The binding of oxygen to heme irons in hemoglobin promotes the binding of nitric oxide (NO) to cysteine␤93, forming S-nitrosohemoglobin. Deoxygenation is accompanied by an allosteric transition in S-nitrosohemoglobin [from the R (oxygenated) to the T (deoxygenated) structure] that releases the NO group. S-nitrosohemoglobin contracts blood vessels and decreases cerebral perfusion in the R structure and relaxes vessels to improve blood flow in the T structure. By thus sensing the physiological oxygen gradient in tissues, hemoglobin exploits conformation-associated changes in the position of cysteine␤93 SNO to bring local blood flow into line with oxygen requirements.Hemoglobin (Hb) is the tetrameric protein in red blood cells (RBCs) that transports oxygen (O 2 ) from the lung to the tissues (1). As RBCs saturated in O 2 migrate through small arteries and resistance arterioles, they are exposed to an O 2 gradient (2). By the time Hb reaches the capillaries, a large fraction (ϳ50 to 65%) of the O 2 has been lost to venous exchange (a functional shunt) (2). Only about 25 to 30% of the O 2 is extracted by the tissues to meet basal metabolic requirements (1-3). Exposed to increasing oxygen tension (PO 2 ) in postcapillary venules and veins (2), Hb is ϳ75% saturated in O 2 (1, 3) upon entering the lung. Thus, on average, only one of four O 2 molecules carried by Hb is used in the respiratory cycle, even though extensive deoxygenation occurs in the flowcontrolling resistance vessels.Hemoglobin exists in two alternative structures, named R (for relaxed, high O 2 affinity) and T (for tense, low O 2 affinity) (4). Hemoglobin assumes the T structure to efficiently release O 2 (4). The allosteric transition in Hb (from R to T) controls the reactivity of two highly conserved cysteines (Cys␤93) that can react with NO or SNO (S-nitrosothiol) (5). Thiol affinity for (S)NO is high in the R structure and low in the T structure. In other words, the NO group is released from thiols of Hb in low PO 2 (5). A major function of (S)NO in the vasculature is to regulate blood flow, which is controlled by the resistance arterioles (6). We therefore proposed that partial deoxygenation of SNO-Hb in these vessels might actually promote O 2 delivery by liberating (S)NO. That is, the allosteric transition in Hb would function to release (S)NO in order to increase blood flow.Hemoglobin is mainly in the R (oxy) structure in both 95% O 2 and 21% O 2 (room air) (4). Hb and SNO-Hb both contract blood vessels in bioassays (7) at these O 2 concentrations (Fig. 1A). That is, their hemes sequester NO from the endothelium. In hypoxia [Ͻ1% O 2 (at a simulated tissue PO 2 of ϳ6 mmHg)], which promotes the T structure (4), Hb strongly contracts blood vessels, whereas SNO-Hb does not (Fig. 1B). NO group release from SNO-Hb is accelerated in RBCs by glutathione (5), which enhances SNO-Hb relaxations through formation of S-nitrosoglutathione (GSNO) (Fig. 1C). The potentiation by glutathione is inversely related to the PO 2 (Fig. 1C), because NO group transfer fr...

Recent studies have underscored questions about the balance of risk and benefit of RBC transfusion. A better understanding of the nature and timing of molecular and functional changes in stored RBCs may provide strategies to improve the balance of benefit and risk of RBC transfusion. We analyzed changes occurring during RBC storage focusing on RBC deformability, RBC-dependent vasoregulatory function, and S-nitrosohemoglobin (SNO-Hb), through which hemoglobin (Hb) O2 desaturation is coupled to regional increases in blood flow in vivo (hypoxic vasodilation). Five hundred ml of blood from each of 15 healthy volunteers was processed into leukofiltered, additive solution 3-exposed RBCs and stored at 1-6°C according to AABB standards. Blood was subjected to 26 assays at 0, 3, 8, 24 and 96 h, and at 1, 2, 3, 4, and 6 weeks. RBC SNO-Hb decreased rapidly (1.2 ؋ 10 ؊4 at 3 h vs. 6.5 ؋ 10 ؊4 (fresh) mol S-nitrosothiol (SNO)/mol Hb tetramer (P ‫؍‬ 0.032, mercuric-displaced photolysis-chemiluminescence assay), and remained low over the 42-day period. The decline was corroborated by using the carbon monoxide-saturated copper-cysteine assay [3.0 ؋ 10 ؊5 at 3 h vs. 9.0 ؋ 10 ؊5 (fresh) mol SNO/mol Hb]. In parallel, vasodilation by stored RBCs was significantly depressed. RBC deformability assayed at a physiological shear stress decreased gradually over the 42-day period (P < 0.001). Time courses vary for several storage-induced defects that might account for recent observations linking blood transfusion with adverse outcomes. Of clinical concern is that SNO levels, and their physiological correlate, RBC-dependent vasodilation, become depressed soon after collection, suggesting that even ''fresh'' blood may have developed adverse biological characteristics.adenosine triphosphate ͉ hemoglobin ͉ nitric oxide ͉ S-nitrosothiols ͉ transfusion

The current perspective of NO biology is formulated predominantly from studies of NO synthesis. The role of S-nitrosothiol (SNO) formation and turnover in governing NO-related bioactivity remains uncertain. We generated mice with a targeted gene deletion of S-nitrosoglutathione reductase (GSNOR), and show that they exhibit substantial increases in whole-cell S-nitrosylation, tissue damage, and mortality following endotoxic or bacterial challenge. Further, GSNOR(-/-) mice have increased basal levels of SNOs in red blood cells and are hypotensive under anesthesia. Thus, SNOs regulate innate immune and vascular function, and are cleared actively to ameliorate nitrosative stress. Nitrosylation of cysteine thiols is a critical mechanism of NO function in both health and disease.