Identification of Free Radicals on Hemoglobin from its Self-peroxidation Using Mass Spectrometry and Immuno-spin Trapping

In an effort to understand the mechanism of radical formation on heme proteins, the formation of radicals on hemoglobin was initiated by reaction with hydrogen peroxide in the presence of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The DMPO nitrone adducts were analyzed by mass spectrometry (MS) and immuno-spin trapping. The spin-trapped protein adducts were then subjected to tryptic digestion and MS analyses. When hemoglobin was reacted with hydrogen peroxide (H2O2) in the presence of DMPO, a DMPO nitrone adduct could be detected by immuno-spin trapping. To verify that DMPO adducts of the protein free radicals had been formed, the reaction mixtures were analyzed by flow injection electrospray ionization mass spectrometry (ESI/MS). The ESI mass spectrum of the hemoglobin/H2O2/DMPO sample shows one adduct each on both the α chain and the β chain of hemoglobin which corresponds in mass to the addition of one DMPO molecule. The nature of the radicals formed on hemoglobin was explored using proteolysis techniques followed by liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry (MS/MS) analyses. The following sites of DMPO addition were identified on hemoglobin: Cys-93 of the β chain, and Tyr-42, Tyr-24, and His-20 of the α chain. Because of the pi-pi interaction of Tyr-24 and His-20, the unpaired electron is apparently delocalized on both the tyrosine and histidine residue (pi-pi stacked pair radical). In an effort to understand the mechanism of radical formation on heme proteins, the formation of radicals on hemoglobin was initiated by reaction with hydrogen peroxide in the presence of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The DMPO nitrone adducts were analyzed by mass spectrometry (MS) and immuno-spin trapping. The spin-trapped protein adducts were then subjected to tryptic digestion and MS analyses. When hemoglobin was reacted with hydrogen peroxide (H2O2) in the presence of DMPO, a DMPO nitrone adduct could be detected by immuno-spin trapping. To verify that DMPO adducts of the protein free radicals had been formed, the reaction mixtures were analyzed by flow injection electrospray ionization mass spectrometry (ESI/MS). The ESI mass spectrum of the hemoglobin/H2O2/DMPO sample shows one adduct each on both the α chain and the β chain of hemoglobin which corresponds in mass to the addition of one DMPO molecule. The nature of the radicals formed on hemoglobin was explored using proteolysis techniques followed by liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry (MS/MS) analyses. The following sites of DMPO addition were identified on hemoglobin: Cys-93 of the β chain, and Tyr-42, Tyr-24, and His-20 of the α chain. Because of the pi-pi interaction of Tyr-24 and His-20, the unpaired electron is apparently delocalized on both the tyrosine and histidine residue (pi-pi stacked pair radical). The role of free radicals in the pathogenesis of human disease has led to an increased interest in the study of free radicals and their reactions. Reactive oxygen metabolites can interact with cellular constituents, including DNA/RNA, proteins, and unsaturated lipids (1Janssen Y.M.W. Van Houten B. Borm P.J.A. Mossman B.T. Lab. Invest. 1993; 69: 261-274PubMed Google Scholar, 2Kappus H. Sies H. Oxidative Stress. Academic Press Inc., London1985: 273-330Crossref Google Scholar, 3Brigelius R. Sies H. Oxidative Stress. Academic Press Inc., London1985: 243-272Crossref Google Scholar, 4Richter C. Gogvadze V. Laffranchi R. Schlapbach R. Schweizer M. Suter M. Walter P. Yaffee M. Biochim. Biophys. Acta. 1995; 1271: 67-74Crossref PubMed Scopus (494) Google Scholar). Previous studies have suggested that hemoproteins may be involved in redox reactions which contribute to tissue and/or organ damage via reaction with hydrogen peroxide (5Alayash A.I. Patel R.P. Cashon R.E. Antioxid. Redox Signal. 