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Reactions of Deoxy-, Oxy-, and Methemoglobin with Nitrogen Monoxide

2003; Elsevier BV; Volume: 278; Issue: 9 Linguagem: Inglês

10.1074/jbc.m210275200

1083-351X

Susanna Herold, Gabriele Röck,

Neonatal Health and Biochemistry

The reaction between hemoglobin (Hb) and NO⋅ has been investigated thoroughly in recent years, but its mechanism is still a matter of substantial controversy. We have carried out a systematic study of the influence of the following factors on the yield of S-nitrosohemoglobin (SNO-Hb) generated from the reaction of NO⋅ with oxy-, deoxy-, and metHb: 1) the volumetric ratio of the protein and the NO⋅ solutions; 2) the rate of addition of the NO⋅ solution to the protein solution; 3) the amount of NO⋅ added; and 4) the concentration of the phosphate buffer. Our results suggest that the highest SNO-Hb yields are mostly obtained by very slow addition of substoichiometric amounts of NO⋅ from a diluted solution. Possible pathways of SNO-Hb formation from the reaction of NO⋅ with oxy-, deoxy-, and metHb are described. Our data strongly suggest that, because of mixing artifacts, care should be taken to use results from in vitro experiments to draw conclusion on the mechanism of the reaction in vivo. The reaction between hemoglobin (Hb) and NO⋅ has been investigated thoroughly in recent years, but its mechanism is still a matter of substantial controversy. We have carried out a systematic study of the influence of the following factors on the yield of S-nitrosohemoglobin (SNO-Hb) generated from the reaction of NO⋅ with oxy-, deoxy-, and metHb: 1) the volumetric ratio of the protein and the NO⋅ solutions; 2) the rate of addition of the NO⋅ solution to the protein solution; 3) the amount of NO⋅ added; and 4) the concentration of the phosphate buffer. Our results suggest that the highest SNO-Hb yields are mostly obtained by very slow addition of substoichiometric amounts of NO⋅ from a diluted solution. Possible pathways of SNO-Hb formation from the reaction of NO⋅ with oxy-, deoxy-, and metHb are described. Our data strongly suggest that, because of mixing artifacts, care should be taken to use results from in vitro experiments to draw conclusion on the mechanism of the reaction in vivo. oxyhemoglobin deoxyhemoglobin iron(III)hemoglobin hemoglobin hemoglobin with the cysteine residue β93 nitrosated iron(II)-nitrosyl complex S-nitrosoglutathione 4,4′-dithiopyridine diethylenetriaminepentaacetic acid myoglobin iron(III)myoglobin The interaction of nitrogen monoxide with hemoproteins, in particular with myoglobin and hemoglobin, is currently an area of intense research (1–15). The reaction between NO⋅ and oxyhemoglobin (oxyHb)1 has repeatedly been suggested to represent the main route for NO⋅depletion in the blood vessels (16Doyle M.P. Hoekstra J.W. J. Inorg. Biochem. 1981; 14: 351-358Crossref PubMed Scopus (541) Google Scholar, 17Chiodi H. Mohler J.G. Environ. 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Curry S.R. Aitken J.F. Mathews A.J. Johnson K.A. Smith R.D. Phillips Jr., G.N. Olson J.S. Biochemistry. 1996; 35: 6976-6983Crossref PubMed Scopus (574) Google Scholar, 23Doherty D.H. Doyle M.P. Curry S.R. Vali R.J. Fattor T.J. Olson J.S. Lemon D.D. Nat. Biotechnol. 1998; 16: 672-676Crossref PubMed Scopus (368) Google Scholar). However, the in vivorelevance of this reaction has recently been questioned (11Pawloski J.R. Hess D.T. Stamler J.S. Nature. 