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Blood, 1 April 2006, Vol. 107, No. 7, pp. 2602-2604. Prepublished online as a Blood First Edition Paper on December 8, 2005; DOI 10.1182/blood-2005-10-4104. This Article Abstract Full Text (PDF) All Versions of this Article: 2005-10-4104v1 107/7/2602    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Huang, K. T. Articles by Kim-Shapiro, D. B. Search for Related Content PubMed PubMed Citation Articles by Huang, K. T. Articles by Kim-Shapiro, D. B. Related Collections Red Cells Brief Reports Related Letters in Blood Online Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  PLENARY PAPERS Lack of allosterically controlled intramolecular transfer of nitric oxide from the heme to cysteine in the subunit of hemoglobin Kris T. Huang, Ivan Azarov, Swati Basu, Jinming Huang, and Daniel B. Kim-Shapiro From the Department of Biomedical Engineering, Wake Forest University School of Medicine; and Department of Physics and Department of Chemistry, Wake Forest University, Winston-Salem, NC.    Abstract Top Abstract Introduction Study design Results and discussion References   The SNO-Hb hypothesis holds that heme-bound nitric oxide (NO) present in the subunits of T-state hemoglobin (Hb) will be transferred to the -93 cysteine upon conversion to R-state Hb, thereby forming SNO-Hb. A deficiency in the ability of Hb to facilitate this intramolecular transfer has recently been purported to play a role in pulmonary hypertension and sickle cell disease. We prepared deoxygenated Hb samples with small amounts of heme-bound NO and then oxygenated the samples. Electron paramagnetic resonance (EPR) spectroscopy was used to (1) determine the concentration of iron nitrosyl Hb (Fe-NO Hb), (2) show that the NO is evenly distributed among and subunits, and (3) show that the Hb undergoes a change in its quaternary state (T to R) upon oxygenation. We did not observe a decrease in the concentration of Fe-NO Hb on oxygenation, which is inconsistent with the prediction of the SNO-Hb hypothesis.    Introduction Top Abstract Introduction Study design Results and discussion References   In 1996, Stamler's group1 published a revolutionary work stating that, rather than simply destroying NO activity through the deoxygenation reaction, hemoglobin (Hb) is capable of preserving, transporting, and exporting NO activity. It has been proposed that these novel activities of Hb are mediated by SNO-Hb, which is stabilized in the (high-oxygen affinity) R-state and destabilized in the (low-oxygen affinity) T-state.1-3 NO binds rapidly to the heme of deoxygenated Hb to form Fe-NO Hb. According to the SNO-Hb hypothesis, when Fe-NO Hb is oxygenated (and undergoes a T to R transition) some of the NO on the heme is transferred to the -93 cysteine to form SNO-Hb.2-4 Upon re-deoxygenation, some of the NO (actually NO+) on the cysteine is transferred back to heme and some is exported by the red blood cell to effect vasodilation.2-5 The amount of heme-bound NO that undergoes allosterically controlled intramolecular transfer is claimed to be dependent on the amount of NO bound per hemoglobin tetramer.2,3 Previously, a decrease in yield of SNO-Hb upon oxygenation of an initially deoxygenated Fe-NO Hb sample was attributed to loss of allosteric control when the NO/Hb ratio was too high.2 More recently, the SNO-Hb hypothesis has been refined wherein it is stated that only NO bound to hemes of the subunits (to form -nitrosyl Hb) can efficiently undergo allosterically controlled intramolecular transfer.3,6 Due to the relative dissociation rates, -nitrosyl Hb is more stable than -nitrosyl Hb.7-9 In the T-state, a percentage of the -nitrosyl proximal histidine bonds break forming pentacoordinate -nitrosyl Hb (as opposed to the hexacoordinate form) that has a characteristic hyperfine splitting signature in electron paramagnetic resonance (EPR).8,10 The efficiency of transfer from heme to thiol was predicted to be -nitrosyl > hexacoordinate -nitrosyl > pentacoordinate -nitrosyl.6 It has recently been suggested that inability of Hb to facilitate allosterically controlled intramolecular transfer contributes to the pathology of certain diseases. The mutation in sickle cell Hb was suggested to impede intramolecular transfer.11 