2001; 3: 313-327Crossref PubMed Scopus (204) Google Scholar, 6Alayash A.I. Nat. Biotechnol. 1999; 17: 545-549Crossref PubMed Scopus (194) Google Scholar, 7Everse J. Hsia N. Free Rad. Biol. Med. 1997; 22: 1075-1099Crossref PubMed Scopus (309) Google Scholar). A recent study has reported evidence for the association of nitrotyrosine (which forms via tyrosyl radical) and coronary artery disease (8Shishehbor M.H. Aviles R.J. Brennan M.-L. Fu X. Goormastic M. Pearce G.L. Gokce N. Keaney Jr, J.F. Penn M.S. Sprecher D.L. Vita J.A. Hazen S.L. J. Am. Med. Assoc. 2003; 289: 1675-1680Crossref PubMed Scopus (409) Google Scholar). To understand the mechanisms of these reactions and their contributions to human diseases, it is important to determine the nature of heme protein radical intermediates involved in these processes. Protein-centered radicals have traditionally been studied through either direct electron spin resonance or by spin trapping (9Hawkins C.L. Davies M.J. Biochim. Biophys. Acta. 2001; 1504: 196-219Crossref PubMed Scopus (625) Google Scholar). Because most radicals are short-lived (i.e. microseconds to minutes), the spin trapping approach has been more widely used. With the spin trapping approach, protein radicals react with a spin trap molecule, such as 2-methyl-2-nitrosopropane or 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 1The abbreviations used are: DMPO, 5,5-dimethyl-1-pyrroline N-oxide; Hb, hemoglobin; oxyHb, oxyferrous Hb; DTPA, diethylenetriamine pentaacetic acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; ESI, electrospray ionization mass spectrometry; LC, liquid chromatography; MS, mass spectrometry. thereby making the radical more stable and, consequently, more long-lived. To take advantage of these more stable, trapped adducts, our research group recently reported the development of an antibody with specificity for the nitrone adduct, which is the final product of trapping radicals with DMPO (10Detweiler C.D. Deterding L.J. Tomer K.B. Chignell C.F. Germolec D. Mason R.P. Free Radic. Biol. Med. 2002; 33: 364-369Crossref PubMed Scopus (107) Google Scholar). For this work, antibodies were raised against a DMPO derivative in which an octanoic acid side chain was conjugated to ovalbumin. Using this DMPO-specific antibody, we have demonstrated that the predominant protein radical formed in rat heart supernatant by hydrogen peroxide is myoglobin-derived (10Detweiler C.D. Deterding L.J. Tomer K.B. Chignell C.F. Germolec D. Mason R.P. Free Radic. Biol. Med. 2002; 33: 364-369Crossref PubMed Scopus (107) Google Scholar). Assignment of the specific amino acid residue or residues that form radical adducts with the spin trap molecule is difficult with ESR or immunostaining. Consequently, researchers have used other biochemical techniques, such as site-directed mutagenesis and amino acid derivatization, to address these questions. These methods, however, suffer from potential drawbacks, such as the protein's stability and conformation can be perturbed by mutations and the formation of the amino acid radical and transfer of the radical is expected to be affected. Recently, mass spectrometry has been utilized to study protein free radicals (11Barr D.P. Gunther M.R. Deterding L.J. Tomer K.B. Mason R.P. J. Biol. Chem. 1996; 271: 15498-15503Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 12Deterding L.J. Barr D.P. Mason R.P. Tomer K.B. J. Biol. Chem. 1998; 1273: 12863-12869Abstract Full Text Full Text PDF Scopus (49) Google Scholar, 13Harris M.N. Burchiel S.W. Winyard P.G. Engen J.R. Mobarak C.D. Timmins G.S. Chem. Res. Toxicol. 2002; 15: 1589-1594Crossref PubMed Scopus (19) Google Scholar, 14Zhang H. He S. Mauk A.G. Biochemistry. 