2001; 409: 622-626Crossref PubMed Scopus (505) Google Scholar, 15Gow A.J. Luchsinger B.P. Pawloski J.R. Singel D.J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9027-9032Crossref PubMed Scopus (378) Google Scholar, 24McMahon T.J. Moon R.E. Luschinger B.P. Carraway M.S. Stone A.E. Stolp B.W. Gow A.J. Pawloski J.R. Watke P. Singel D.J. Piantadosi C.A. Stamler J.S. Nat. Med. 2002; 8: 711-717Crossref PubMed Scopus (410) Google Scholar). Indeed, it has been proposed that in vivo NO⋅preferentially binds to the very small amount of deoxygenated heme of hemoglobin that is present under physiological conditions (about 1%) to yield an iron(II)-nitrosyl complex (HbFe(II)NO) (15Gow A.J. Luchsinger B.P. Pawloski J.R. Singel D.J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9027-9032Crossref PubMed Scopus (378) Google Scholar). This proposal is based on the hypothesis that NO⋅ binds R-state hemoglobin at least 100 times faster than T-state hemoglobin (25Gross S.S. Lane P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9967-9969Crossref PubMed Scopus (92) Google Scholar). It has further been suggested that the "NO group" can be transferred intramolecularly from the heme (HbFe(II)NO) to the conserved cysteine residue β93, producing S-nitrosohemoglobin (SNO-Hb) (26Gow A.J. Stamler J.S. Nature. 1998; 391: 169-173Crossref PubMed Scopus (517) Google Scholar). This process has been proposed to be driven thermodynamically, since the S-nitrosocysteine of SNO-Hb is significantly more stable when Hb is in the R-state. When the red blood cells reach hypoxic tissues, O2 is delivered from SNO-Hb, triggering the conversion of Hb to the T-state (26Gow A.J. Stamler J.S. Nature. 1998; 391: 169-173Crossref PubMed Scopus (517) Google Scholar). Because of steric interactions with nearby amino acid residues, the S-nitrosocysteine of SNO-Hb is highly destabilized in the deoxyHb T-state (27Chan N.-L. Rogers P.H. Arnone A. Biochemistry. 1998; 37: 16459-16464Crossref PubMed Scopus (105) Google Scholar). Thus, transnitrosation from SNO-Hb to other thiols within the red blood cell is favored. In particular, it has been suggested that the "NO group" is transferred to a cysteine residue of the band 3 anion exchange membrane protein and finally transported out of the red blood cell by a still unidentified route (11Pawloski J.R. Hess D.T. Stamler J.S. Nature. 2001; 409: 622-626Crossref PubMed Scopus (505) Google Scholar, 24McMahon T.J. Moon R.E. Luschinger B.P. Carraway M.S. Stone A.E. Stolp B.W. Gow A.J. Pawloski J.R. Watke P. Singel D.J. Piantadosi C.A. Stamler J.S. Nat. Med. 2002; 8: 711-717Crossref PubMed Scopus (410) Google Scholar). One of the central points of this hypothetical mechanism is that the delivery of both O2 and NO⋅ to tissues is regulated allosterically; cooperative liberation of NO⋅ and O2, transported under physiological conditions by hemoglobin, should thus allow for very efficient delivery of dioxygen to peripheral respiring tissues (11Pawloski J.R. Hess D.T. Stamler J.S. Nature. 2001; 409: 622-626Crossref PubMed Scopus (505) Google Scholar, 24McMahon T.J. Moon R.E. Luschinger B.P. Carraway M.S. Stone A.E. Stolp B.W. Gow A.J. Pawloski J.R. Watke P. Singel D.J. Piantadosi C.A. Stamler J.S. Nat. Med. 2002; 8: 711-717Crossref PubMed Scopus (410) Google Scholar). An increasing amount of evidence has recently been published that challenges this fascinating hypothesis. Huang et al. (28Huang Z. Louderback J.G. Goyal M. Azizi F. King B. Kim-Shapiro D.B. Biochim. Biophys. Acta. 2001; 1568: 252-260Crossref PubMed Scopus (80) Google Scholar) have shown that there is a linear correlation between the oxygen saturation of Hb and the yield of HbFe(II)NO, indicating that the rate of NO⋅ binding to deoxyHb is independent of oxygen saturation. In another work, the same group has shown that the rate constant for NO⋅ binding to R-state hemoglobin is (2.1 ± 0.1) × 107m−1 s−1, nearly identical to that reported for NO⋅ binding to T-state Hb (2.6 × 107m−1s−1) (29Cassoly R. Gibson Q.H. J. Mol. Biol. 1975; 91: 301-313Crossref PubMed Scopus (211) Google Scholar). Gladwin et al. (12Gladwin M.T. Ognibene F.P. Pannell L.K. Nichols J.S. Pease-Fye M.E. Shelhamer J.H. Schechter A.