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• W1977644100 abstract "The human myoglobin (Mb) sequence is similar to other mammalian Mb sequences, except for a unique cysteine at position 110. Reaction of wild-type recombinant human Mb, the C110A variant of human Mb, or horse heart Mb with H2O2(protein/H2O2 = 1:1.2 mol/mol) resulted in formation of tryptophan peroxyl (Trp-OO⋅) and tyrosine phenoxyl radicals as detected by EPR spectroscopy at 77 K. For wild-type human Mb, a second radical (g ∼ 2.036) was detected after decay of Trp-OO⋅ that was not observed for the C110A variant or horse heart Mb. When the spin trap 5,5-dimethyl-1-pyrrolineN-oxide (DMPO) was included in the reaction mixture at protein/DMPO ratios ≤1:10 mol/mol, a DMPO adduct exhibiting broad absorptions was detected. Hyperfine couplings of this radical indicated a DMPO-thiyl radical. Incubation of wild-type human Mb with thiol-blocking reagents prior to reaction with peroxide inhibited DMPO adduct formation, whereas at protein/DMPO ratios ≥1:25 mol/mol, DMPO-tyrosyl radical adducts were detected. Mass spectrometry of wild-type human Mb following reaction with H2O2demonstrated the formation of a homodimer (mass of 34,107 ± 5 atomic mass units) sensitive to reducing conditions. The human Mb C110A variant afforded no dimer under identical conditions. Together, these data indicate that reaction of wild-type human Mb and H2O2 differs from the corresponding reaction of other myoglobin species by formation of thiyl radicals that lead to a homodimer through intermolecular disulfide bond formation. The human myoglobin (Mb) sequence is similar to other mammalian Mb sequences, except for a unique cysteine at position 110. Reaction of wild-type recombinant human Mb, the C110A variant of human Mb, or horse heart Mb with H2O2(protein/H2O2 = 1:1.2 mol/mol) resulted in formation of tryptophan peroxyl (Trp-OO⋅) and tyrosine phenoxyl radicals as detected by EPR spectroscopy at 77 K. For wild-type human Mb, a second radical (g ∼ 2.036) was detected after decay of Trp-OO⋅ that was not observed for the C110A variant or horse heart Mb. When the spin trap 5,5-dimethyl-1-pyrrolineN-oxide (DMPO) was included in the reaction mixture at protein/DMPO ratios ≤1:10 mol/mol, a DMPO adduct exhibiting broad absorptions was detected. Hyperfine couplings of this radical indicated a DMPO-thiyl radical. Incubation of wild-type human Mb with thiol-blocking reagents prior to reaction with peroxide inhibited DMPO adduct formation, whereas at protein/DMPO ratios ≥1:25 mol/mol, DMPO-tyrosyl radical adducts were detected. Mass spectrometry of wild-type human Mb following reaction with H2O2demonstrated the formation of a homodimer (mass of 34,107 ± 5 atomic mass units) sensitive to reducing conditions. The human Mb C110A variant afforded no dimer under identical conditions. Together, these data indicate that reaction of wild-type human Mb and H2O2 differs from the corresponding reaction of other myoglobin species by formation of thiyl radicals that lead to a homodimer through intermolecular disulfide bond formation. myoglobin bovine serum albumin diethylenetriaminepentaacetic acid 2,2-dithiopyridine 5,5′-dithiobis(2-nitrobenzoic acid) 5,5-dimethyl-1-pyrroline N-oxide dithiothreitol electrospray ionization mass spectrometry polyacrylamide gel electrophoresis milliwatts Protein-centered free radicals are now well recognized as normal intermediates for an increasing number of enzymes (1.Stubbe J. van der Donk W.A. Chem. Rev. 1998; 98: 705-762Crossref PubMed Scopus (1341) Google Scholar). Examples of such radical intermediates include the thiyl (2.Licht S. Gerfen G.J. Stubbe J. Science. 1996; 271: 477-481Crossref PubMed Scopus (271) Google Scholar) and tyrosyl (3.Stubbe J. Biochemistry. 1988; 27: 3893-3900Crossref PubMed Scopus (77) Google Scholar) radical centers of ribonucleotide reductase, the tyrosyl radical of prostaglandin H synthase (4.Lassmann G. Odenwaller R. Curtis J.F. DeGray J.A. Mason R.P. Marnett L.J. Eling T.E. J. Biol. Chem. 1991; 266 (Correction (1992) J. Biol. Chem. 267, 6449): 20045-20055Abstract Full Text PDF PubMed Google Scholar), the glycyl radical of pyruvate formate-lyase (5.Wagner A.F. Frey M. Neugebauer F.A. Schafer W. Knappe J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 996-1000Crossref PubMed Scopus (301) Google Scholar), and the tryptophan radical of cytochromec peroxidase (6.Sivaraja M. Goodin D.B. Smith M. Hoffman B.M. Science. 