Nitric oxide is consumed, rather than conserved, by reaction with oxyhemoglobin under physiological conditions

Abstract

Although irreversible reaction of NO with the oxyheme of hemoglobin (producing nitrate and methemoglobin) is extremely rapid, it has been proposed that, under normoxic conditions, NO binds preferentially to the minority deoxyheme to subsequently form S-nitrosohemoglobin (SNOHb). Thus, the primary reaction would be conservation, rather than consumption, of nitrogen oxide. Data supporting this conclusion were generated by using addition of a small volume of a concentrated aqueous solution of NO to a normoxic hemoglobin solution. Under these conditions, however, extremely rapid reactions can occur before mixing. We have thus compared bolus NO addition to NO generated homogeneously throughout solution by using NO donors, a more physiologically relevant condition. With bolus addition, multiple hemoglobin species are formed (as judged by visible spectroscopy) as well as both nitrite and nitrate. With donor, only nitrate and methemoglobin are formed, stoichiometric with the amount of NO liberated from the donor. Studies with increasing hemoglobin concentrations reveal that the nitrite-forming reaction (which may be NO autoxidation under these conditions) competes with reaction with hemoglobin. SNOHb formation is detectable with either bolus or donor; however, the amounts formed are much smaller than the amount of NO added (less than 1%). We conclude that the reaction of NO with hemoglobin under normoxic conditions results in consumption, rather than conservation, of NO. O ne of the most important experimental findings that led to the postulate that the endothelium-derived relaxing factor (EDRF) is identical to nitric oxide (nitrogen monoxide, NO) was the demonstration that endothelium-dependent relaxation is exquisitely sensitive to inhibition by hemoglobin, which rapidly reacts with NO (1). Curiously, however, even though very small concentrations of hemoglobin are quite potent in preventing EDRF-dependent relaxation, little attention was initially paid to the problem this raises with the NO͞EDRF hypothesis, namely, that in vivo NO is produced immediately adjacent to a pool of very high (mM) concentrations of hemoglobin, which will act as a potent sink for the NO, thus decreasing its concentration at all locations and preventing it from reaching a physiologically functional level. Mathematical modeling has illustrated the validity of this concept (2-4).There have emerged two hypotheses to explain how NO might still function as EDRF. According to one (5-10), rather than irreversibly consuming NO, hemoglobin actually conserves it, in the form of a nitrosothiol moiety, and the linkage of the accessibility͞ reactivity of the thiol group involved (␤cys93) to the oxygenationdependent allosteric transitions in hemoglobin establishes a respiratory cycle whereby NO and O 2 are simultaneously taken up in the lung and then delivered to the tissue during the arterial͞venous transit. Thus, hemoglobin would deliver to oxygen-deficient vascular beds not only the oxygen required for sustained metabolism but also a vasodilator (NO).Th...

Routes to S -nitroso-hemoglobin formation with heme redox and preferential reactivity in the β subunits

“…The latter authors also measure SNO production and obtain nearly equivalent yields of Fe(II)NO and SNO-Hb under a variety of conditions. In related experiments, Joshi et al (30) fail to detect Fe(II)NO at very high oxygenation and nonphysiological NO levels. Notably, they do observe the production of SNO-Hb in yields that are similar to those reported by Gow et al (3).…”

Nitric oxide red blood cell membrane permeability at high and low oxygen tension

“…Briefly, NO buffer was added to either Hb or whole blood at various oxygen saturations and the HbNO yield (amount of HbNO formed/amount of NO added) was determined using electron paramagnetic resonance (EPR). To insure that previously published results were not compromised due to a bolus effect (where concentrated NO in saturated buffer exhausts local heme sites before it can be properly mixed so that secondary chemistry can occur [31][32][33]) we performed additional experiments using an NO donor, ProliNO (Cayman Chemical (Ann Arbor, MI)). A stock solution of ProliNO was first prepared in pH 10, 0.05 M Trizma base buffer (Sigma Chemicals, St. Louis, MO) and a couple of microliters of the stock was diluted into 800 μL of Hb at a concentration of 3.4 mM in pH 7.4, 0.1 M sodium phosphate buffer to give a final concentration of ProliNO of 50 μM.…”

Control of Plasma Nitric Oxide Bioactivity by Perfluorocarbons

“…15 The apparent limitation for plasma NO to exert its biological function is the constant presence of abundant hemoglobin (10 mmol/L) from red blood cells that converts NO into inactive metabolites at near-diffusion-limited rates. 16 Moreover, NO is unable to react directly with thiols. It must be oxidized first to form nitrosative agents, such as N 2 O 3 .…”