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• W2061156310 abstract "Our previous results run counter to the hypothesis that S-nitrosohemoglobin (SNO-Hb) serves as anin vivo reservoir for NO from which NO release is allosterically linked to oxygen release. We show here that SNO-Hb undergoes reductive decomposition in erythrocytes, whereas it is stable in purified solutions and in erythrocyte lysates treated with an oxidant such as ferricyanide. Using an extensively validated methodology that eliminates background nitrite and stabilizes erythrocyte S-nitrosothiols, we find the levels of SNO-Hb in the basal human circulation, including red cell membrane fractions, were 46 ± 17 nm in human arterial erythrocytes and 69 ± 11 nm in venous erythrocytes, incompatible with the postulated reservoir function of SNO-Hb. Moreover, we performed experiments on human red blood cells in which we elevated the levels of SNO-Hb to 10,000 times the normal in vivo levels. The elevated levels of intra-erythrocytic SNO-Hb fell rapidly, independent of oxygen tension and hemoglobin saturation. Most of the NO released during this process was oxidized to nitrate. A fraction (25%) was exported as S-nitrosothiol, but this fraction was not increased at low oxygen tensions that favor the deoxy (T-state) conformation of Hb. Results of these studies show that, within the redox-active erythrocyte environment, the β-globin cysteine 93 is maintained in a reduced state, necessary for normal oxygen affinity, and incapable of oxygen-linked NO storage and delivery. Our previous results run counter to the hypothesis that S-nitrosohemoglobin (SNO-Hb) serves as anin vivo reservoir for NO from which NO release is allosterically linked to oxygen release. We show here that SNO-Hb undergoes reductive decomposition in erythrocytes, whereas it is stable in purified solutions and in erythrocyte lysates treated with an oxidant such as ferricyanide. Using an extensively validated methodology that eliminates background nitrite and stabilizes erythrocyte S-nitrosothiols, we find the levels of SNO-Hb in the basal human circulation, including red cell membrane fractions, were 46 ± 17 nm in human arterial erythrocytes and 69 ± 11 nm in venous erythrocytes, incompatible with the postulated reservoir function of SNO-Hb. Moreover, we performed experiments on human red blood cells in which we elevated the levels of SNO-Hb to 10,000 times the normal in vivo levels. The elevated levels of intra-erythrocytic SNO-Hb fell rapidly, independent of oxygen tension and hemoglobin saturation. Most of the NO released during this process was oxidized to nitrate. A fraction (25%) was exported as S-nitrosothiol, but this fraction was not increased at low oxygen tensions that favor the deoxy (T-state) conformation of Hb. Results of these studies show that, within the redox-active erythrocyte environment, the β-globin cysteine 93 is maintained in a reduced state, necessary for normal oxygen affinity, and incapable of oxygen-linked NO storage and delivery. iron-nitrosyl-hemoglobin S-nitroso hemoglobin N-ethylmaleimide phosphate-buffered saline analysis of variance nitrite and nitrate diethylenetriaminepentaacetic acid Nitric oxide (NO) is a soluble gas that is continuously synthesized in endothelial cells and is a critical endogenous vasodilator (1Furchgott R.F. Zawadzki J.V. Nature. 1980; 288: 373-376Crossref PubMed Scopus (10026) Google Scholar, 2Ignarro L.J. Buga G.M. Wood K.S. Byrns R.E. Chaudhuri G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 9265-92659Crossref PubMed Scopus (4363) Google Scholar, 3Palmer R.M. Ferrige A.G. Moncada S. Nature. 