In addition, sustained hypoxia purported to be associated with pulmonary hypertension was suggested to result in the build up of -nitrosyl species with concomitant loss of -nitrosyl Hb and SNO-Hb.6 Working together with other laboratories on oxygen cycling-induced changes in heme nitrosylation/S-nitrosation, we did not find evidence for allosterically controlled intramolecular transfer.12 Our observations refuting the idea of allosterically controlled intramolecular transfer were criticized based on there being (at least in some cases) unequal distribution of NO among Hb tetramers and prolonged incubations, which would tend to result in a buildup of -nitrosyl species.3 The current study was designed to test whether allosterically controlled intramolecular transfer occurs when samples are prepared to maximize the amount of -nitrosyl Hb and to equally distribute small amounts of NO among Hb tetramers.    Study design Top Abstract Introduction Study design Results and discussion References   Normal adult Hb from red blood cells was obtained as described previously.13,14 Hb samples were deoxygenated by gentle flow of an inert gas and stirring. All experiments were performed at room temperature. Determination of percentages of oxygenated, deoxygenated, and methemoglobin was performed using near infrared spectroscopy.13 SNO-Hb standards for validation studies were prepared as described previously.15 The CO/CuCl/cysteine (3C) assay was also used for validation studies as described.16 The NO donor ProliNO (Cayman Chemical, Ann Arbor, MI) was prepared in 0.01 M NaOH and a small aliquot was rapidly mixed with the deoxygenated Hb for a final concentration of NO of about 5 µM. The half-life of ProliNO is 2 seconds at 37°C, which allowed NO to be evenly distributed among Hb tetramers in our experiments. After 1 minute, aliquots of the sample were frozen for EPR analysis and prepared for measurements of SNO-Hb using SNO stabilization solution and the tri-iodide chemiluminescence method as described previously.12,17 EPR using a Bruker EMX 10/12 (Bruker, Billerica, MA) was performed as described previously.18 Samples were oxygenated by exposure to air and gentle stirring for 5 minutes after which they were assayed for oxygen saturation, Fe-NO Hb, and SNO-Hb. The amount of Fe-NO Hb and its distribution among hexa/pentacoordinate species and subunits was analyzed by fitting to basis spectra and performing a double integral as described previously.19    Results and discussion Top Abstract Introduction Study design Results and discussion References   Although EPR is widely accepted as a sensitive and specific method to measure Fe-NO Hb, there has been some disagreement over the accuracy of the tri-iodide method in determining the concentration of SNO-Hb.20 Figure 1A shows that the tri-iodide method compares well with another recently developed assay.16 In Figure 1B, we show that the tri-iodide method is capable of accurately measuring SNO-Hb in the range of concentrations and NO/Hb ratios expected in our oxygenation studies as previously demonstrated.15 View larger version (19K): [in this window] [in a new window]   Figure 1.. Validation of the tri-iodide method. (A) A SNO-Hb standard sample that was 125 µM in heme and 59 µM in nitrosated thiol was serially diluted 1:1 reaching a final concentration of 0.9 µM (SNO). For each concentration, the sample was split in 2 and assayed by the tri-iodide method () or the 3C method (). The measured concentration of SNO is plotted against the expected amount based on the amount that the initial sample was diluted. The solid line shows the predicted amount. The initial concentration of SNO-Hb was taken as the average of that using the 3C and tri-iodide method (57 µM and 60 µM). (B) A SNO-Hb standard sample (57 µM in SNO and 1 mM in heme) was spiked into high concentrations of NO-free Hb with a heme concentration 5 mM to give expected final concentrations of SNO between 1 and 12 µM (SNO/heme between 0.00025 and 0.003). The amount of SNO-Hb in the spiked samples was measured using the tri-iodide method and plotted against the expected values.   