Hemoglobin conformation couples erythrocyte S-nitrosothiol content to O2 gradients

Allan Doctor, Ruth Platt, Mary Lynn Sheram, Anne Eischeid, Timothy McMahon, Thomas Maxey, Joseph Doherty, Mark Axelrod, Jaclyn Kline, Matthew Gurka, Andrew Gow, Benjamin Gaston

Research output: Contribution to journalArticle

144 Citations (Scopus)

Abstract

It is proposed that the bond between nitric oxide (NO) and the Hb thiol Cys-β93 (SNOHb) is favored when hemoglobin (Hb) is in the relaxed (R, oxygenated) conformation, and that deoxygenation to tense (T) state destabilizes the SNOHb bond, allowing transfer of NO from Hb to form other (vasoactive) S-nitrosothiols (SNOs). However, it has not previously been possible to measure SNOHb without extensive Hb preparation, altering its allostery and SNO distribution. Here, we have validated an assay for SNOHb that uses carbon monoxide (CO) and cuprous chloride (CuCl)-saturated Cys. This assay is specific for SNOs and sensitive to 2-5 pmol. Uniquely, it measures the total SNO content of unmodified erythrocytes (RBCs) (SNORBC), preserving Hb allostery. In room air, the ratio of SNORBC to Hb in intact RBCs is stable over time, but there is a logarithmic loss of SNORBC with oxyHb desaturation (slope, 0.043). This decay is accelerated by extraerythrocytic thiol (slope, 0.089; P <0.001). SNORBC stability is uncoupled from O2 tension when Hb is locked in the R state by CO pretreatment. Also, SNORBC is increased ≈20-fold in human septic shock (P = 0.002) and the O2-dependent vasoactivity of RBCs is affected profoundly by SNO content in a murine lung bioassay. These data demonstrate that SNO content and O2 saturation are tightly coupled in intact RBCs and that this coupling is likely to be of pathophysiological significance.

Original languageEnglish
Pages (from-to)5709-5714
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume102
Issue number16
DOIs
Publication statusPublished - 19 Apr 2005
Externally publishedYes

Fingerprint

S-Nitrosothiols
Hemoglobins
Erythrocytes
Carbon Monoxide
Sulfhydryl Compounds
Septic Shock
Biological Assay
Air
Lung

Keywords

  • Nitric oxide
  • Sepsis
  • Vascular physiology

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Hemoglobin conformation couples erythrocyte S-nitrosothiol content to O2 gradients. / Doctor, Allan; Platt, Ruth; Sheram, Mary Lynn; Eischeid, Anne; McMahon, Timothy; Maxey, Thomas; Doherty, Joseph; Axelrod, Mark; Kline, Jaclyn; Gurka, Matthew; Gow, Andrew; Gaston, Benjamin.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 102, No. 16, 19.04.2005, p. 5709-5714.

Research output: Contribution to journalArticle

Doctor, A, Platt, R, Sheram, ML, Eischeid, A, McMahon, T, Maxey, T, Doherty, J, Axelrod, M, Kline, J, Gurka, M, Gow, A & Gaston, B 2005, 'Hemoglobin conformation couples erythrocyte S-nitrosothiol content to O2 gradients', Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 16, pp. 5709-5714. https://doi.org/10.1073/pnas.0407490102
Doctor, Allan ; Platt, Ruth ; Sheram, Mary Lynn ; Eischeid, Anne ; McMahon, Timothy ; Maxey, Thomas ; Doherty, Joseph ; Axelrod, Mark ; Kline, Jaclyn ; Gurka, Matthew ; Gow, Andrew ; Gaston, Benjamin. / Hemoglobin conformation couples erythrocyte S-nitrosothiol content to O2 gradients. In: Proceedings of the National Academy of Sciences of the United States of America. 2005 ; Vol. 102, No. 16. pp. 5709-5714.
@article{c65049e448ad4190983befcabee6419f,
title = "Hemoglobin conformation couples erythrocyte S-nitrosothiol content to O2 gradients",
abstract = "It is proposed that the bond between nitric oxide (NO) and the Hb thiol Cys-β93 (SNOHb) is favored when hemoglobin (Hb) is in the relaxed (R, oxygenated) conformation, and that deoxygenation to tense (T) state destabilizes the SNOHb bond, allowing transfer of NO from Hb to form other (vasoactive) S-nitrosothiols (SNOs). However, it has not previously been possible to measure SNOHb without extensive Hb preparation, altering its allostery and SNO distribution. Here, we have validated an assay for SNOHb that uses carbon monoxide (CO) and cuprous chloride (CuCl)-saturated Cys. This assay is specific for SNOs and sensitive to 2-5 pmol. Uniquely, it measures the total SNO content of unmodified erythrocytes (RBCs) (SNORBC), preserving Hb allostery. In room air, the ratio of SNORBC to Hb in intact RBCs is stable over time, but there is a logarithmic loss of SNORBC with oxyHb desaturation (slope, 0.043). This decay is accelerated by extraerythrocytic thiol (slope, 0.089; P <0.001). SNORBC stability is uncoupled from O2 tension when Hb is locked in the R state by CO pretreatment. Also, SNORBC is increased ≈20-fold in human septic shock (P = 0.002) and the O2-dependent vasoactivity of RBCs is affected profoundly by SNO content in a murine lung bioassay. These data demonstrate that SNO content and O2 saturation are tightly coupled in intact RBCs and that this coupling is likely to be of pathophysiological significance.",
keywords = "Nitric oxide, Sepsis, Vascular physiology",
author = "Allan Doctor and Ruth Platt and Sheram, {Mary Lynn} and Anne Eischeid and Timothy McMahon and Thomas Maxey and Joseph Doherty and Mark Axelrod and Jaclyn Kline and Matthew Gurka and Andrew Gow and Benjamin Gaston",
year = "2005",
month = "4",
day = "19",
doi = "10.1073/pnas.0407490102",
language = "English",
volume = "102",
pages = "5709--5714",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "16",