1989; 245: 738-740Crossref PubMed Scopus (469) Google Scholar). On the other hand, detrimental protein-centered radicals have been implicated in oxidative stress associated with pathology of inflammatory disease(s) and possibly in aging (7.Dean R.T. Fu S. Stocker R. Davies M.J. Biochem. J. 1997; 324: 1-18Crossref PubMed Scopus (1438) Google Scholar, 8.Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. 2nd Ed. Clarendon Press, Oxford1992Google Scholar). At least some of the toxic effects of protein radicals, as exemplified by initiation of lipid peroxidation (9.Witting P.K. Willhite C.A. Davies M.J. Stocker R. Chem. Res. Toxicol. 1999; 12: 1173-1181Crossref PubMed Scopus (29) Google Scholar, 10.Moore K.P. Holt S.G. Patel R.P. Svistunenko D.A. Zackert W. Goodier D. Reeder B.J. Clozel M. Anand R. Cooper C.E. Morrow J.D. Wilson M.T. Darley-Usmar V. Roberts L.J., II J. Biol. Chem. 1998; 273: 31731-31737Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 11.Miller Y.I. Altamentova S.M. Shaklai N. Biochemistry. 1997; 36: 12189-12198Crossref PubMed Scopus (166) Google Scholar, 12.Miller Y.I. Felikman Y. Shaklai N. Arch. Biochem. Biophys. 1996; 326: 252-260Crossref PubMed Scopus (72) Google Scholar), involve both intramolecular (13.Wilks A. Ortiz de Montellano P.R. J. Biol. Chem. 1992; 267: 8827-8833Abstract Full Text PDF PubMed Google Scholar) and intermolecular (14.Rao S.I. Wilks A. Ortiz de Montellano P.R. J. Biol. Chem. 1993; 268: 803-809Abstract Full Text PDF PubMed Google Scholar) translocation of radical centers from one amino acid residue to another. One particularly well studied example of radical translocation is provided by the protein-centered radical formed by myoglobin upon reaction with hydrogen peroxide. Myoglobin (Mb)1 has a limited ability to reduce H2O2 to water with the concomitant formation of ferryl (Fe(IV)=O) heme. At protein/H2O2 ratios ≤1:5 mol/mol, reaction of metmyoglobin with H2O2 yields both ferryl (Fe(IV)=O) Mb and protein radicals (globin⋅). Although the ferryl Mb is stable for hours at room temperature (15.George P. Irvine D.H. Biochem. J. 1952; 52: 511-517Crossref PubMed Scopus (200) Google Scholar), the identity of the Mb residue(s) that form radicals in the presence of H2O2 has been the subject of some controversy. Globin⋅ radicals have been localized to tyrosine (16.Tew D. Ortiz de Montellano P.R. J. Biol. Chem. 1988; 263: 17880-17886Abstract Full Text PDF PubMed Google Scholar, 17.Davies M.J. Biochim. Biophys. Acta. 1991; 1077: 86-90Crossref PubMed Scopus (141) Google Scholar) and/or tryptophan residues (18.Gunther M.R. Tschirret-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 (133) Google Scholar). Additionally, globin⋅ radicals may undergo subsequent chemistry (18.Gunther M.R. Tschirret-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 (133) Google Scholar) and are capable of oxidizing a variety of biological molecules (19.Kelman D.J. DeGray J.A Mason R.P. J. Biol. Chem. 1994; 269: 7458-7463Abstract Full Text PDF PubMed Google Scholar). Hydroxyl radicals, however, do not appear to be involved in the transfer of oxidative damage (20.Davies M.J. Free Radic. Res. Commun. 1990; 10: 361-370Crossref PubMed Scopus (104) Google Scholar,21.Turner J.J. Rice-Evans C.A. Davies M.J. Newman E.S. Biochem. J. 1991; 277: 833-837Crossref PubMed Scopus (75) Google Scholar). Human Mb is similar in sequence to other mammalian myoglobins. One significant difference, however, is the presence of Cys110. No other species of known mammalian Mb possesses a cysteine residue (22.Hubbard S.R. Hendrickson W.A. Lambright D.G. Boxer S.G. J. Mol. Biol. 1990; 213: 215-218Crossref PubMed Scopus (75) Google Scholar). As Mb is released under some pathological situations such as ischemia/reperfusion of the heart (23.Galaris D. Eddy L. Arduini A. Cadenas E. Hochstein P. Biochem. Biophys. Res. Commun. 