View larger version (21K): [in this window] [in a new window]   Figure 2.. Lack of allosterically controlled intramolecular transfer. (A) Typical EPR spectra of Hb before and after oxygenation. The triplet hyperfine structure characteristic of pentacoordinate -nitrosyl Hb present in the deoxygenated sample (deoxy Hb) disappears upon oxygenation (oxyHb). For this sample, the total Hb concentration was 4.5 mM and was 99.6% deoxygenated when ProliNO was added. Before oxygenation the total Fe-NO Hb was 3.8 µM, of which 53% was on the subunits and 24% was of the pentacoordinate -nitrosyl form. After oxygenation, the total Fe-NO Hb was measured to be 4.1 µM, of which 47% was on the subunits and 0% was of the pentacoordinate -nitrosyl form. (B) Measured changes in Fe-NO Hb and SNO-Hb versus those predicted by the SNO-Hb hypothesis. The predictions are shown assuming 80% transfer from heme to thiol. Thus, the prediction is that the amount of Fe-NO Hb would go from 3.7 µM to 0.74 µM upon oxygenation, giving a percentage change of 2.96/3.7 = 80%. The SNO-Hb is predicted to go from 0.66 µM to 3.62 µM (2.96 µM is transferred from the heme iron), giving a percentage change of 2.96/0.66 = 448%. These predictions are based on data presented by Gow and Stamler that measured the amount of SNO-Hb yield as a function of the NO/Hb ratio.2 Below a NO/Hb ratio of 1, as the NO/Hb ratio increased, the SNO-Hb yield decreased.2 At the lowest NO/Hb ratio tested of 0.01, the reported yield was 80%.2 Our samples had an NO/Hb ratio below 0.001. Error bars shown in measured values correspond to 1 standard deviation.   Figure 2A shows typical EPR spectra from a partially nitrosylated sample before and after oxygenation. The deoxygenated sample (deoxy Hb) presents a triplet hyperfine structure characteristic of pentacoordinate -nitrosyl Hb. This hyperfine structure disappears upon oxygenation as expected due to a change in the quaternary state of the Hb (T to R). The SNO-Hb hypothesis predicts that, upon the oxygen-induced quaternary change, the total amount of nitrosyl Hb should be substantially reduced. This prediction is not observed. Table 1 summarizes the characteristics of the partially nitrosylated Hb samples prepared under deoxygenated conditions followed by oxygenation. Because the total amount of NO released is so small (NO/Hb = 0.00085) and the NO donor is mixed before it releases NO, there should be virtually no Hb tetramers with more than one NO bound. As expected by the equal association rate of NO with hemes and subunits,21 the initial sample contains 50% nitrosylation on the subunits—the most one can expect for a deoxygenated sample. A small amount of SNO-Hb was detected by chemiluminescence in the initial samples, perhaps due to a small amount of nitrite contamination.22 Upon oxygenation, contrary to the prediction of the SNO-Hb hypothesis, the concentration of Fe-NO Hb did not decrease. The slight increase observed may have been due to the reaction of deoxygenated Hb with the small amount of nitrite present (the concentration of nitrite was 2.0 ± 1.1 µM before oxygenation and 1.3 ± 0.6 µM afterward).22 Effective conversion from the T to R quaternary states is expected for the percentages of oxygenation and deoxygenation and low amounts of methemoglobin (MetHb) shown in Table 1. This allosteric transition is confirmed by the disappearance of the pentacoordinate -nitrosyl species upon oxygenation, which is destabilized in the R-state. Overall, the data shown in Figure 2 and Table 1 show that we did not observe the loss of Fe-NO Hb and gain in SNO-Hb predicted by the SNO-Hb hypothesis. View this table: [in this window] [in a new window]   Table 1.. Hemoglobin properties before and after oxygenation   Figure 2B presents a summary of the measured percentage changes in Fe-NO Hb and SNO-Hb upon oxygenation. In addition, the predicted percentage changes based on an 80% transfer efficiency2 are also shown. It has been almost 10 years since the SNO-Hb hypothesis was originally proposed,1 yet no conclusive evidence using the widely accepted technique of EPR has been put forth to support the notion of an allosterically controlled intramolecular transfer mechanism. Results have been presented using EPR to support the intramolecular transfer but most of these have included nitrite in the Hb preparations3,4 and we have previously suggested that the added nitrite was potentially responsible for