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9943-9948Crossref PubMed Scopus (238) Google Scholar) found a strong correlation between metHb and plasma nitrate in the venous blood from volunteers inhaling 80 ppm of NO⋅ gas and concluded that the main reaction of NO⋅ with Hb in the red blood cells of arterial blood leads to NO⋅ depletion, not conservation of its bioactivity. In addition, the same group determined that SNO-Hb is present in the low nm range in the blood, but they did not find a significant arterial-venous gradient (30Gladwin M.T. Shelhamer J.H. Schechter A.N. Pease-Fye M.E. Waclawiw M.A. Panza J.A. Ognibene F.P. Cannon III, R.O. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11482-11487Crossref PubMed Scopus (405) Google Scholar, 31Cannon III, R.O. Schechter A.N. Panza J.A. Ognibene F.P. Pease-Fye M.E. Waclawiw M.A. Shelhamer J.H. Gladwin M.T. J. Clin. Invest. 2001; 108: 279-287Crossref PubMed Scopus (250) Google Scholar, 32Gladwin M.T. Wang X. Reiter C.D. Yang B.K. Vivas E.X. Bonaventura C. Schechter A.N. J. Biol. Chem. 2002; 277: 27818-27828Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Such a gradient should be found if NO⋅ was released in the capillaries after O2 dissociation. Finally, in a recent paper Gladwinet al. (32Gladwin M.T. Wang X. Reiter C.D. Yang B.K. Vivas E.X. Bonaventura C. Schechter A.N. J. Biol. Chem. 2002; 277: 27818-27828Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) showed that SNO-Hb is intrinsically unstable in the reductive environment of the red blood cells and thus concluded that SNO-Hb cannot accumulate in the red blood cells to form a reservoir of bioactive NO⋅. Taken together, these results suggest that SNO-Hb cannot play a role in the regulation of blood flow under normal physiological conditions. In addition, while this work was in progress, three papers have appeared that address the problem of mixing artifacts from the addition of an NO⋅ bolus to oxyHb (33Han T.H. Hyduke D.R. Vaughn M.W. Fukuto J.M. Liao J.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7763-7768Crossref PubMed Scopus (92) Google Scholar, 34Joshi M.S. Ferguson Jr., T.B. Han T.H. Hyduke D.R. Liao J.C. Rassaf T. Bryan N. Feelisch M. Lancaster Jr., J.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10341-10346Crossref PubMed Scopus (186) Google Scholar) and oxymyoglobin (oxyMb) (35Zhang Y. Hogg N. Free Radical Biol. Med. 2002; 32: 1212-1219Crossref PubMed Scopus (53) Google Scholar) solutions. The common conclusion of the authors of the three mentioned papers (33Han T.H. Hyduke D.R. Vaughn M.W. Fukuto J.M. Liao J.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7763-7768Crossref PubMed Scopus (92) Google Scholar, 34Joshi M.S. Ferguson Jr., T.B. Han T.H. Hyduke D.R. Liao J.C. Rassaf T. Bryan N. Feelisch M. Lancaster Jr., J.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10341-10346Crossref PubMed Scopus (186) Google Scholar, 35Zhang Y. Hogg N. Free Radical Biol. Med. 2002; 32: 1212-1219Crossref PubMed Scopus (53) Google Scholar) is that the addition of a small volume of a saturated (2 mm) NO⋅ solution to a diluted oxyHb or oxyMb/GSH solution may lead to artifactual generation of high SNO-Hb/HbFe(II)NO or S-nitrosoglutathione (GSNO)/MbFe(II)NO yields, respectively. Thus, some of the high SNO-Hb yields reported in previousin vitro studies (15Gow A.J. Luchsinger B.P. Pawloski J.R. Singel D.J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9027-9032Crossref PubMed Scopus (378) Google Scholar, 26Gow A.J. Stamler J.S. Nature. 