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-beta(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 approximately 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.
authors.affiliation: Department of Pediatric Critical Care, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Allan
authors.initials: A
authors.lastname: Doctor
authors.forename: Ruth
authors.initials: R
authors.lastname: Platt
authors.forename: Mary Lynn
authors.initials: ML
authors.lastname: Sheram
authors.forename: Anne
authors.initials: A
authors.lastname: Eischeid
authors.forename: Timothy
authors.initials: T
authors.lastname: McMahon
authors.forename: Thomas
authors.initials: T
authors.lastname: Maxey
authors.forename: Joseph
authors.initials: J
authors.lastname: Doherty
authors.forename: Mark
authors.initials: M
authors.lastname: Axelrod
authors.forename: Jaclyn
authors.initials: J
authors.lastname: Kline
authors.forename: Matthew
authors.initials: M
authors.lastname: Gurka
authors.forename: Andrew
authors.initials: A
authors.lastname: Gow
authors.forename: Benjamin
authors.initials: B
authors.lastname: Gaston
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods; D002248:Carbon Monoxide / Q000378:metabolism; D003300:Copper / Q000737:chemistry; D004912:Erythrocytes / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008163:Luminescent Measurements; D008168:Lung / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D010100:Oxygen / Q000378:metabolism*; D011487:Protein Conformation; D015203:Reproducibility of Results; D012128:Respiratory Distress Syndrome / Q000378:metabolism; D026403:S-Nitrosothiols / Q000737:chemistry* / Q000378:metabolism; D018805:Sepsis / Q000378:metabolism; D018746:Systemic Inflammatory Response Syndrome / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D006454:Hemoglobins; D026403:S-Nitrosothiols; D003300:Copper; D002248:Carbon Monoxide; C028419:cuprous chloride; D010100:Oxygen