1989; 160: 1162-1168Crossref PubMed Scopus (105) Google Scholar) and H2O2is produced continuously in vivo (24.Chance B. Sies H. Boveris A. Physiol. Rev. 1979; 59: 527-605Crossref PubMed Scopus (4764) Google Scholar), the reaction of human Mb and H2O2 may afford a physiologically relevant oxidant. Although the reactions of horse heart Mb and sperm whale Mb with H2O2 have been studied extensively, the corresponding reaction of human Mb has not been investigated previously. In this work, we have studied the reaction of human Mb and its C110A variant to evaluate the possible role of the reactive thiol group in the reaction of this protein with hydrogen peroxide through the combined use of EPR spectroscopy and electrospray mass spectrometry. Horse heart Mb, reduced glutathione, iodoacetamide, bovine serum albumin (BSA), trypsin (type III; 10,200 unit/mg of protein), bovine liver catalase (40,000 unit/mg of protein), urea, ascorbate, EDTA, TEMPO, DTPA, 2,2-dithiodipyridine (DTP), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) were obtained from Sigma. DMPO was purified by stirring solutions (1 m in 50 mm phosphate buffer, pH 7.4) with activated charcoal (100 mg/ml) in the dark. After 30 min, the solution was filtered, and portions were stored at −80 °C prior to use (25.Kotake Y. Reineke L.A. Tanigawa T. Koshida H. Free Radic. Biol. Med. 1994; 17: 215-223Crossref PubMed Scopus (34) Google Scholar). Tryptone and yeast extract were from Becton Dickinson (Sparks, MD). Dithiothreitol (DTT) and NaCl were obtained from Fisher. H2O2was from Bio-Rad. Buffers were prepared from either glass-distilled water or glass-distilled water purified further by passage through a Barnstead Nanopure system. All buffers were stored over Chelex 100® (Bio-Rad) at 4 °C for at least 24 h to remove contaminating transition metals as verified by ascorbate autoxidation analysis (26.Buettner G.R. Methods Enzymol. 1990; 186: 125-127Crossref PubMed Scopus (77) Google Scholar). Organic solvents and all other chemicals employed were of the highest quality available. Transformed bacteria (strain AR68) containing plasmids for both wild-type recombinant human myoglobin and the C110A variant (27.Ragavan V. Szabo A. Boxer S.G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5681-5684Crossref PubMed Scopus (97) Google Scholar) were obtained from Prof. Steven G. Boxer. The cells were grown at 28 °C in 10 × 2-liter flasks containing 2YT Superbroth (1 liter/flask: Tryptone (16 g/liter), yeast extract (10 g/liter), and NaCl (5 g/liter)) toA 600 nm = 1.2. The expression of recombinant myoglobins was induced by immersing each flask in a water bath (55 °C) for 5 min and then transferring the flask to an incubator operating at 42 °C. Cultures were incubated for a further 6–7 h, and the cells were harvested. Myoglobin (isolated as a fusion product) was purified by anion-exchange chromatography (Whatman DE52 resin) as described (27.Ragavan V. Szabo A. Boxer S.G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5681-5684Crossref PubMed Scopus (97) Google Scholar). Rapidly increasing the culture temperature in this manner was essential as simply increasing incubation temperature from 28 to 42 °C failed to induce protein expression. The partially purified fusion protein was reconstituted with excess hemin (heme/protein ∼ 1:1.5; Porphyrin Products, Logan, UT), treated with trypsin (to cleave the fusion segment) and then further purified as described (27.Ragavan V. Szabo A. Boxer S.G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5681-5684Crossref PubMed Scopus (97) Google Scholar). Under these conditions, recombinant myoglobin was isolated as metmyoglobin. When required, proteins were concentrated by centrifugal ultrafiltration (Centriprep-10 concentrators, Millipore Corp.). X-band EPR spectra (293 or 77 K) were obtained with a Bruker ESP 300e spectrometer equipped with a Hewlett-Packard microwave frequency counter. Mb solutions (∼1 mm in 50 mm sodium phosphate buffer, pH 7.4) were treated with H2O2 at molar ratios of 1:1.2–5 in both the presence and absence of DMPO (protein/DMPO = 1:5–100 mol/mol). For low temperature studies, a sample of the reaction mixture (250 μl) was placed into a standard 3-mm quartz cell (Wilmad, Buena, NJ), snap-frozen in liquid nitrogen, and transferred to a finger Dewar insert (77 K) for EPR analyses. Analyses of DMPO spin-trapped adducts were performed with samples (50 μl) of the