1998; 391: 169-173Crossref PubMed Scopus (517) Google Scholar) may be artifacts arising from the chosen experimental conditions. However, the data reported in these three papers (33Han T.H. Hyduke D.R. Vaughn M.W. Fukuto J.M. Liao J.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7763-7768Crossref PubMed Scopus (92) Google Scholar, 34Joshi M.S. Ferguson Jr., T.B. Han T.H. Hyduke D.R. Liao J.C. Rassaf T. Bryan N. Feelisch M. Lancaster Jr., J.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10341-10346Crossref PubMed Scopus (186) Google Scholar, 35Zhang Y. Hogg N. Free Radical Biol. Med. 2002; 32: 1212-1219Crossref PubMed Scopus (53) Google Scholar) are not always consistent, and contrasting mechanisms have been proposed to explain similar observations. In particular, from their studies of the reaction between oxyHb and NO⋅ in the presence of cyanide, Han et al. (33Han T.H. Hyduke D.R. Vaughn M.W. Fukuto J.M. Liao J.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7763-7768Crossref PubMed Scopus (92) Google Scholar) concluded that, when an NO⋅ bolus is added to an oxyHb solution, SNO-Hb is formed through a pathway that does not include NO+ generated from oxidation of NO⋅ by metHb. In contrast, they suggest that SNO-Hb is possibly formed by reaction of Cys-β93 with N2O3. According to their mathematical simulations, formation of N2O3after the addition of NO⋅ to oxyHb under aerobic conditions is not possible kinetically, and thus they suggested that N2O3 must be present in the NO⋅ stock solution. This suggestion is highly unlikely, since N2O3 rapidly hydrolyzes to nitrite in aqueous solutions. Indeed, it has been shown that the addition of unpurified gaseous NO⋅ (directly from the gas tank) to GSH under anaerobic conditions leads to the formation of GSNO (36Kharitonov V.G. Sundquist A.R. Sharma V.S. J. Biol. Chem. 1995; 270: 28158-28164Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). In contrast, GSNO was not detected when a solution of unpurified NO⋅ was added to the GSH solution (under anaerobic conditions) (36Kharitonov V.G. Sundquist A.R. Sharma V.S. J. Biol. Chem. 1995; 270: 28158-28164Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). A final observation of Han et al. (33Han T.H. Hyduke D.R. Vaughn M.W. Fukuto J.M. Liao J.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7763-7768Crossref PubMed Scopus (92) Google Scholar) is that if NO⋅ is delivered with the NO⋅ donor 2-(N,N-diethylamino)diazenolate 2-oxide (DEA), oxyHb is converted exclusively to metHb and nitrate, and no detectable amounts of SNO-Hb are formed. Joshi et al. (34Joshi M.S. Ferguson Jr., T.B. Han T.H. Hyduke D.R. Liao J.C. Rassaf T. Bryan N. Feelisch M. Lancaster Jr., J.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10341-10346Crossref PubMed Scopus (186) Google Scholar) also studied the reaction of NO⋅with oxyHb. From their results, the authors of this paper also concluded that SNO-Hb is generated from the reaction of Cys-β93 with N2O3. However, Joshi et al. (34Joshi M.S. Ferguson Jr., T.B. Han T.H. Hyduke D.R. Liao J.C. Rassaf T. Bryan N. Feelisch M. Lancaster Jr., J.