Changes in mean arterial pressure were monitored in rats following 50% isovolemic exchange transfusion with solutions of chemically modified hemoglobins. Blood pressure responses fall into three categories: 1) an immediate and sustained increase, 2) an immediate yet transient increase, or 3) no significant change either during or subsequent to exchange transfusion. The reactivities of these hemoglobins with nitric monoxide ( ⅐ NO) were measured to test the hypothesis that different blood pressure responses to these solutions result from differences in ⅐ NO scavenging reactions. All hemoglobins studied exhibited a value of 30 M ؊1 s ؊1 for both ⅐ NO bimolecular association rate constants and the rate constants for ⅐ NO-induced oxidation in vitro. Only the ⅐ NO dissociation rate constants and, thus, the equilibrium dissociation constants varied. Values of equilibrium dissociation constants ranged from 2 to 14 pM and varied inversely with vasopressor response. Hemoglobin solutions that exhibited either transient or no significant increase in blood pressure showed tighter ⅐ NO binding affinities than hemoglobin solutions that exhibited sustained increases. These results suggest that blood pressure increases observed upon exchange transfusion with cell-free hemoglobin solutions can not be the result of ⅐ NO scavenging reactions at the heme, but rather must be due to alternative physiologic mechanisms.Control of blood pressure and resistance to blood flow is achieved by a dynamic constriction and relaxation of smooth muscle tissue which surrounds all blood vessels except capillaries. Vascular smooth muscle tension is continually adjusted by a complex system that causes either vasoconstriction or vasodilation, depending on metabolic need (1). Research performed over the last decade has established that endotheliumderived nitric oxide ( ⅐ NO) 1 can cause vasodilation. ⅐ NO is produced by endothelial cells that lie between the intravascular space and the surrounding smooth muscle. Among the findings was the demonstration that ⅐ NO donors (e.g. nitroprusside, nitroglycerin) lead to vasorelaxation through activation of guanylate cyclase, whereas inhibitors of ⅐ NO synthesis (e.g. N Gmonomethyl-L-arginine) or scavengers (e.g. hemoglobin) cause vasoconstriction (for reviews, see Refs. 2 and 3).Since cell-free hemoglobin is being developed as a red cell substitute (4), reactions between hemoglobin and ⅐ NO are of potential importance in maintenance of microvascular blood flow and O 2 delivery. Despite the wide variation that exists in the physical properties (O 2 affinity, molecular mass, and solution properties) of different cell-free hemoglobins, it appears vasoconstriction is a feature common to many hemoglobin solutions (for reviews, see Refs. 2, 3, and 5). It is tempting to conclude that ⅐ NO scavenging is the principal, if not sole mechanism for vasoconstriction associated with cell-free hemoglobin. However, it is well established that multiple factors contribute to the physiological control of vascular smooth muscle to...

The distal E7 histidine in vertebrate myoglobins and haemoglobins has been strongly conserved during evolution and is thought to be important in fine-tuning the ligand affinities of these proteins. A hydrogen bond between the N epsilon proton of the distal histidine and the second oxygen atom may stabilize O2 bound to the haem iron. The proximity of the imidazole side chain to the sixth coordination position, which is required for efficient hydrogen bonding, has been postulated to inhibit sterically the binding of CO and alkyl isocyanides. To test these ideas, engineered mutants of sperm whale myoglobin and the alpha- and beta-subunits of human haemoglobin were prepared in which E7 histidine was replaced by glycine. Removal of the distal imidazole in myoglobin and the alpha-subunits of intact, R-state haemoglobin caused significant changes in the affinity for oxygen, carbon monoxide and methyl isocyanide; in contrast, the His-E7 to Gly substitution produced little or no effect on the rates and extents of O2, CO and methyl isocyanide binding to beta-chains within R-state haemoglobin. In the beta-subunit the distal histidine seems to be less significant in regulating the binding of ligands to the haem iron in the high affinity quaternary conformation. Structural differences in the oxygen binding pockets shown by X-ray crystallographic studies account for the functional differences of these proteins.

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