reaction mixture transferred into capillary tubes with a glass pipette. The capillary was then placed into a quartz EPR tube, and the tube was transferred to the cavity for EPR analyses at 293 K. The limit of detection of a stable nitroxide (TEMPO) under identical conditions was determined to be ∼50 nm. Unless indicated otherwise, the time between removal of the sample, transfer to the appropriate cell, and tuning the spectrometer was consistently <30 s. EPR spectra were obtained as an average of three to five scans with a modulation frequency of 100 kHz and a sweep time of 84 s. Microwave power, modulation amplitude, and scan range used for each analysis varied appropriately as indicated in the figure legends. Hyperfine couplings were obtained by spectral simulation using the simplex algorithm (28.Duling D.R. J. Magn. Reson. 1994; 104B: 105-110Crossref Scopus (885) Google Scholar) provided in the WINSIM program (NIEHS, National Institutes of Health). All hyperfine couplings are expressed in units of milliteslas. Simulations were considered acceptable if they produced correlation factors of R > 0.85. Peak area was estimated by integration using standard WINEPR software. Where possible, peak areas were standardized against a freshly prepared solution of TEMPO (5 μm in 50 mm phosphate buffer, pH 7.4) measured under identical spectrometer conditions; concentrations are expressed as moles of spins/mol of Mb protein. DTPA (100 μm) was included in all Mb solutions prior to addition of H2O2 to minimize the possibility of free transition metal-mediated decomposition of peroxide by Fenton chemistry. For the analyses of power saturation data, a plot of log(I/P 0.5) versus logP, where I represents intensity and Prepresents the microwave power, was chosen, as this results in a line that is parallel to the abscissa so long as the signal is not saturated and that slopes downward with increasing saturation. The saturation data (29.Rajagopalan K.V. Handler P. Palmer G. Beinert H. J. Biol. Chem. 1968; 243: 3784-3796Abstract Full Text PDF PubMed Google Scholar, 30.Rupp H. Rao K.K. Hall D.O. Cammack R. Biochim. Biophys. Acta. 1978; 537: 255-260Crossref PubMed Scopus (158) Google Scholar) were fitted to the relationship I ∝ (P 0.5)/(1 +P/P 12)0.5 b, whereb ranges from 1 (for an inhomogeneous case) to 2 (for a homogeneous case). The curve of the fitted line tends to a slope of 0.5b under conditions of power saturation, andP 12 is the half-saturation power obtained from the intersection of the two straight line segments of the line of best fit. Electrospray ionization mass spectrometry (ESI/MS) was performed with a triple quadrupole mass spectrometer described in detail elsewhere (31.Hunter C.L. Mauk A.G. Douglas D.J. Biochemistry. 1997; 36: 1018-1025Crossref PubMed Scopus (92) Google Scholar). Samples of myoglobin/H2O2 reaction mixtures were diluted to a final concentration of 10 μm in heme (using methanol/water (1:10, v/v)) and were infused continuously (flow rate of 1 μl/min) into the ion source at 20 °C. Experiments were performed without an internal standard. Multiply charged gas-phase proteins were generated by pneumatically assisted ESI. Depending on the voltage difference between the ion sampling orifice and skimmer, the heme-protein interactions can be disrupted, and free heme can be completely dissociated from the heme-protein complex (31.Hunter C.L. Mauk A.G. Douglas D.J. Biochemistry. 1997; 36: 1018-1025Crossref PubMed Scopus (92) Google Scholar). For this study, the voltage difference between the orifice and skimmer was set at +100–110 V, such that dissociation of heme from the heme-protein complex could occur, and mass peaks were obtained largely for the apoprotein (apoMb) form of the protein. This dissociation of heme from the native protein was necessary to reduce the complexity of species analyzed where a homodimer was obtained (see below), although some contamination from intact protein (or holoprotein) was detected. Estimates of mass for holoMb, however, were performed with a voltage difference of 50–60 V to preserve the heme-protein complex and to afford charge-state distributions largely for the holoMb form of the protein. The ESI/MS system was calibrated with solutions of CsI, and horse