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10341-10346Crossref PubMed Scopus (186) Google Scholar) have proposed that, when NO⋅ is added as a bolus, it is possible that substantial amounts of N2O3 are formed from the reaction of NO⋅ with O2. Nevertheless, this conclusion contrasts with their further observation that the SNO-Hb yields were nearly identical when NO⋅ was added as a bolus or with the NO⋅ donor (Z)-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazenium 1,2-diolate (NHMA). Finally, from their experiments with oxyMb and NO⋅ in the presence of GSH, Zhang et al. (35Zhang Y. Hogg N. Free Radical Biol. Med. 2002; 32: 1212-1219Crossref PubMed Scopus (53) Google Scholar) also concluded that the addition of a bolus of NO⋅ causes mixing artifacts that facilitate the NO⋅/O2 reaction. Thus, analogously to Joshi et al. (34Joshi M.S. Ferguson Jr., T.B. Han T.H. Hyduke D.R. Liao J.C. Rassaf T. Bryan N. Feelisch M. Lancaster Jr., J.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10341-10346Crossref PubMed Scopus (186) Google Scholar), they suggest that GSNO is generated from the reaction of the nitrosating species N2O3with GSH. This conclusion is supported by the observation that, in contrast to the data reported by Joshi et al. (34Joshi M.S. Ferguson Jr., T.B. Han T.H. Hyduke D.R. Liao J.C. Rassaf T. Bryan N. Feelisch M. Lancaster Jr., J.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10341-10346Crossref PubMed Scopus (186) Google Scholar) but in analogy to those described in Han et al. (33Han T.H. Hyduke D.R. Vaughn M.W. Fukuto J.M. Liao J.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7763-7768Crossref PubMed Scopus (92) Google Scholar), the slow addition of NO⋅ with the NO⋅-donor 2-(N,N-diethylamino)diazenolate 2-oxide to an oxyMb/GSH solution did not result in detectable amounts of GSNO. In the present work, we carried out a systematic study of the influence of the following factors on the percentage of SNO-Hb generated relative to the amount of NO⋅ added to oxy-, deoxy-, and metHb solutions: 1) the volumetric ratio of the protein and the NO⋅ solutions; 2) the rate of addition of the NO⋅ solution to the protein solution; 3) the amount of NO⋅ added; and 4) the concentration of the phosphate buffer (0.1 versus 0.01m). For comparison, in some of the reactions, we substituted Hb with equimolar amounts of Mb mixed with 0.5 eq of GSH, to mimic the heme/Cys-β93 ratio in Hb. To be able to directly compare our results, we chose to keep the protein concentration constant in most of our experiments (final concentration 50 μm) and generally added 1 or 0.1 eq of NO⋅. Three different techniques were used to add the NO⋅ solution to the protein solution: the addition of a small volume of a saturated (2 mm) NO⋅solution (Method 1), the addition of an equivalent volume of a diluted NO⋅ solution (Method 2), either as fast as possible or very slowly, within 1–2 min. Buffer solutions (0.1 or 0.01 m) were prepared from K2HPO4/KH2PO4 (Fluka) with deionized Milli-Q water and always contained 0.1 mm diethylenetriaminepentaacetic acid (DTPA; Sigma). Sodium nitrite, sodium nitrate, sodium dithionite, potassium hexacyanoferrate(III), potassium cyanide, potassium permanganate, sulfanilamide,N-(1-naphthyl)-ethylenediaminedihydrochloride, ammonium sulfamate, and glutathione were obtained from Fluka. 4,4′-Dithiopyridine (4-PDS) was purchased from Aldrich. GSNO was prepared by the method of Hart (37Hart T.W. Tetrahedron Lett. 1985; 26: 2013-2016Crossref Scopus (386) Google Scholar). Briefly, reduced glutathione was allowed to react with sodium nitrite under acidic conditions at 0 °C, followed by the addition of cold acetone. The resulting precipitate was filtered off, washed, and dried under vacuum in the dark. The pink solid of GSNO, which had similar visible absorption maxima and extinction coefficients as reported by Hart (37Hart T.W. Tetrahedron Lett. 1985; 26: 2013-2016Crossref Scopus (386) Google Scholar), was stored at −80 °C in the dark. Nitrogen monoxide was obtained from Linde and passed through a NaOH solution as well as a column of NaOH pellets to remove higher nitrogen oxides before use. A saturated NO⋅ solution was prepared by degassing water for at least 1 h with argon and then saturating it with NO⋅ for at least 1 h. When needed, the obtained stock solution (∼2 mm) was diluted with degassed buffer in gas-tight SampleLock Hamilton syringes. The final NO⋅ concentrations were measured with an ANTEK Instruments nitrogen monoxide analyzer, with a chemiluminescence detector. Horse heart myoglobin was purchased from Sigma. metMb was prepared by oxidizing the protein with a small amount of potassium hexacyanoferrate(III). The solution was then purified over a Sephadex G-25 column by using a 0.1 mphosphate buffer solution (pH 7.0) as the eluant. The concentration of the metMb solutions was determined by measuring the absorbances at 408, 502, and/or 630 nm (ε408 = 188 mm−1 cm−1, ε502 = 10.2 mm−1 cm−1, and ε630 = 3.9 mm−1cm−1) (38Antonini E. Brunori M. Hemoglobin and Myoglobin in Their Reactions with Ligands. North-Holland, Amsterdam1971Google Scholar). oxyMb was prepared by reducing metMb with a slight excess of sodium dithionite. The solution was purified chromatographically on a Sephadex G-25 column by using a 0.1m phosphate buffer solution (pH 7.0) as the eluant. The concentration of the oxyMb solutions was determined by measuring the absorbances at 417, 542, and/or 580 nm (ε417 = 128 mm−1 cm−1, ε542 = 13.9 mm−1 cm−1, and ε580 = 14.4 mm−1cm−1) (38Antonini E. Brunori M. Hemoglobin and Myoglobin in Their Reactions with Ligands. North-Holland, Amsterdam1971Google Scholar). Purified human oxyHb stock solution (57 mg/ml solution of HbA0 with ∼1.1% metHb) was a kind gift from APEX Bioscience, Inc. The obtained solution was frozen in small aliquots (0.5–1 ml) and stored at −80 °C. oxyHb solutions were prepared by diluting the stock solution with buffer, and concentrations (always expressed per heme) were determined by measuring the absorbance at 415, 541, and/or 577 nm (ε415 = 125 mm−1 cm−1, ε541 = 13.8 mm−1 cm−1, and ε577 = 14.6 mm−1cm−1) (38Antonini E. Brunori M. Hemoglobin and Myoglobin in Their Reactions with Ligands. North-Holland, Amsterdam1971Google Scholar). metHb solutions were prepared by oxidizing oxyHb with a slight excess of potassium hexacyanoferrate(III). The solution was purified chromatographically on a Sephadex G-25 column by using a 0.1 m phosphate buffer solution (pH 7.0) as the eluant. The concentration of the metHb solutions (always expressed per heme) was determined by measuring the absorbances at 405, 500, and/or 631 nm (ε405 = 179 mm−1 cm−1, ε500 = 10.0 mm−1 cm−1, and ε631 = 4.4 mm−1cm−1) (38Antonini E. Brunori M. Hemoglobin and Myoglobin in Their Reactions with Ligands. North-Holland, Amsterdam1971Google Scholar). HbFe(II) (deoxyHb) solutions were prepared by thoroughly degassing oxyHb solutions with argon for at least 1 h without causing foaming of the solution. The concentration of the HbFe(II) solutions was determined by measuring the absorbance at 430 and/or 555 nm (ε430 = 133 mm−1cm−1 and ε555 = 12.5 mm−1 cm−1) (38Antonini E. Brunori M. Hemoglobin and Myoglobin in Their Reactions with Ligands. North-Holland, Amsterdam1971Google Scholar). metHbCN solutions were prepared by treating metHb with a slight excess of KCN. The solution was purified chromatographically on a Sephadex G-25 column by using a 0.1 m phosphate buffer solution (pH 7.0) as the eluant. The concentration of the metHbCN solutions (always expressed per heme) was determined by measuring the