title: S-Nitrosohemoglobin is unstable in the reductive erythrocyte environment and lacks O2/NO-linked allosteric function.
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.
authors.affiliation: Critical Care Medicine Department, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892-1662, USA. mgladwin@nih.gov
authors.forename: Mark T
authors.initials: MT
authors.lastname: Gladwin
authors.forename: Xunde
authors.initials: X
authors.lastname: Wang
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Reiter
authors.forename: Benjamin K
authors.initials: BK
authors.lastname: Yang
authors.forename: Esther X
authors.initials: EX
authors.lastname: Vivas
authors.forename: Celia
authors.initials: C
authors.lastname: Bonaventura
authors.forename: Alan N
authors.initials: AN
authors.lastname: Schechter
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D002417:Cattle; D004355:Drug Stability; D004912:Erythrocytes / Q000378:metabolism*; D006454:Hemoglobins / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009569:Nitric Oxide / Q000097:blood*; D009573:Nitrites / Q000097:blood; D010100:Oxygen / Q000097:blood*; D012709:Serum Albumin / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism
chemical_list: D006454:Hemoglobins; D009573:Nitrites; C103574:S-nitrosohemoglobin; D012709:Serum Albumin; D012710:Serum Albumin, Bovine; D009569:Nitric Oxide; D010100:Oxygen