heart Mb (mass of the apoprotein predicted from the amino acid sequence of 16,950 atomic mass units) was employed as a standard to test the mass accuracy of the system prior to use. Mass values were obtained by standard fitting analyses of the variousm/z distributions using BioMultiView software (Perkin-Elmer Sciex Instruments, Foster City, CA). Mass determinations were performed for two or more independent protein preparations on different days. Mass values reported here refer to the means ± S.D. from five or more analyses. Electronic absorption spectra were obtained with a Cary 3E UV/visible spectrophotometer. Mb concentrations were determined for solutions prepared in 50 mm sodium phosphate buffer, pH 7.4, assuming ε408 nm ∼ 188,000m−1 cm−1. Reactive thiol content was determined by incubating protein samples (1–5 μm in 50 mm sodium phosphate buffer, pH 7.4) with either DTP or DTNB (1:10 molar ratio of protein to reagent). Absorbance was then measured at 325 or 446 nm, respectively (32.Garel M.C. Beuzard Y. Thillet J. Domenget C. Martin J. Galacteros F. Rosa J. Eur. J. Biochem. 1982; 123: 513-519Crossref PubMed Scopus (47) Google Scholar, 33.Lynch S.M. Frei B. J. Lipid Res. 1993; 34: 1745-1753Abstract Full Text PDF PubMed Google Scholar), and concentrations of free thiol were estimated by comparison with GSH standards. For hemeproteins, the absorbance of the DTNB adduct (421 nm) is nearly coincident with the heme absorption at 408 nm. To avoid this interference, TNB− anion formation was measured at 446 nm and used to estimate protein thiol content (34.Kelman D.J. Mason R.P. Arch. Biochem. Biophys. 1993; 306: 439-442Crossref PubMed Scopus (16) Google Scholar). In some experiments, wild-type human Mb was incubated with iodoacetamide (protein/thiol-blocking reagent = 1:10 mol/mol) prior to addition of DTP or DTNB. The stability of mixed disulfides (i.e. from the reaction of reduced GSH with DTNB or DTP) to reduction by ascorbate was evaluated at 25 °C at variable concentrations (0.1–1 molar eq) of ascorbate by monitoring the spectrum between 250 and 500 nm following addition of ascorbate. Cross-linking experiments were performed by incubating solutions of either wild-type human Mb or the C110A variant (65 μm in 50 mm sodium phosphate buffer, pH 7.4) with H2O2 (325 μm final concentration) and DTPA (100 μm) at 37 °C. Samples of the reaction mixture were removed periodically and diluted immediately with the same buffer with or without added DTT. In some cases, wild-type human Mb was incubated with iodoacetamide prior to addition of H2O2. Samples were then heated at 100 °C for 5 min and analyzed by SDS-PAGE (35.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar) after staining with Coomassie Blue. Statistics were performed with Student's t test available in Microsoft Excel, and significant difference was accepted at p < 0.05. The reactive thiol content of wild-type human Mb and the C110A variant and that of horse heart Mb and BSA are shown in TableI. BSA was found to possess a single reactive thiol that was accessible to both DTP and DTNB, consistent with previous reports (36.Gatti R.M. Radi R. Ohara A. FEBS Lett. 1994; 348: 287-290Crossref PubMed Scopus (146) Google Scholar, 37.Silvester J.A. Timmins G.S. Davies M.J. Free Radic. Biol. Med. 1998; 24: 754-766Crossref PubMed Scopus (54) Google Scholar). As expected from their amino acid sequences, wild-type human Mb also exhibited a single reactive thiol, whereas horse heart Mb and the C110A variant of the human protein contained no free thiol irrespective of treatment (Table I). Treatment of wild-type human Mb with iodoacetamide prior to free thiol determination decreased the thiol content of this protein significantly as expected following carboxymethylation of Cys110, indicating that the thiol had been effectively blocked by this treatment.Table IFree thiol content of human myoglobins and bovine serum albuminProteinReagentFree thiol contentmol/molWild-type human MbDTP1.11 ± 0.01DTNB0.94 ± 0.03Human Mb C110A variantDTP0.02 ± 0.01DTNB0.00 ± 0.03Horse heart MbDTP0.07 ± 0.00DTNB0.01 ± 0.20BSADTP1.07 ± 0.02DTNB0.96 ± 0.10Thiol-blocked wild-type human Mb aThiol-blocked wild-type human Mb was prepared by modification with iodoacetamide immediately