1987; 327: 524-526Crossref PubMed Scopus (9366) Google Scholar). Although it is generally believed that endothelium-derived NO is the primary determinant of NO-mediated control of basal blood flow in humans (4Gladwin 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, 5Cannon 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), considerable recent interest and controversy have focused on the role of intravascular NO-derived molecules that could stabilize NO bioactivity and contribute to blood flow and oxygen delivery. Such molecules include high and low molecular weight S-nitrosothiols in plasma (6Stamler J.S. Jaraki O. Osborne J. Simon D.I. Keaney J. Vita J. Singel D. Valeri C.R. Loscalzo J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7674-7677Crossref PubMed Scopus (1133) Google Scholar, 7Fang K. Ragsdale N.V. Carey R.M. MacDonald T. Gaston B. Biochem. Biophys. Res. Commun. 1998; 252: 535-540Crossref PubMed Scopus (111) Google Scholar, 8Gladwin 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, 9Jourd'heuil D. Gray L. Grisham M.B. Biochem. Biophys. Res. Commun. 2000; 273: 22-26Crossref PubMed Scopus (78) Google Scholar, 10Marley R. Feelisch M. Holt S. Moore K. Free Radical Res. 2000; 32: 1-9Crossref PubMed Scopus (152) Google Scholar) and nitrite (4Gladwin 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, 11Li H. Samouilov A. Liu X. Zweier J.L. J. Biol. Chem. 2001; 276: 24482-24489Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). In addition, NO reacts reversibly with hemoglobin to form an NO-heme adduct, iron-nitrosyl-hemoglobin (HbFeIINO),1 and can also nitrosate a surface thiol on cysteine 93 of the β-globin chain to form S-nitrosohemoglobin (SNO-Hb). The potential role of S-nitrosated hemoglobin as an NO transporter is particularly appealing, because the environment of β-cysteine 93 is sensitive to the R ↔ T conformational equilibrium of hemoglobin. The conformational transition from the R- to T-state could thus promote the allosteric delivery of both oxygen and NO to regions with low oxygen tension. Two central observations supporting this SNO-Hb hypothesis are reports of observed arterial-venous gradients of SNO-Hb in the rat (suggesting a dynamic cycle) and evidence that delivery of oxygen and NO are allosterically coupled events (12Jia L. Bonaventura C. Bonaventura J. Stamler J.S. Nature. 1996; 380: 221-226Crossref PubMed Scopus (1480) Google Scholar, 13Stamler J.S. Jia L., Eu, J.P. McMahon T.J. Demchenko I.T. Bonaventura J. Gernert K. Piantadosi C.A. Science. 1997; 276: 2034-2037Crossref PubMed Scopus (959) Google Scholar, 14Gow A.J. Stamler J.S. Nature. 1998; 391: 169-173Crossref PubMed Scopus (517) Google Scholar). However, the evidence for a dynamic cycle is brought into question by widely varying reports for the basal levels of intracellular SNO-Hb in arterial and venous blood. The reported levels vary from 200 nm to 5 μm. These levels were determined using a variety of different assays, mostly based on photolysis or chemically mediated release of NO gas and subsequent detection by the ozone-based chemiluminescent analyzer (8Gladwin 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, 9Jourd'heuil D. Gray L. Grisham M.B. Biochem. Biophys. Res. Commun. 2000; 273: 22-26Crossref PubMed Scopus (78) Google Scholar, 12Jia L. Bonaventura C. Bonaventura J. Stamler J.S. Nature. 1996; 380: 221-226Crossref PubMed Scopus (1480) Google Scholar, 15Funai E.F. Davidson A. Seligman S.P. Finlay T.H. Biochem. Biophys. Res. Commun. 1997; 239: 875-877Crossref PubMed Scopus (67) Google Scholar). We have developed methodologies to selectively oxidize the NO first from iron-nitrosyl-hemoglobin followed by reduction of the S-NO bond from hemoglobin in solutions of I3−, releasing NO gas from the cysteine for ozone-based chemiluminescent detection (4Gladwin 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, 5Cannon 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, 8Gladwin 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), whereas other laboratories first cleave theS-NO linkage with mercury and then measure the NO levels (with and without mercury) using ultraviolet light photolysis (12Jia L. Bonaventura C. Bonaventura J. Stamler J.S. Nature. 