2002; 41: 13507-13513Crossref PubMed Scopus (26) Google Scholar, 15Wright P.J. English A.M. J. Am. Chem. Soc. 2003; 125: 8655-8665Crossref PubMed Scopus (90) Google Scholar). The spin trap molecules form a covalent bond with the protein resulting in a new nitroxide radical, which may be stable under electrospray ionization mass spectrometry conditions. The one-electron oxidation product of this nitroxide is a stable nitrone with a covalent bond between DMPO and the original free radical (Scheme 1). In addition, the use of peptide mapping methodologies with mass spectrometry allows for the unequivocal assignment of the trapped radical on the protein. We are, therefore, using a combination of peptide mapping by mass spectrometry and the immuno-spin trapping technique to investigate the formation and structures of the protein radicals generated on heme-containing proteins. In a previous report using the immuno-spin trapping approach (16Ramirez D.C. Chen Y.-R. Mason R.P. Free Radic. Biol. Med. 2003; 34: 830-839Crossref PubMed Scopus (75) Google Scholar), we demonstrated that hemoglobin reacted with H2O2 in the presence of DMPO and suggested that the tyrosine residues and possibly a cysteine residue of hemoglobin are the sites of formation of the radical-derived nitrone adducts. The exposure of hemoproteins, such as hemoglobin (Hb) and myoglobin (Mb), to hydrogen peroxide has been shown to initially produce a porphyrin cation radical and ferryl ion. The porphyrin cation radical oxidizes one or more residues to form globin-centered radical(s) (16Ramirez D.C. Chen Y.-R. Mason R.P. Free Radic. Biol. Med. 2003; 34: 830-839Crossref PubMed Scopus (75) Google Scholar, 17Tew D. Ortiz de Montellano P.R. J. Biol. Chem. 1988; 263: 17880-17886Abstract Full Text PDF PubMed Google Scholar, 18McArthur K.M. Davies M.J. Biochim. Biophys. Acta. 1993; 1202: 173-181Crossref PubMed Scopus (84) Google Scholar, 19Maples K.R. Kennedy C.H. Johnson S.J. Mason R.P. Arch. Biochem. Biophys. 1990; 277: 402-409Crossref PubMed Scopus (95) Google Scholar, 20Gunther M.R. Twchirret-Guth R.A. Witkowska H.E. Fann Y.C. Barr D.P. Ortiz de Montellano P.R. Mason R.P. Biochem. J. 1998; 330: 1293-1299Crossref PubMed Scopus (134) Google Scholar, 21Witting P.K. Mauk A.G. J. Biol. Chem. 2001; 276: 16540-16547Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The exact site of this radical in hemoglobin has been speculated to be Tyr-42 on the α chain, based on x-ray data and the inhibition of the radical adduct signal by iodination of the tyrosine (18McArthur K.M. Davies M.J. Biochim. Biophys. Acta. 1993; 1202: 173-181Crossref PubMed Scopus (84) Google Scholar). Maples et al. (19Maples K.R. Kennedy C.H. Johnson S.J. Mason R.P. Arch. Biochem. Biophys. 1990; 277: 402-409Crossref PubMed Scopus (95) Google Scholar) reported the detection of a peroxide-dependent hemoglobinthiyl radical in rat and human hemoglobin and concluded, based on ESR parameters and the effect of thiol blocking agents, that the radical adduct was a thiyl radical adduct. In the presence of a spin trap, such as DMPO, the Hb-centered radical(s) yield a nitroxide radical adduct(s), which decays on the order of 1 min (22Kim Y.-M. Jeong S-H. Yamazaki I. Piette L.H. Han S. Hong S.-J. Free Radic. Res. 1995; 22: 11-21Crossref PubMed Scopus (15) Google Scholar). This decay is the result of the oxidation of the nitroxide radical adduct to the corresponding globin radical-derived nitrone adduct by the ferryl moiety (Scheme 1). The globin radical-derived nitrone adduct can be investigated by immuno-spin trapping (16Ramirez D.C. Chen Y.-R. Mason R.P. Free Radic. Biol. Med. 2003; 34: 830-839Crossref PubMed Scopus (75) Google Scholar). In the absence of a spin trap, the globin-centered radical(s) can decay by formation of cross links (23Thillet J. Michelson A.M. Free Radic. Res. Commun. 