some of the phenomena attributed to intramolecular transfer.12 One set of EPR data has been presented in the absence of nitrite, yet the growth in Fe-NO Hb presented was said to be due to slow loss of SNO-Hb on aging without indication to its relation to allosteric control.3 Other evidence for intramolecular transfer has relied on detection using photolysis chemiluminescence, but this technique reports levels of Fe-NO Hb in human blood that are at least 10 times that measured by EPR.4,6,23 We conclude that allosterically controlled intramolecular transfer from heme to thiol does not occur under normal conditions and therefore a defect in this processing of NO by Hb does not contribute to pathologic states.    Acknowledgements   We thank Dr Neil Hogg for critical reading of a draft of this manuscript.    Footnotes   Submitted October 14, 2005; accepted November 28, 2005. Prepublished online as Blood First Edition Paper, December 8, 2005; DOI 10.1182/blood-2005-10-4104. Supported by National Institutes of Health grant HL58091 (D.B.K.-S.). Further support is acknowledged through Career Award K02 HL078706 (D.B.K.-S.). I.A. and K.T.H. worked out details of the experiments and performed some of them; S.B. and J.H. performed some of the experiments; and D.B.K.-S. designed the experiments and wrote the paper. K.T.H. and I.A. contributed equally to this work. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Daniel B. Kim-Shapiro, Department of Physics, Wake Forest University, Winston-Salem, NC 27109; e-mail: shapiro{at}wfu.edu.    References Top Abstract Introduction Study design Results and discussion References   Jia L, Bonaventura C, Bonaventura J, Stamler JS. S-nitrosohaemoglobin: a dynamic activity of blood involved in vascular control. Nature. 1996;380: 221-226.[CrossRef][Medline] [Order article via Infotrieve] Gow AJ, Stamler JS. Reactions between nitric oxide and haemoglobin under physiological conditions. Nature. 1998;391: 169-173.[CrossRef][Medline] [Order article via Infotrieve] Singel DJ, Stamler JS. Chemical physiology of blood flow regulation by red blood cells: the role of nitric oxide and S-nitrosohemoglobin. Annu Rev Physiol. 2005;67: 99-145.[CrossRef][Medline] [Order article via Infotrieve] McMahon TJ, Moon RE, Luschinger BP, et al. Nitric oxide in the human respiratory cycle. Nat Med. 2002;8: 711-717.[Medline] [Order article via Infotrieve] Pawloski JR, Hess DT, Stamler JS. Export by red blood cells of nitric oxide bioactivity. Nature. 2001;409: 622-626.[CrossRef][Medline] [Order article via Infotrieve] McMahon TJ, Ahearn GS, Moya MP, et al. A nitric oxide processing defect of red blood cells created by hypoxia: deficiency of S-nitrosohemoglobin in pulmonary hypertension. Proc Natl Acad Sci U S A. 2005;102: 14801-14806.[Abstract/Free Full Text] Huang TH. Nmr-studies of the quaternary structure and heterogeneity of nitrosylhemoglobin and methemoglobin. J Biol Chem. 1979;254: 1467-1474. Hille R, Olson JS, Palmer G. Spectral transitions of nitrosyl hemes during ligand-binding to hemoglobin. J Biol Chem. 1979;254: 2110-2120. Perutz MF, Kilmartin JV, Nagai K, Szabo A, Simon SR. Influence of globin structures on state of heme-ferrous low-spin derivatives. Biochemistry-US. 1976;15: 378-387. Yonetani T, Tsuneshige A, Zhou YX, Chen XS. Electron paramagnetic resonance and oxygen binding studies of alpha-nitrosyl hemoglobin—a novel oxygen carrier having no-assisted allosteric functions. J Biol Chem. 1998;273: 20323-20333.[Abstract/Free Full Text] Pawloski JR, Hess DT, Stamler JS. Impaired vasodilation by red blood cells in sickle cell disease. Proc Natl Acad Sci U S A. 2005;102: 2531-2536.[Abstract/Free Full Text] Xu XL, Cho M, Spencer NY, et al. Measurements of nitric oxide on the heme iron and beta-93 thiol of human hemoglobin during cycles of oxygenation and deoxygenation. Proc Natl Acad Sci U S A. 2003;100: 11303-11308.[Abstract/Free Full Text] Huang Z, Louderback JG, Goyal M, Azizi F, King SB, Kim-Shapiro DB. Nitric oxide binding to oxygenated hemoglobin under physiological conditions. Biochim Biophys Acta. 2001;1568: 252-260.