1996; 380: 221-226Crossref PubMed Scopus (1480) Google Scholar, 16McMahon T.J. Exton Stone A. Bonaventura J. Singel D.J. Stamler J. J. Biol. Chem. 2000; 275: 16738-16745Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). In addition to liberating NO gas from both S-nitrosothiols and iron-nitrosyls, both systems reduce nitrite to NO, requiring extensive treatment of samples through molecular sizing columns. However, hemoglobin possesses anion binding sites that may retain nitrite, raising concerns that the measured NO levels may be overestimated as a result of conversion of hemoglobin-bound nitrite to NO (17Imai K. Imai K. Allosteric Effects in Haemoglobin. Cambridge University Press, Cambridge, United Kingdom1982: 39-45Google Scholar). In addition, there have been a number of challenges to the second core principle of the SNO-Hb hypothesis, that NO is released during the oxygen-linked conformational shift of hemoglobin from its R- to T-state. Although both kinetic and thermodynamic arguments have been made to support the allosterically mediated release of NO from SNO-Hb (12Jia L. Bonaventura C. Bonaventura J. Stamler J.S. Nature. 1996; 380: 221-226Crossref PubMed Scopus (1480) Google Scholar, 14Gow A.J. Stamler J.S. Nature. 1998; 391: 169-173Crossref PubMed Scopus (517) Google Scholar, 16McMahon T.J. Exton Stone A. Bonaventura J. Singel D.J. Stamler J. J. Biol. Chem. 2000; 275: 16738-16745Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), the physiological relevance of this possible linkage has been challenged on the basis of the very high oxygen affinity of SNO-Hb, potentially limiting its role in basal regulation of NO/oxygen delivery (18Bonaventura C. Ferruzzi G. Tesh S. Stevens R.D. J. Biol. Chem. 1999; 274: 24742-24748Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 19Patel R.P. Hogg N. Spencer N.Y. Kalyanaraman B. Matalon S. Darley-Usmar V.M. J. Biol. Chem. 1999; 274: 15487-15492Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 20Wolzt M. MacAllister R.J. Davis D. Feelisch M. Moncada S. Vallance P. Hobbs A.J. J. Biol. Chem. 1999; 274: 28983-28990Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), and the oxygen-independent kinetics of the reaction of SNO-Hb with millimolar levels of glutathione, present at such concentration in erythrocytes (16McMahon T.J. Exton Stone A. Bonaventura J. Singel D.J. Stamler J. J. Biol. Chem. 2000; 275: 16738-16745Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 19Patel R.P. Hogg N. Spencer N.Y. Kalyanaraman B. Matalon S. Darley-Usmar V.M. J. Biol. Chem. 1999; 274: 15487-15492Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 20Wolzt M. MacAllister R.J. Davis D. Feelisch M. Moncada S. Vallance P. Hobbs A.J. J. Biol. Chem. 1999; 274: 28983-28990Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 21Deem S. Gladwin M.T. Berg J.T. Kerr M.E. Swenson E.R. Am. J. Respir. Crit. Care Med. 2001; 163: 1164-1170Crossref PubMed Scopus (24) Google Scholar). In the present studies we present a methodology for the stabilization SNO-Hb in red cell lysates, the elimination of all background nitrite signal, and the specific detection of SNO-Hb in human blood down to 5 nm concentration (0.00005% SNO/heme). Very low levels of SNO-Hb were found in both arterial and venous erythrocytes with this improved detection method, and no significant arterial-venous gradients were observed. To evaluate the allosteric properties of SNO-Hb within intact erythrocytes, we modified human red cells so that they contained up to 10,000 times the normal in vivo levels of SNO-Hb and evaluated the effects of hemoglobin oxygen saturation on its levels and on NO export. In these experiments we found that, within the redox-active erythrocyte environment, the β-globin cysteine 93 is maintained in a reduced state, which may be necessary for normal oxygen affinity, and that SNO-Hb is rapidly degraded independent of hemoglobin oxygen saturation. The reductive decomposition of SNO-Hb in the erythrocytes provides an explanation for why SNO-Hb levels in human blood are low. Because the lifetime of SNO-Hb in erythrocytes is both transient and insensitive to oxygen tension, its participation in NO storage, delivery, and regulation of human blood flow under normal physiological conditions appears unlikely. It remains possible that SNO-Hb may participate in NO-dependent events in vivo during pathological