}

TY - JOUR

T1 - Hemoglobin conformation couples erythrocyte S-nitrosothiol content to O2 gradients

AU - Doctor, Allan

AU - Platt, Ruth

AU - Sheram, Mary Lynn

AU - Eischeid, Anne

AU - McMahon, Timothy

AU - Maxey, Thomas

AU - Doherty, Joseph

AU - Axelrod, Mark

AU - Kline, Jaclyn

AU - Gurka, Matthew

AU - Gow, Andrew

AU - Gaston, Benjamin

PY - 2005/4/19

Y1 - 2005/4/19

N2 - It is proposed that the bond between nitric oxide (NO) and the Hb thiol Cys-β93 (SNOHb) is favored when hemoglobin (Hb) is in the relaxed (R, oxygenated) conformation, and that deoxygenation to tense (T) state destabilizes the SNOHb bond, allowing transfer of NO from Hb to form other (vasoactive) S-nitrosothiols (SNOs). However, it has not previously been possible to measure SNOHb without extensive Hb preparation, altering its allostery and SNO distribution. Here, we have validated an assay for SNOHb that uses carbon monoxide (CO) and cuprous chloride (CuCl)-saturated Cys. This assay is specific for SNOs and sensitive to 2-5 pmol. Uniquely, it measures the total SNO content of unmodified erythrocytes (RBCs) (SNORBC), preserving Hb allostery. In room air, the ratio of SNORBC to Hb in intact RBCs is stable over time, but there is a logarithmic loss of SNORBC with oxyHb desaturation (slope, 0.043). This decay is accelerated by extraerythrocytic thiol (slope, 0.089; P <0.001). SNORBC stability is uncoupled from O2 tension when Hb is locked in the R state by CO pretreatment. Also, SNORBC is increased ≈20-fold in human septic shock (P = 0.002) and the O2-dependent vasoactivity of RBCs is affected profoundly by SNO content in a murine lung bioassay. These data demonstrate that SNO content and O2 saturation are tightly coupled in intact RBCs and that this coupling is likely to be of pathophysiological significance.

AB - It is proposed that the bond between nitric oxide (NO) and the Hb thiol Cys-β93 (SNOHb) is favored when hemoglobin (Hb) is in the relaxed (R, oxygenated) conformation, and that deoxygenation to tense (T) state destabilizes the SNOHb bond, allowing transfer of NO from Hb to form other (vasoactive) S-nitrosothiols (SNOs). However, it has not previously been possible to measure SNOHb without extensive Hb preparation, altering its allostery and SNO distribution. Here, we have validated an assay for SNOHb that uses carbon monoxide (CO) and cuprous chloride (CuCl)-saturated Cys. This assay is specific for SNOs and sensitive to 2-5 pmol. Uniquely, it measures the total SNO content of unmodified erythrocytes (RBCs) (SNORBC), preserving Hb allostery. In room air, the ratio of SNORBC to Hb in intact RBCs is stable over time, but there is a logarithmic loss of SNORBC with oxyHb desaturation (slope, 0.043). This decay is accelerated by extraerythrocytic thiol (slope, 0.089; P <0.001). SNORBC stability is uncoupled from O2 tension when Hb is locked in the R state by CO pretreatment. Also, SNORBC is increased ≈20-fold in human septic shock (P = 0.002) and the O2-dependent vasoactivity of RBCs is affected profoundly by SNO content in a murine lung bioassay. These data demonstrate that SNO content and O2 saturation are tightly coupled in intact RBCs and that this coupling is likely to be of pathophysiological significance.

KW - Nitric oxide

KW - Sepsis

KW - Vascular physiology

UR - http://www.scopus.com/inward/record.url?scp=20244377241&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=20244377241&partnerID=8YFLogxK

U2 - 10.1073/pnas.0407490102

DO - 10.1073/pnas.0407490102

M3 - Article

C2 - 15824313

AN - SCOPUS:20244377241

VL - 102

SP - 5709

EP - 5714

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 16

ER -