1. Hemoglobin conformation couples erythrocyte S -nitrosothiol content to O 2 gradients
- Author
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Anne Eischeid, Benjamin Gaston, Joseph Doherty, Mark J. Axelrod, Allan Doctor, Ruth Platt, Thomas S. Maxey, Jaclyn Kline, Andrew J. Gow, Matthew J. Gurka, Mary Lynn Sheram, and Timothy J. McMahon
- Subjects
Erythrocytes ,Protein Conformation ,Allosteric regulation ,Nitric oxide ,Hemoglobins ,Mice ,chemistry.chemical_compound ,Sepsis ,Animals ,Humans ,Lung ,Deoxygenation ,Fluorescent Dyes ,S-Nitrosothiols ,chemistry.chemical_classification ,Carbon Monoxide ,Respiratory Distress Syndrome ,Multidisciplinary ,Reproducibility of Results ,Biological Sciences ,Systemic Inflammatory Response Syndrome ,Oxygen ,chemistry ,Biochemistry ,Luminescent Measurements ,Thiol ,Biophysics ,Room air distribution ,Biological Assay ,Hemoglobin ,Copper ,Carbon monoxide - Abstract
It is proposed that the bond between nitric oxide (NO) and the Hb thiol Cys-β 93 (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) (SNO RBC ), preserving Hb allostery. In room air, the ratio of SNO RBC to Hb in intact RBCs is stable over time, but there is a logarithmic loss of SNO RBC with oxyHb desaturation (slope, 0.043). This decay is accelerated by extraerythrocytic thiol (slope, 0.089; P < 0.001). SNO RBC stability is uncoupled from O 2 tension when Hb is locked in the R state by CO pretreatment. Also, SNO RBC is increased ≈20-fold in human septic shock ( P = 0.002) and the O 2 -dependent vasoactivity of RBCs is affected profoundly by SNO content in a murine lung bioassay. These data demonstrate that SNO content and O 2 saturation are tightly coupled in intact RBCs and that this coupling is likely to be of pathophysiological significance.
- Published
- 2005
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