conditions associated with red cell oxidation or under circumstances where NO generation is increased in response to infection or pharmacological NO treatment. All protocols were approved by the Institutional Review Board of the NHLBI (National Institutes of Health, Bethesda, MD) following formal scientific review. All subjects signed an informed consent. All chemicals and NO gas, unless otherwise stated, were obtained from Sigma-Aldrich. Hemolysates of cells containing native human hemoglobin (Hb A0) were used to prepare purified Hb by the ammonium sulfate method with chromatographic purification with a fast protein liquid chromatography system. Pure red cell lysates from freshly obtained venous or arterial blood were used when indicated. S-Nitrosothiol reagents were prepared by reaction of the acidified amino acid with nitrite, neutralized to pH 7.4 using 5 n NaOH and measured by both the I3− (22Samouilov A. Zweier J.L. Anal. Biochem. 1998; 258: 322-330Crossref PubMed Scopus (110) Google Scholar) and Cu+/l-cysteine (7Fang K. Ragsdale N.V. Carey R.M. MacDonald T. Gaston B. Biochem. Biophys. Res. Commun. 1998; 252: 535-540Crossref PubMed Scopus (111) Google Scholar) chemiluminescence assays, and compared with SNO-glutathione (Calbiochem) and nitrite standards. Oxyhemoglobin was equilibrated in 2% borate buffer at pH 9.2 or in PBS at pH 7.4, temperature 4 °C, and then treated with S-nitrosocysteine at a ratio of 10:1 (CysNO to heme) for 30 min, followed by extensive dialysis or Sephadex G25 desalting (8Gladwin 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, 18Bonaventura C. Ferruzzi G. Tesh S. Stevens R.D. J. Biol. Chem. 1999; 274: 24742-24748Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 23McMahon T.J. Stamler J.S. Methods Enzymol. 1999; 301: 99-114Crossref PubMed Scopus (69) Google Scholar). Human and bovine albumin (Sigma; 40 mg/ml with 0.5 μm EDTA in PBS) was reduced with dithiothreitol (3–5 mm); passed through Sephadex G25 columns to remove dithiothreitol, nitrite, and small thiols; and incubated with S-nitrosocysteine (final concentration at 2 mm) for 30 min at room temperature in the dark to form SNO-albumin. This solution was then dialyzed at 4 °C against 3 × 3 liters of 0.5 μm diethylenetriaminepentaacetic acid (DTPA) in PBS for 24 h. Whole blood was collected from normal volunteers in EDTA followed by an overnight incubation in room air with a 10% volume of 100 ml of phosphate-buffered saline with citrate, dextrose, and adenine (CPD-A solution; Baxter, Covina, CA). Blood samples were centrifuged at 750 × g for 5 min and adjusted to a 50% hematocrit by removal of small amounts of plasma. To elevate intra-erythrocytic SNO-Hb levels, S-nitrosocysteine (10 mm final concentration) was incubated with these whole blood samples for 3 h at 4 °C (on ice). To ensure the removal of all of theS-nitrosocysteine, the cells were centrifuged, plasma and buffy coat discarded, and cells then washed with a 25-fold excess 4 °C PBS for 5 min. The PBS was removed and the wash repeated for a total of five 5-min washes. The final wash PBS was collected, and the residual S-nitrosothiol in the wash was less than 50 nm. Red blood cells prepared by this methodology contained 1.1 ± 0.2% SNO/heme (220 μm intracellular SNO-Hb), 0% iron-nitrosyl-hemoglobin, and 8.7% methemoglobin. To measureS-nitrosothiol and nitrite levels, standards of SNO-Hb or SNO-albumin (prepared as described above) were added to 1.7-ml reaction tubes cooled on ice. After cooling, 100 μl of sulfanilamide/HgCl2 solution (16% sulfanilamide, 0.2% HgCl2 in 2 m HCl), or sulfanilamide alone, was added to the reaction mixtures followed by a balance of PBS/DTPA (100 μm) to make a final volume of 1100 μl. After 5 min of incubation in the dark at room temperature, 100 μl of 1.6%N-(1-naphthyl)ethenediamine in 2 m HCl was added to the reaction mixture and again the sample was allowed to incubate in the dark for 5 min. Following the second incubation, the reaction mixtures were centrifuged at 17,900 × g for 5 min, and 1 ml of supernatants were transferred to optical cuvettes to