1985; 1: 89-100Crossref PubMed Scopus (7) Google Scholar), such as dityrosine (24Guilivi C. Davies K.J.A. J. Biol. Chem. 2001; 276: 24129-24136Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) and possibly other cross-links. To identify the precise amino acid residues trapped by the DMPO, we have utilized mass spectrometry-based sequencing. This approach includes proteolysis of the hemoglobin-derived DMPO adducts by trypsin, followed by MS peptide mapping, and finally MS/MS analyses of the peptides. Using this approach, we have identified the specific location of the DMPO adducts on hemoglobin and report our results here. Materials—Pure human oxyferrous Hb (oxyHb) was a kind gift of Apex Biosciences Inc. (Research Triangle Park, NC). Diethylenetriamine pentaacetic acid (DTPA) was purchased from Sigma. Beef liver catalase was purchased from Roche Applied Science and used as received. The spin trap DMPO was purchased from Alexis Biochemicals (San Diego, CA) and purified by double distillation at room temperature. The DMPO concentration was measured at 228 nm, assuming a molar absorption coefficient of 7800 m-1 cm-1 (25Buettner G.R. Free Radic. Res. Commun. 1990; 10: 11-15Crossref PubMed Scopus (59) Google Scholar). Reagent grade 30% H2O2 was obtained from Fisher Scientific Co. The H2O2 concentration was verified using ϵ240 nm m-1 were at for followed by the addition of to reactions. human was in with and the was through a with For the and the reactions of with H2O2 molar to were in the presence of DMPO in at for were by of of H2O2 by Because mass and the formation of adducts, the for the MS analyses were in of the and the reactions were by and mixtures were with with the addition of and at for to room of heme was in each in and by were on each The were using as by the the were on a with a in for min at room and then with a of the to at a of was for 1 were detected by exposure of the to for hemoglobin sample and the hemoglobin DMPO H2O2 reaction sample in were subjected to tryptic was to a of the at a of The reactions were to at tandem mass C. Deterding L.J. Tomer K.B. J. 1999; PubMed Scopus Google Scholar) was used for the of the electrospray ionization mass and tandem mass This is with a electrospray and of a mass and an mass The was and the was for the MS analyses. for flow injection analyses were with a of acid and the mass at using a injection For the a of and a was used to the of were and a of min was used for the The used was a CA) at a flow of the data was For these the can from the MS to the MS/MS and then to the MS based on or such as and The advantage of this is that both MS and MS/MS data can be from a of the The used for these was to the and the of the as from a was with a data and by the of Protein-centered on the the formation of radicals on hemoglobin has been demonstrated (16Ramirez D.C. Chen Y.-R. Mason R.P. Free Radic. Biol. Med. 2003; 34: 830-839Crossref PubMed Scopus (75) Google Scholar), hemoglobin is reacted with hydrogen radicals are formed which can be detected using immuno-spin trapping with an these studies (16Ramirez D.C. Chen Y.-R. Mason R.P. Free Radic. Biol. Med. 2003; 34: 830-839Crossref PubMed Scopus (75) Google Scholar), tyrosine and possibly cysteine residues were to be involved in the formation of radicals in In the ESR spin trapping the formation of DMPO radical adducts on both tyrosyl and amino on hemoglobin in the presence of have been (16Ramirez D.C. Chen Y.-R. Mason R.P. Free Radic. Biol. Med. 2003; 34: 830-839Crossref PubMed Scopus (75) Google Scholar, 19Maples K.R. Kennedy C.H. Johnson S.J. Mason R.P. Arch. Biochem. Biophys. 1990; 277: 402-409Crossref PubMed Scopus (95) Google Scholar). To the hemoglobin hemoglobin was reacted with hydrogen peroxide in the presence of DMPO spin The and of the reaction mixtures is shown in The exposure of human to hydrogen peroxide hemoglobin and order 1). DMPO the Hb from hydrogen by trapping the radicals protein damage and that protein radical-derived nitrone adducts was and used to myoglobin radical-derived nitrone adducts in the and in the supernatant of rat heart (10Detweiler C.D. Deterding L.J. Tomer K.B. Chignell C.F. Germolec D. Mason R.P. Free Radic. Biol. Med. 2002; 33: 364-369Crossref PubMed Scopus (107) Google Scholar). Recently, this was shown to be in hemoglobin radical-derived nitrone adducts by exposure of to a of H2O2 (16Ramirez D.C. Chen Y.