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**, and
- Benjamin Gaston§,††
- Departments of *Pediatric Critical Care, §Pediatric Respiratory Medicine, ‡Surgery, ¶Chemistry, and ∥Biostatistics, University of Virginia, Charlottesville, VA 22908; †Pulmonary and Critical Care, Duke University, Durham, NC 27708; **Stokes Research Institute, University of Pennsylvania, Philadelphia, PA 19104
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Edited by Irwin Fridovich, Duke University Medical Center, Durham, NC, and approved February 25, 2005 (received for review October 19, 2004)
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.
Footnotes
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↵ †† To whom correspondence should be addressed at: Pediatric Respiratory Medicine, Box 386, University of Virginia Health System, Charlottesville, VA 22908. E-mail: bmg3g{at}virginia.edu.
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Author contributions: A.D., T. McMahon, A.G., and B.G. designed research; A.D., R.P., M.L.S., A.E., T. Maxey, J.D., M.A., J.K., and B.G. performed research; A.D., R.P., M.L.S., A.E., T. McMahon, T. Maxey, J.D., M.A., J.K., M.G., A.G., and B.G. analyzed data; and A.D. wrote the paper.
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This paper was submitted directly (Track II) to the PNAS office.
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Abbreviations: R, relaxed; T, tense; SNO, S-nitrosothiol; CSNO, S-nitrosocysteine; SIRS, systemic inflammatory response syndrome; ARDS, acute respiratory distress syndrome; GSNO, S-nitrosoglutathione; HPV, hypoxic pulmonary vasoconstriction; PA, pulmonary artery; PAP, PA pressure; 3C, CuCl/Cys/CO.
- Copyright © 2005, The National Academy of Sciences