measure absorbance at 540 nm. Subtraction of appropriate blanks and of values for samples treated without HgCl2 determined the levels ofS-nitrosothiol without nitrite. The concentration ofS-nitrosothiol or nitrite was calculated using the extinction coefficient of 50,000 m−1cm−1 and with standards of known concentrations of GSNO. Different chemical reagents are mixed in a glass purge vessel. Helium gas is bubbled sequentially through the purge vessel, through 15 ml of 1 mNaOH, and then into the chemiluminescent nitric oxide analyzer (model 280 NO analyzer; Sievers, Boulder, CO), which can detect upward from 0.3 pmol of NO gas. The following chemicals were placed in the purge vessel for the experiments described herein. The method for the measurement of nitrite and S-nitrosothiols by reaction with I3− to release NO gas (22Samouilov A. Zweier J.L. Anal. Biochem. 1998; 258: 322-330Crossref PubMed Scopus (110) Google Scholar) was applied to hemoglobin (8Gladwin 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). In brief, 7 ml of glacial acetic acid and 2 ml of distilled water were mixed with 100 mg of KI. A crystal of I2 was added to yield a concentration of ∼20–30 mm. Alternatively, to allow for the injection of large volumes of solutions with high protein concentration, a stock solution of I3− was prepared each day. Serial injections of S-nitrosoglutathione and nitrite were used over 4 h to document the stability of the I3− stock solution exposed to air. There was no decrease in NO release from S-nitrosothiols over this time period. This allowed us to change the reagent prior to each injection of hemoglobin solutions and removed variability produced by increasing protein concentrations and foaming. To specifically identify nitrite versus S-nitrosothiol, samples were incubated with and without acidified sulfanilamide (as described below) and reacted in the I3− reductant. Specific treatment of red cell and hemoglobin samples prior to reaction in the I3− reductant is described below. Nitrate was measured by reduction in vanadium(III) at 90 °C (24Ewing J.F. Janero D.R. Free Radical Biol. Med. 1998; 25: 621-628Crossref PubMed Scopus (60) Google Scholar). To reduce foaming during the analysis of nitrate levels in plasma, the samples were treated with a 2:1 volume of cold ethanol and centrifuged at 17,900 × g for 5 min. The value for nitrate was determined by subtraction of the nitrite and S-nitrosothiol values, derived by I3− assay, from the result derived from vanadium assay. To eliminate nitrite contamination from human hemoglobin samples, Sephadex G25 desalted samples were reacted with and without 5 mmHgCl2 and then treated with a 10% volume of 5% sulfanilamide in 1 n HCl as described under “Results” (10Marley R. Feelisch M. Holt S. Moore K. Free Radical Res. 2000; 32: 1-9Crossref PubMed Scopus (152) Google Scholar). These solutions completely abolished contaminating nitrite signal, whereas purified SNO-Hb was completely stable. The levels of SNO-Hb after acidified sulfanilamide treatment as measured by the I3− chemiluminescent assay were the same as the levels measured by the Griess-Saville assay after subtraction of the nitrite background, suggesting that some nitrite is associated with preparations of SNO-Hb even after extensive dialysis or column separation. Red blood cell pellet samples (100 μl) were lysed in 900 μl of PBS with KCN/K3FeIII(CN)6 (4 mm), NEM (10 mm), DTPA (100 μm), and 1% Nonidet P-40 (to solubilize membrane, which may containS-nitrosated anion exchange protein 1 (Ref. 25Pawloski J.R. Hess D.T. Stamler J.S. Nature. 2001; 409: 622-626Crossref PubMed Scopus (505) Google Scholar)). The pH of this solution was 7.2. Lysed red blood cell samples were incubated in this solution at room temperature for 5 min and 500 μl passed through a prerinsed 9.5-ml bed volume Sephadex G25 column. The hemoglobin aliquots from 3–3.7 ml were collected, separated into two fractions (270 μl each), and reacted with and without HgCl2 (final concentration 5 mm) for 2 min. 10% volume of 5% sulfanilamide in 1 n HCl was added to the hemoglobin solutions and incubated for 5 min (final concentration of 0.5% sulfanilamide in 0.1 n HCl). 