-R. Mason R.P. Free Radic. Biol. Med. 2003; 34: 830-839Crossref PubMed Scopus (75) Google Scholar) and on Hb H2O2 was generated by of D.C. C.D. Mason R.P. Chignell C.F. 2003; PubMed Scopus Google Scholar). The covalent of DMPO to the hemoglobin the presence of H2O2 that this by a mechanism (Scheme D.C. Chen Y.-R. Mason R.P. Free Radic. Biol. Med. 2003; 34: 830-839Crossref PubMed Scopus (75) Google Scholar, S. Biochemistry. 2002; 41: PubMed Scopus Google Scholar, and N. R. C.D. Mason R.P. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). hydrogen and DMPO were to hemoglobin-derived nitrone adducts of DMPO on verify that DMPO had been trapped on the hemoglobin the reaction mixtures were analyzed by flow injection The resulting ESI mass for the hemoglobin are shown in In the ESI mass spectrum of hemoglobin of the α chain and the of of and of are as as forms of these In the absence of hydrogen peroxide the mass spectrum is to that of hemoglobin In the absence of DMPO spin trap in the reaction forms of the protein are with and which of oxygen the protein by the reaction of free radicals with The electrospray mass spectrum of the reaction and DMPO shows molecules of α chain of α chain one DMPO molecule, β chain of and β chain one DMPO molecule. In addition, of forms of these are These data that a DMPO molecule is trapped on each of the hemoglobin of these were in of the These data that the formation of the DMPO adduct on hemoglobin is on the presence of both the hydrogen peroxide and the DMPO spin trap in the reaction In the hemoglobin is reacted with hydrogen peroxide in the absence of DMPO as the spin trap forms of the protein are these form from the reaction of radicals with The hydrogen oxygen in is protein free as demonstrated by inhibition with DMPO DMPO is to be an that reactions of radicals with oxygen by trapping The by DMPO of Hb against is in the immuno-spin trapping and These data between oxygen and DMPO for the radical and MS/MS of of DMPO on of the specific location of the DMPO adducts on hemoglobin was by peptide mapping and MS analyses. To determine which amino in hemoglobin a trapped DMPO molecule, hemoglobin and the reaction were subjected to tryptic digestion and by Because of the of the digestion was used in with the MS analyses. The data were using the data which the to from the MS to the MS/MS and then to the MS based on by the The resulting MS/MS data were then using In the DMPO was as a on the amino The then for tryptic by the addition of adducts of The corresponding MS/MS be on the of the presence of a DMPO adduct and which amino acid by DMPO the MS/MS With the amino acid residues were identified as a DMPO molecule in the reaction of these were in the of the hemoglobin tryptic These to tryptic peptide and tryptic peptide acid residues of β chain DMPO as as tryptic peptide and tryptic peptide of α chain The MS/MS data the DMPO to the Cys-93 of the β chain, Tyr-42 of the α chain, and Tyr-24 of the α chain. These data were by in the MS/MS to the using the shows the MS/MS spectrum of the tryptic peptide of α chain. A of both and P. J. PubMed Scopus Google Scholar, 1988; Scopus Google Scholar) are which to the peptide In addition, the of from is The result from peptide and the result from The mass of the corresponds to amino a In addition, the of the and the data to the location of the DMPO adduct. The mass between the and corresponds to the mass of a tyrosine residue the mass of one DMPO molecule. These the assignment of the DMPO molecule to the Tyr-42 residue of tryptic peptide of α spectrum of tryptic peptide of the β chain of The reaction of human hemoglobin with hydrogen peroxide in the presence of DMPO was with and analyzed by with an to the of DMPO and from the through For identified are on the spectrum of tryptic peptide of the α chain of The reaction of human hemoglobin with hydrogen peroxide in the presence of DMPO was with and analyzed by are with the DMPO on with an be for by a adduct. are with the DMPO on The as corresponds to the for a histidine residue For identified