The binding of oxygen to heme irons in hemoglobin promotes the binding of nitric oxide (NO) to cysteineβ93, 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 cysteineβ93 SNO to bring local blood flow into line with oxygen requirements.

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) O ₂ 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.

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.