300-μl volumes of the hemoglobin solutions, with and without HgCl2 treatment (to specify S-nitrosothiol), were injected into 8 ml of I3− solution in the reaction chamber (see “I3− Reagent”). The I3− was replaced for each injection from a stock solution prepared fresh each day and no antifoam agent was used. The quantity of NO released was determined by calculation of the area under the curve (see “Statistical Analysis”), corrected for the dilution of added HgCl2 and acidified sulfanilamide, and the value divided by the concentration of heme measured spectrally in Drabkin's reagent (prior to addition of HgCl2 and acidified sulfanilamide). For the determination of the in vivolevels of red blood cell SNO, whole blood was drawn from 8 normal volunteers from the artery and vein into vacuum containers with sodium heparin (to preserve physiological arterial and venous hemoglobin oxygen saturations). Samples were spun at 750 × g for 5 min and plasma discarded. 100 μl of the red cell pellet below the buffy coat was removed and immediately lysed in 900 μl of PBS with 1% Nonidet P-40, KCN/K3FeIII(CN)6(4 mm), NEM (10 mm), and DTPA (100 μm). The pH of this solution was 7.2. Following Sephadex G25 desalting, hemoglobin concentration was measured in Drabkin's reagent, and samples were reacted with and without 5.0 mmHgCl2 for 2 min, then with 10% volume of 5% sulfanilamide in 1 n HCl for 3 min, followed by NO measurement by I3− chemiluminescent assay. Erythrocytes containing elevated levels of SNO-Hb were prepared as described above. Only samples with less than 10% methemoglobin were used. One ml of pre-agitated (to ensure mixing) erythrocyte suspension was added to 9 ml of buffer in two tonometers. As described under “Results,” the tonometers alternatively contained human plasma collected from the same individual who donated the red cells, PBS with 24 mm NaHCO3 (distilled water added to keep sodium concentration 140 mm), and PBS with 24 mm NaHCO3 and 400 μm GSH. GSH was added as a thiol acceptor for potential erythrocyte-derived trans-nitrosation reactions (26Lipton A.J. Johnson M.A. Macdonald T. Lieberman M.W. Gozal D. Gaston B. Nature. 2001; 413: 171-174Crossref PubMed Scopus (286) Google Scholar). One tonometer was pre-equilibrated for 60 min with 21% oxygen and 5% CO2 gases and the second tonometer with 0% oxygen and 5% CO2. The tonometers were protected from light and agitated on a rocker platform. Following the addition of cells, 1-ml samples were withdrawn every 5 min using Hamilton syringes. Samples were added to 8 mm NEM and 100 μm DTPA to stabilize S-nitrosothiols (10Marley R. Feelisch M. Holt S. Moore K. Free Radical Res. 2000; 32: 1-9Crossref PubMed Scopus (152) Google Scholar) and centrifuged at 750 × g for 5 min. The cells and buffer were separated and flash-frozen on dry ice. The buffer nitrite, nitrate, and S-nitrosothiol content and red cell SNO-Hb was subsequently measured by chemiluminescence (as described above). Samples were also withdrawn at the same 5-min intervals for determination of hemoglobin saturation (by co-oximetry), pO2, pCO2, and pH (i-STAT Corp., East Windsor, NJ). Methemoglobin concentrations were measured by absorption spectroscopy at 700, 630, 576, and 560 nm using the Winterbourn relationship (27Winterbourn C.C. Methods Enzymol. 1990; 186: 265-272Crossref PubMed Scopus (398) Google Scholar). Oxygen dissociation curve measurements of SNO-Hb-containing red blood cells were performed using the Hemox-Analyzer (TCS Scientific Corp., New Hope, PA) (28Gladwin M.T. Schechter A.N. Shelhamer J.H. Pannell L.K. Conway D.A. Hrinczenko B.W. Nichols J.S. Pease-Fye M.E. Noguchi C.T. Rodgers G.P. Ognibene F.P. J. Clin. Invest. 