before thiol determination (see “Experimental Procedures”).DTP0.22 ± 0.03 bValues are significantly different from corresponding values obtained for unmodified wild-type human Mb (p < 0.05).DTNB0.04 ± 0.04 bValues are significantly different from corresponding values obtained for unmodified wild-type human Mb (p < 0.05).Data represent the means ± S.D. of at least three separate determinations. All reactions were monitored until steady state was achieved, and then absorbance was measured. Absorbance was measured at 325 and 446 nm for adducts of DTP and DTNB, respectively (see “Experimental Procedures”).a Thiol-blocked wild-type human Mb was prepared by modification with iodoacetamide immediately before thiol determination (see “Experimental Procedures”).b Values are significantly different from corresponding values obtained for unmodified wild-type human Mb (p < 0.05). Open table in a new tab Data represent the means ± S.D. of at least three separate determinations. All reactions were monitored until steady state was achieved, and then absorbance was measured. Absorbance was measured at 325 and 446 nm for adducts of DTP and DTNB, respectively (see “Experimental Procedures”). EPR spectra (77 K) for various mammalian myoglobins reacted with H2O2 at a protein/H2O2 molar ratio of 1:1.2 and frozen and recorded immediately after mixing are shown in Fig.1. For horse heart Mb, the C110A variant of human Mb, and the wild-type human protein, weak EPR signals were detected at g ∼ 2.036, 2.013, and 2.006, indicating that the initial globin⋅ formed under these reaction conditions was the same for each protein. Upon incubation of the reaction mixture for wild-type human Mb at 77 K, the signal at g ∼ 2.036 decayed to the base line within 5 min. Subsequently, a second signal appeared at an identical g-value. This latter species was stable at 77 K for at least 15 min (Fig. 2). During this time, the signal at g ∼ 2.011 both increased in intensity and broadened in appearance, such that the features noted earlier were no longer distinguishable. The decay of the g ∼ 2.036 signal and the subsequent broadening of the g ∼ 2.011 signal were also observed for horse heart Mb and the human C110A variant; however, the signal at g ∼ 2.036 did not reappear in either case, even after 30 min of incubation at 77 K (data not shown). The latter observation is consistent with that described for the decay of Trp-OO⋅ in horse heart Mb (38.Irwin J.A. Ostdal H. Davies M.J. Arch. Biochem. Biophys. 1999; 362: 94-104Crossref PubMed Scopus (93) Google Scholar).Figure 2Changes in EPR spectral features of globin⋅ following the reaction of wild-type human Mb and H2O2. Reaction conditions and EPR parameters were as described for Fig. 1. Where indicated (arrows), g-values correspond to gx ∼ 2.036, gy ∼ 2.013, and gz ∼ 2.006. Spectra were obtained after 0 (A), 5 (B), 10 (C), and 30 (D) min. The EPR signal at g = 2.036 decayed to below the detection limit over 0–5 min and was detected again after a further 10 min at 77 K. The maximum concentrations of radicals detected were 0.26 ± 0.01 and 0.44 ± 0.01 mol of spins/mol of human Mb for signals at g ∼ 2.036 and 2.011, respectively, whereas the concentration of radicals measured at g ∼ 2.036 after 15 min was determined to be 0.20 ± 0.01 mol of spins/mol of human Mb. Data represent three or more experiments with different Mb preparations. mT, milliteslas.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To obtain further information concerning the identity of the protein radicals generated by the reaction of wild-type human Mb with H2O2, the spin trap DMPO was added to the reaction mixture prior to addition of peroxide. After 2 min at 20 °C, reaction mixtures treated with DMPO afforded a radical with a four-line EPR spectrum (Fig.3 A). The signal was relatively broad, with the outer most line at a lower g-value broadened almost to the base line. The broad nature of the EPR signal is consistent with a radical exhibiting restricted rotational motion on the EPR time scale and is indicative of DMPO trapping a radical on a large molecule (i.e. a protein). No EPR signal was detected in the absence of protein, DMPO, or H2O2 (Fig. 3,C–E, respectively). Treatment of the wild-type protein with