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Cited by 160Hemoglobin structure and functionNitric Oxide and Endothelin EffectsHeme Oxygenase-1 and Carbon Monoxide

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S-Nitrosohemoglobin Is Unstable in the Reductive Erythrocyte Environment and Lacks O2/NO-linked Allosteric Function

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Gladwin, Mark T., Wang, Xunde, Reiter, Christopher D., Yang, Benjamin K., Vivas, Esther X., Bonaventura, Celia, Schechter, Alan N.·2002·Journal of Biological Chemistry
DOI 10.1074/jbc.m203236200
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Abstract

Our previous results run counter to the hypothesis that S-nitrosohemoglobin (SNO-Hb) serves as an in 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 beta-globin cysteine 93 is maintained in a reduced state, necessary for normal oxygen affinity, and incapable of oxygen-linked NO storage and delivery.

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Crossref · 10.1074/jbc.m203236200

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Figures

FIG. 1 Reduction of S -nitrosothiols result in rapid and complete release of NO from S -nitrosohemoglobin in solutions of I3 H11546 . Preparations of SNO-Hb ( panel A ) and SNO-albumin ( panel B ) analyzed by I 3 -chemiluminescent assay ( closed circles ) and by the Griess-Saville assay ( open squares ). Panel C , SNO-Hb standards in lysed red cell pellet at concentrations from 200 nM (0.002% S -NO/heme) to 3,200 nM (0.032% S -NO/heme); n = 4. Panel D , a representative set of nitric oxide analyzer readouts from one experiment is shown.
FIG. 1 Reduction of S -nitrosothiols result in rapid and complete release of NO from S -nitrosohemoglobin in solutions of I3 H11546 . Preparations of SNO-Hb ( panel A ) and SNO-albumin ( panel B ) analyzed by I 3 -chemiluminescent assay ( closed circles ) and by the Griess-Saville assay ( open squares ). Panel C , SNO-Hb standards in lysed red cell pellet at concentrations from 200 nM (0.002% S -NO/heme) to 3,200 nM (0.032% S -NO/heme); n = 4. Panel D , a representative set of nitric oxide analyzer readouts from one experiment is shown.CC-BY · From the PDF
FIG. 2 Stability of SNO-Hb in KCN/K3Fe III (CN) 6 . Panel A , standard samples of SNO-Hb, nitrite, nitrate, and HbFe II NO were injected
FIG. 2 Stability of SNO-Hb in KCN/K3Fe III (CN) 6 . Panel A , standard samples of SNO-Hb, nitrite, nitrate, and HbFe II NO were injectedCC-BY · From the PDF
Image 3
CC-BY · From the PDF
Image 4
CC-BY · From the PDF
FIG. 3 Stability of SNO-Hb under denaturing (detergent treatment) conditions and in red cell lysates. Panel A , standards of
FIG. 3 Stability of SNO-Hb under denaturing (detergent treatment) conditions and in red cell lysates. Panel A , standards ofCC-BY · From the PDF
Image 6
CC-BY · From the PDF
FIG. 5 Arterial and venous red blood cell S -nitrosothiol levels (SNO-Hb and membrane SNO compounds) in the human circulation. Panel A , NO release (in mV) following the injection of 300 𝛍 l of red cell lysates treated with Nonidet P-40, KCN/K 3 Fe III (CN)6, NEM, and DTPA; lysates were from arterial and venous blood obtained from 8 normal volunteers as described under ' Experimental Procedures. ' Panel B , mean of data ± S.E. for arterial and venous red blood cell total S -nitrosothiol levels in 8 normal volunteers.
FIG. 5 Arterial and venous red blood cell S -nitrosothiol levels (SNO-Hb and membrane SNO compounds) in the human circulation. Panel A , NO release (in mV) following the injection of 300 𝛍 l of red cell lysates treated with Nonidet P-40, KCN/K 3 Fe III (CN)6, NEM, and DTPA; lysates were from arterial and venous blood obtained from 8 normal volunteers as described under ' Experimental Procedures. ' Panel B , mean of data ± S.E. for arterial and venous red blood cell total S -nitrosothiol levels in 8 normal volunteers.CC-BY · From the PDF
FIG. 6 Effects of hypoxia on the stability of chemically produced SNO-Hb-containing human erythrocytes and on NO export. Red cells were incubated in tonometers at 10% hematocrit with 21% oxygen and 0% oxygen as described under ' Experimental Procedures. ' Panel A , hemoglobin saturations in normoxia ( solid line ) and hypoxia ( dashed line ). Panel B , SNO-Hb levels in normoxia ( solid line ) and hypoxia ( dashed line ). Panels C -E , S -nitrosothiol, nitrite, and nitrate export in normoxia ( solid lines ) and hypoxia ( dashed lines ). Panel F , total SNO-Hb degradation versus NOx formation during normoxia ( open bars ) and hypoxia ( hatched bars ).
FIG. 6 Effects of hypoxia on the stability of chemically produced SNO-Hb-containing human erythrocytes and on NO export. Red cells were incubated in tonometers at 10% hematocrit with 21% oxygen and 0% oxygen as described under ' Experimental Procedures. ' Panel A , hemoglobin saturations in normoxia ( solid line ) and hypoxia ( dashed line ). Panel B , SNO-Hb levels in normoxia ( solid line ) and hypoxia ( dashed line ). Panels C -E , S -nitrosothiol, nitrite, and nitrate export in normoxia ( solid lines ) and hypoxia ( dashed lines ). Panel F , total SNO-Hb degradation versus NOx formation during normoxia ( open bars ) and hypoxia ( hatched bars ).CC-BY · From the PDF

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Research works printed but not linked: 1

  1. Garel, 1984, Mol. Pharmacol., 26, 559

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Metadata

Authors
Gladwin, M. T., Wang, X., Reiter, C. D., Yang, B. K., Vivas, E. X., Bonaventura, C., & Schechter, A. N.
Affiliations
  • National Institutes of Health (US)
  • Duke University (US)
Corresponding author
Gladwin, Mark T.
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2002-08
Volume
277
Issue
31
Pages
27818-27828
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.m203236200
OpenAlex
W2061156310
Cited by
160
References
45
Citations by year
2026: 1 · 2024: 2 · 2023: 3 · 2022: 3 · 2021: 1 · 2020: 1 · 2019: 1 · 2018: 4 · 2017: 4 · 2016: 3 · 2015: 1 · 2014: 3 · 2013: 4 · 2012: 5
FWCI
7.92
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Hemoglobin structure and function
  • Health Sciences › Medicine › Physiology (medical) › Nitric Oxide and Endothelin Effects
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Heme Oxygenase-1 and Carbon Monoxide
Keywords
S-nitrosohemoglobin, nitric oxide storage, erythrocytes, S-nitrosothiols, reductive decomposition, oxygen affinity, allosteric regulation, nitrate formation, redox conditions, nitric oxide delivery
MeSH terms
Allosteric Regulation, Animals, Cattle, Drug Stability, Erythrocytes, Hemoglobins, Humans, Kinetics, Nitric Oxide, Nitrites, Oxygen, Serum Albumin, Serum Albumin, Bovine

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