are on the The MS/MS spectrum of tryptic peptide residues of β chain is shown in of is which corresponds in mass to the of a DMPO molecule and the of from the ion. A of through and through are as as the of DMPO and from the through with an The mass between and corresponds to the mass of a cysteine These data the unequivocal assignment of a DMPO molecule to Cys-93 of tryptic peptide of β chain. The MS/MS spectrum of the at which corresponds in mass to tryptic peptide of α chain a DMPO molecule was and the resulting MS/MS spectrum in which are to the is shown in on the of these the tyrosine residue in this peptide was identified as the site of the DMPO adduct. The corresponding MS/MS spectrum and are shown in A of are as as the and ion. The mass between the of and the of corresponds to the mass of a tyrosine residue a of the MS/MS however, are in the of the spectrum that be for by a adduct, at with an In addition, in the are which to a a DMPO molecule. For the of corresponds in mass to a DMPO and the of corresponds in mass to a These data the presence of a DMPO adduct on a amino acid in the tryptic of these data that is a DMPO adduct on the histidine residue The mass between the of as in and the of as corresponds to the mass of a histidine residue a These data the presence of a DMPO molecule on His-20 of from α chain. which the presence of DMPO on the histidine residue are as through through and for a histidine residue in The of therefore, is a of tryptic one with a DMPO at His-20 and one with a DMPO at The of the corresponding one to the His-20 DMPO adduct is in a the Tyr-24 DMPO adduct. is evidence for the presence of a sites of radical formation were identified on the α chain of hemoglobin Tyr-24, and is evidence that sites are trapped by DMPO at the In the electrospray mass spectrum of the and DMPO reaction that in mass to the α and β as as that to the addition of a DMPO molecule to each of these is For are which to the α chain DMPO The amino acid residues that have been identified by mass spectrometry as a site for DMPO spin trapping in hemoglobin are shown in The was from the x-ray of the hemoglobin B. J. Biol. PubMed Scopus Google Scholar). The of the α and β of hemoglobin are as the trapped amino and are shown as are shown in His-20, Tyr-24, and Tyr-42 on the α chain are shown in and Cys-93 on the β chain is shown in on previous it has been that the product between the reaction of heme and hydrogen peroxide is an heme of a ferryl and a porphyrin cation radical to as Inc., Scholar). electron is then from an amino acid residue to the These electron can either or L.J. Barr D.P. Mason R.P. Tomer K.B. J. Biol. Chem. 1998; 1273: 12863-12869Abstract Full Text Full Text PDF Scopus (49) Google Scholar). In the absence of DMPO, the hydrogen peroxide-dependent oxidation of hemoglobin to the of oxygen In it is that Tyr-24 and His-20 of the α chain of hemoglobin are to one Because of the pi-pi interaction of Tyr-24 and His-20, the unpaired electron is apparently delocalized on both the tyrosine and histidine residue (pi-pi stacked radical as in the radical J. D.C. M.S. S. PubMed Scopus Google Scholar, Biochim. Biophys. 2001; PubMed Scopus Google Scholar). one radical is trapped at a To our this is the report of a radical in a histidine is a of the redox in such and has been reported in the M.R. J.A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). The pi-pi of histidine with tyrosine the oxidation of histidine the oxidation potential of histidine is that of or S. J. Chem. Soc. 1998; Scopus Google Scholar). In using peptide mapping and MS the DMPO radical adducts on hemoglobin were mass spectrometry as to the location of the DMPO molecules in the was that Cys-93 on β chain, Tyr-42, His-20, and Tyr-24 on the α chain of hemoglobin form a DMPO adduct. peptide mapping in combination with mass spectrometry is an technique for the location of radical adducts in proteins. We C. and for of the

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