). As HOHICA (I in Fig. 1b) has been characterized as a bicyclic compound (7Jin F. Leitich J. von Sonntag C. J. Chem. Soc. Perkin Trans. II. 1993; : 1583-1588Crossref Scopus (138) Google Scholar), we conclude that YG dioxide has a hydroperoxide on the Tyr ring that has undergone the same conjugate addition (Fig. 1b, reaction 5). Gly-Tyr—Although GY gave no detectable hydroperoxide in Fig. 2b, low yields of dioxide and monoxide derivatives were detected by LC/MS (Fig. 3c). The fragmentation patterns (Fig. S2) show a hydroperoxide group, or corresponding -OH, located on the Tyr, and also suggest (as discussed below for peptides containing Met) that the Tyr ring is modified by conjugate addition of the amide nitrogen. The situation was different for peptides containing Met. We investigated dipeptides MY, YM, as well as Met-Enk and Gly-Met-Enk. These peptides produced dimers when exposed to the XO/HRP system, and dimerization was enhanced when O2⋅− was removed with SOD (Table 1 and Fig. 2a). LC/MS analysis (Fig. 5, a–d, and Table 1) showed that a dioxygenated M + 32 + H+ species was produced from all the peptides. Formation of the dioxygenated species required O2⋅− as it was suppressed by SOD. However, none of the peptides gave a positive response in the FOX assay (Table 1 and Fig. 2b), implying that the two-oxygen addition products were not hydroperoxides.TABLE 1Methionyl peptides examined for dioxide formationAmino acid sequenceMolecular massRTaRT retention time. Conditions as described under "Experimental Procedures." of [M + H]+RT of dioxideDimerHydroperoxide (H2O2 eq. μm)No SODSODg/molminμmYM312.417.912.7, 14.03.8 ± 0.17.0 ± 0.10.23 ± 0.006MY312.417.412.6, 14.75.41 ± 0.16.9 ± 0.2<0.2YGGFM573.716.111.3, 12.53.3 ± 0.18.6 ± 0.1<0.2GYGGFM630.713.310.2, 11.08.0 ± 0.410.5 ± 0.4<0.2Boc-YGGFM673.814.19.65.8 ± 0.211.4 ± 0.5<0.2RFYVVM814.08.4, 10.67.5, 9.610.7 ± 0.817.6 ± 0.4<0.2YSFKDMGLGR1244.410.79.41.0 ± 0.22.7 ± 0.2<0.2MEVDPIGHLY1173.418.612.33.3 ± 0.37.8 ± 0.3<0.2YM-S = O361.115.9313.53, 14.072.55 ± 0.094.15 ± 0.050.32 ± 0.002a RT retention time. Conditions as described under "Experimental

Referência(s)