[Medline] [Order article via Infotrieve] Geraci G, Parkhurst LJ, Gibson QH. Preparation and properties of - and -chains from human hemoglobin. J Biol Chem. 1969;17: 4664-4667. Gladwin MT, Wang XD, Reiter CD, et al. S-nitrosohemoglobin is unstable in the reductive erythrocyte environment and lacks O-2/NO-linked allosteric function. J Biol Chem. 2002;277: 27818-27828.[Abstract/Free Full Text] Doctor A, Platt R, Sheram ML, et al. Hemoglobin conformation couples erythrocyte S-nitrosothiol content to O-2 gradients. Proc Natl Acad Sci U S A. 2005;102: 5709-5714.[Abstract/Free Full Text] Yang BK, Vivas EX, Reiter CD, Gladwin MT. Methodologies for the sensitive and specific measurement of S-nitrosothiols, iron-nitrosyls, and nitrite in biological samples. Free Radic Res. 2003;37: 1-10.[Medline] [Order article via Infotrieve] Huang KT, Keszler A, Patel N, et al. The reaction between nitrite and deoxyhemoglobin: reassessment of reaction kinetics and stoichiometry. J Biol Chem. 2005;280: 31126-31131.[Abstract/Free Full Text] Xu XL, Lockamy VL, Chen KJ, et al. Effects of iron nitrosylation on sickle cell hemoglobin solubility. J Biol Chem. 2002;277: 36787-36792.[Abstract/Free Full Text] Giustarini D, Milzani A, Colombo R, Dalle-Donne I, Rossi R. Nitric oxide, S-nitrosothiols and hemoglobin: is methodology the key? Trends Pharmacol Sci. 2004;25: 311-316.[CrossRef][Medline] [Order article via Infotrieve] Hille R, Palmer G, Olson JS. Chain equivalence in reaction of nitric-oxide with hemoglobin. J Biol Chem. 1977;252: 403-405.[Abstract/Free Full Text] Cosby K, Partovi KS, Crawford JH, et al. Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation. Nat Med. 2003;9: 1498-1505.[CrossRef][Medline] [Order article via Infotrieve] Piknova B, Gladwin MT, Schechter AN, Hogg N. Electron paramagenetic resonance analysis of nitrosylhemoglobin in humans during NO inhalation. J Biol Chem. 2005;280: 40583-40588.[Abstract/Free Full Text] CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? Related Letters in Blood Online: NO, SNO, and hemoglobin: lessons in complexity Andrew J. Gow and David Singel Blood 2006 108: 3224-3225. [Full Text] [PDF] Detecting physiologic fluctuations in the S-nitrosohemoglobin micropopulation: triiodide versus 3C Allan Doctor, Benjamin Gaston, and Daniel B. Kim-Shapiro Blood 2006 108: 3225-3227. [Full Text] [PDF] This article has been cited by other articles: M. T. Gladwin and D. B. Kim-Shapiro The functional nitrite reductase activity of the heme-globins Blood, October 1, 2008; 112(7): 2636 - 2647. [Abstract] [Full Text] [PDF] S. C. Rogers, A. Khalatbari, B. N. Datta, S. Ellery, V. Paul, M. P. Frenneaux, and P. E. James NO metabolite flux across the human coronary circulation Cardiovasc Res, July 15, 2007; 75(2): 434 - 441. [Abstract] [Full Text] [PDF] R. Grubina, Z. Huang, S. Shiva, M. S. Joshi, I. Azarov, S. Basu, L. A. Ringwood, A. Jiang, N. Hogg, D. B. Kim-Shapiro, et al. Concerted Nitric Oxide Formation and Release from the Simultaneous Reactions of Nitrite with Deoxy- and Oxyhemoglobin J. Biol. Chem., April 27, 2007; 282(17): 12916 - 12927. [Abstract] [Full Text] [PDF] A. J. Gow and D. Singel NO, SNO, and hemoglobin: lessons in complexity. Blood, November 1, 2006; 108(9): 3224 - 3225. [Full Text] [PDF] A. Doctor, B. Gaston, and D. B. Kim-Shapiro Detecting physiologic fluctuations in the S-nitrosohemoglobin micropopulation: triiodide versus 3C. Blood, November 1, 2006; 108(9): 3225 - 3227. [Full Text] [PDF] M. T. Gladwin, N. J. H. Raat, S. Shiva, C. Dezfulian, N. Hogg, D. B. Kim-Shapiro, and R. P. Patel Nitrite as a vascular endocrine nitric oxide reservoir that contributes to hypoxic signaling, cytoprotection, and vasodilation Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2026 - H2035. [Abstract] [Full Text] [PDF] B. W. Allen and C. A. Piantadosi How do red blood cells cause hypoxic vasodilation? The SNO-hemoglobin paradigm Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H1507 - H1512. [Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) All Versions of this Article: 2005-10-4104v1 107/7/2602    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Huang, K. T. Articles by Kim-Shapiro, D. B. Search for Related Content PubMed PubMed Citation Articles by Huang, K. T. Articles by Kim-Shapiro, D. B. Related Collections Red Cells Brief Reports Related Letters in Blood Online Social Bookmarking                   What's this?

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