1999; 104: 937-945Crossref PubMed Scopus (97) Google Scholar). We have found that the Sievers analytical software (model 280 NO analyzer) produces some error when measuring the area under the curve for samples with 0.3–5 pmol of NO. We therefore transferred raw data from the Sievers program to Origin (Microcal Software, Inc., Northampton, MA). The data were smoothed using the Savitzky-Golay filter method provided with the software (symmetric, 21-point window; polynomial degree = 2). Two-tailed paired t tests were used to analyze differences in the values of paired samples before, during, and after experiments. For time-course experiments, the data were analyzed by repeated measures analysis of variance (ANOVA). Data are reported as the mean ± the standard error of the mean. To validate our standards and improved chemiluminescent methodologies, preparations of SNO-Hb and SNO-albumin were analyzed by both the I3− chemiluminescent assay and by the Griess-Saville assay. As shown in Fig. 1(A and B), the I3−chemiluminescent assay compared favorably with the well established Griess-Saville assay over a wide range of concentrations for both SNO-Hb and SNO-albumin. To determine the limits of sensitivity, purified samples of synthesized SNO-Hb were added to freshly drawn, washed (in PBS), lysed erythrocyte pellets at concentrations from 200 nm (0.002% S-NO/heme) to 3,200 nm(0.032% S-NO/heme). Following treatment with ferricyanide and cyanide (described below) to eliminate iron-nitrosyl signal and to stabilize SNO-Hb in red blood cell lysates, the hemoglobin samples were passed through a prerinsed G25 Sephadex sizing column and injected into the I3− reagent. The NO recovery was stoichiometric with an r 2 value of 0.996 (Fig.1, C and D; p < 0.001;n = 4). Levels of SNO-Hb measured by other laboratories using UV photolysis have been reported to be as high as 0.001/hemoglobin tetramer or 2.5 μm in whole blood. As can be seen in Fig. 1 (C and D), such levels would be readily measured using our assay. We have used solutions of KCN and K3FeIII(CN)6to selectively oxidize the NO from the heme while preserving theS-nitrosothiol linkage. This sample treatment allows us to first remove the NO bound to the heme group so that we can specifically measure the NO release from cysteine 93 in the I3− chemiluminescence-based assay. The standards of SNO-Hb shown in Fig. 1 (C and D) were pretreated with KCN/K3FeIII(CN)6 prior to reaction in the I3− chemiluminescence-based assay (8Gladwin 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). SNO-Hb is stable in a large molar excess of KCN and K3FeIII(CN)6. This was shown using three approaches. In the first experiment (Fig.2 A), 10-μl volumes of solutions containing SNO-Hb (0.9 mm SNO), nitrite (10 mm), nitrate (10 mm), and HbFeIINO (0.67 mm) were injected into a purge vessel containing 0.2m KCN/K3FeIII(CN)6 in PBS. The reaction chamber was purged with helium in-line with the chemiluminescent NO analyzer to detect released NO gas. As shown in Fig. 2 A, SNO-Hb, nitrite, and nitrate are stable in this solution (i.e. do not release appreciable amounts of NO), whereas NO gas is released from HbFeIINO. The small NO signal from the injection of SNO-Hb derives from small amounts of iron-nitrosyl-hemoglobin formed in the synthesis of SNO-Hb (see small mercury-stable peak in Fig. 2 B). In the second set of experiments, SNO-Hb was measured using the I3−chemiluminescence-based assay with and without cyanide/ferricyanide and HgCl2 pretreatments. Briefly, 10 μl of 0.9 mmSNO-Hb was incubated for 30 min in 90 μl of PBS, 90 μl of 0.2m KCN/K3FeIII(CN)6 in PBS, or 90 μl of 0.5 mm HgCl2 and then passed through a Sephadex G25 sizing column. The samples were then injected into a reaction vessel containing the I3−reagent. The reaction chamber was purged with helium in-line with the chemiluminescent NO analyzer to detect released NO gas. As shown in Fig. 2 B, there is no loss of SNO-Hb that has been incubated in a large molar excess of KCN/K3FeIII(CN)6, in the absence of HgCl2, whereas it is completely decomposed by incubation with HgCl2. In the final experiment, SNO-H" @default.
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• W2061156310 title "S-Nitrosohemoglobin Is Unstable in the Reductive Erythrocyte Environment and Lacks O2/NO-linked Allosteric Function" @default.
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