absorbances at 419 and/or 540 nm (ε419 = 124 mm−1cm−1 and ε540 = 12.5 mm−1 cm−1) (38Antonini E. Brunori M. Hemoglobin and Myoglobin in Their Reactions with Ligands. North-Holland, Amsterdam1971Google Scholar). SNO-Hb (36 mg/ml with an S-nitroso content of 850 μm;i.e. about 74% of Cys-β93 and metHb content of 10.7%) was a kind gift from APEX Bioscience, Inc. Absorption spectra were collected in 1 cm cells on a UVIKON 820 or on an Analytik Jena Specord 200. All reactions were carried out in phosphate buffer (0.1 or 0.01 m), pH 7.2, at room temperature. For Method 1, 2 ml of a protein solution (mostly ∼50 μm in 0.1 m phosphate buffer, pH 7.2, containing 0.1 mm DTPA) were placed in a 5-ml round bottom flask, which was then closed with a rubber septum. If anaerobic conditions were required, the solutions were thoroughly degassed with argon for 45–60 min. Depending on the equivalents of NO⋅required (mostly 1, 0.5, or 0.1 eq) 5–50 μl of a NO⋅-saturated solution were rapidly added to the oxyHb solution under constant stirring by using a Hamilton gas-tight syringe. For the experiments under aerobic conditions, particular care was taken to add the NO⋅ solution at the bottom of the flask to avoid any contact of NO⋅ with the oxygen present in the head space of the flask. Alternatively, the reaction was carried out in a sealable cell for anaerobic applications (Hellma). In this case, the required amount of a saturated NO⋅ solution (5–50 μl) was added to 2 ml of a protein solution while vortex-mixing. Method 2 used the same procedure as described for Method 1with the difference that the volumetric ratio of the protein and the NO⋅ solutions was always 1. In a typical experiment, 2 ml of a 100 μm protein solution (in 0.1 m phosphate buffer, pH 7.2, containing 0.1 mm DTPA) were mixed under constant stirring with 2 ml of a diluted NO⋅ solution (10–100 μm depending on the required equivalents). The diluted NO⋅ solutions were prepared by mixing in a gas-tight SampleLock Hamilton syringe the NO⋅-saturated solution with the required amounts of degassed buffer. In most cases, each experiment was carried out by adding the NO⋅ solution in two different ways: fast(in one shot as fast as possible) or slow (as slowly as possible, within 1–2 min). The reactions were carried out under aerobic conditions according to Method 1or 2. After the addition of the NO⋅ solution, the reaction mixtures were stirred for 10 min and then analyzed forS-nitrosated Cys-β93 and free Cys-β93, and, in some cases, a UV-visible spectrum was recorded. A control experiment was carried out to check that no artifactual S-nitrosation was taking place under the acidic conditions of the Saville assay. For this purpose, after treating the oxyHb solution with NO⋅, 100 μl of a 10 mm N-ethylmaleimide (NEM) solution in H2O were added and allowed to react for 20 min. Finally, the amount of S-nitrosated Cys-β93 was quantified. A series of experiments was carried out in the presence of an excess KCN. For this purpose, a solution containing oxyHb and either 10 or 100 eq of KCN (relative to the oxyHb concentration) was treated with 1 eq of NO⋅ according to Method 1 exactly as described above for the reaction in the absence of KCN. Most of the reactions were carried out in a sealable cell for anaerobic applications. The oxyHb solutions were thoroughly degassed for about 30 min, until the recorded UV-visible spectrum indicated the complete formation of deoxyHb. Alternatively, the reactions were carried out in round bottom flasks under anaerobic conditions according to Method 1 or2. After the addition of the NO⋅ solution, the reaction mixtures were stirred for 10 min. Then