iodoacetamide prior to addition of peroxide inhibited the formation of the radical adduct (Fig. 3 F). Simulation of the EPR signal from the DMPO adduct with WINSIM software (Fig. 3 B) indicated hyperfine couplings from single nitrogen (AN) and hydrogen (AH) atoms. Consistent with a previous report on DMPO trapping of protein thiyl radicals (39.Yeong-Renn C. Gunther M.R. Mason R.P. J. Biol. Chem. 1999; 274: 3308-3314Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), addition of iodoacetamide after formation of the DMPO adduct did not significantly affect the EPR signal of the radical adduct on wild-type recombinant human Mb (data not shown). Hyperfine couplings obtained from these simulations together with reported values for relevant DMPO adducts are summarized in Table II. The couplings obtained from the quartet signal in this work are similar to those previously reported for the glutathione-derived thiyl radical (Table II). Importantly, hyperfine coupling of the free electron to the β-hydrogen in the DMPO adduct detected here is also similar to that reported for DMPO-thiyl radicals derived from both BSA and cytochromec oxidase (Table II). The magnitude of the hydrogen coupling is typical of a heteroatom-centered radical (40.Buettner G.R. Free Radic. Biol. Med. 1987; 3: 259-303Crossref PubMed Scopus (1496) Google Scholar). These data, together with the EPR spectra of the reaction mixtures reported above, suggest that a thiyl radical is produced on wild-type human Mb upon reaction with hydrogen peroxide.Table IIEPR hyperfine coupling constants for various DMPO adductsThiol sourceSolventANAHRef.GSHH2O1.51.649BSA aThe EPR signal of the DMPO adduct in these studies was attributed to that from coupling to single nitrogen and hydrogen atoms; however, only AH was reported as coupling to the β-hydrogen in the EPR analyses of the DMPO adduct was the only coupling clearly resolved.Sodium phosphate buffer (50 mm, pH 7.4)1.636, 37CcOSodium phosphate buffer (50 mm, pH 7.4)1.51.639Human MbSodium phosphate buffer (50 mm, pH 7.4)1.4 ± 0.11.6 ± 0.2This workHyperfine coupling constants are given as data from a single literature source or mean of literature values (where applicable) or were derived from this study. All values are given in milliteslas. Hyperfine values determined in this study were optimized by simulation using WINSIM software (28.Duling D.R. J. Magn. Reson. 1994; 104B: 105-110Crossref Scopus (885) Google Scholar), accepting correlation coefficients of R> 0.85. Data shown represent the means ± S.D. of three independent studies using different preparations of wild-type human Mb. CcO, cytochrome c oxidase.a The EPR signal of the DMPO adduct in these studies was attributed to that from coupling to single nitrogen and hydrogen atoms; however, only AH was reported as coupling to the β-hydrogen in the EPR analyses of the DMPO adduct was the only coupling clearly resolved. Open table in a new tab Hyperfine coupling constants are given as data from a single literature source or mean of literature values (where applicable) or were derived from this study. All values are given in milliteslas. Hyperfine values determined in this study were optimized by simulation using WINSIM software (28.Duling D.R. J. Magn. Reson. 1994; 104B: 105-110Crossref Scopus (885) Google Scholar), accepting correlation coefficients of R> 0.85. Data shown represent the means ± S.D. of three independent studies using different preparations of wild-type human Mb. CcO, cytochrome c oxidase. DMPO adducts derived from tyrosine phenoxyl radicals have been reported for other mammalian myoglobins (18.Gunther M.R. Tschirret-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 (133) Google Scholar, 19.Kelman D.J. DeGray J.A Mason R.P. J. Biol. Chem. 1994; 269: 7458-7463Abstract Full Text PDF PubMed Google Scholar). Therefore, we next in" @default.
• W1977644100 created "2016-06-24" @default.
• W1977644100 creator A5019945539 @default.
• W1977644100 creator A5073987688 @default.
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• W1977644100 date "2000-07-01" @default.
• W1977644100 modified "2023-10-01" @default.
• W1977644100 title "Reaction of Human Myoglobin and H2O2" @default.
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