the flasks or the cells were opened, and the solutions were analyzed forS-nitrosated Cys-β93 (under aerobic conditions) immediately and/or after 10 min. In a control experiment, theS-nitrosated Cys-β93 content was determined under anaerobic conditions by carrying out the analysis in a sealable cell with thoroughly degassed Saville reagents. A series of experiments was carried out by adding an excess KCN before exposing the reaction mixtures to air. For this purpose, to 2 ml of deoxyHb (50 μm in 0.1 m phosphate buffer) we first added 100 μl of a NO⋅-saturated solution and, immediately after, 100 μl of a 10 mm KCN solution in water. The reaction mixture was stirred for 10 min, exposed to air, and, after 10 more min, analyzed with the Saville assay. An oxyHb solution (2 ml, 50 μm in 0.1m phosphate buffer, pH 7.2, containing 0.1 mmDTPA) was placed in a sealable cell and degassed for ∼30 min, until the recorded UV-visible spectrum indicated the formation of deoxyHb. Then 110 or 130 μl of an O2-saturated H2O solution were added until the recorded UV-visible spectrum matched a spectrum calculated for a 1:1 or a 3:1 mixture, respectively. The concentration of the two hemoglobin species present in solution was also controlled by using the equation given in Ref. 39Benesch R.E. Benesch R. Yung S. Anal. Biochem. 1973; 55: 245-248Crossref PubMed Scopus (368) Google Scholar. The solutions were then treated with a concentrated NO⋅ solution according toMethod 1. The reaction mixtures were stirred for 10 min. Finally, the cell was opened, and the solution was immediately analyzed for S-nitrosated Cys-β93 under aerobic conditions. The reactions were carried out both under aerobic and under anaerobic conditions according to Method 1 or 2. After the addition of the NO⋅ solution, the reaction mixtures were stirred for 30 min, degassed for 30–60 min (only for the anaerobic experiments), and analyzed for S-nitrosated Cys-β93 under aerobic conditions. Solutions containing twice the amount of Hb (50 μm oxyHb and 50 μm metHb) were mixed under aerobic conditions with 50 or 5 μm NO⋅ according to either Method 1 or Method 2 (fast and slow). The reaction mixtures were stirred for 30 min and then analyzed forS-nitrosated Cys-β93. To 2 ml of a solution containing 50 μm oxyHb and 50 μm metMb, 1 or 0.1 eq of NO⋅ were added from a saturated solution (Method 1) under aerobic conditions. The reaction mixture was stirred for 30 min and then analyzed forS-nitrosated Cys-β93. The addition of NO⋅ was performed under strictly anaerobic conditions according to Method 1 or 2. After 10 min, the solution was thoroughly degassed for 1 h and analyzed forS-nitrosated Cys-β93 under aerobic conditions. metMb was mixed with 0.5 eq of GSH (relative to the metMb concentration), treated with NO⋅, and analyzed exactly as described above for the reaction of metHb with NO⋅. oxyMb was mixed with 0.5 eq of GSH (relative to the oxyMb concentration), treated with NO⋅, and analyzed exactly as described above for the reaction of oxyHb with NO⋅. The S-nitrosothiol content was determined by the method of Saville (40Saville B. Analyst. 1958; 83: 670-672Crossref Google Scholar). Our NO⋅ solutions always contained variable amounts of nitrite (variable amounts between 0.1 and 1 eq, relative to the NO⋅ concentration). Thus, since the analysis of the S-nitrosothiol content is carried out under acidic conditions, we first eliminated excess nitrite by the addition of ammonium sulfamate (40Saville B. Analyst. 1958; 83: 670-672Crossref Google Scholar). For the analysis of theS-nitrosothiol content, two different sets of reagents were prepared. Diluted reagents include solution