Rate of reaction with nitric oxide determines the hypertensive effect of cell-free hemoglobin.

Administration of extracellular hemoglobin-based oxygen carriers often induces mild increases in blood pressure. In order to test whether nitric oxide (NO) scavenging is responsible for the hypertensive effect, we constructed and tested a set of recombinant hemoglobins that vary in rates of reaction with NO. The results suggest that the rapid reactions of oxy- and deoxyhemoglobin with nitric oxide are the fundamental cause of the hypertension. The magnitude of the blood-pressure effect correlates directly with the in vitro rate of NO oxidation. Hemoglobins with decreased NO-scavenging activity may be more suitable for certain therapeutic applications than those that cause depletion of nitric oxide.

Quaternary structure regulates hemin dissociation from human hemoglobin.

Rate constants for hemin dissociation from the alpha and beta subunits of native and recombinant human hemoglobins were measured as a function of protein concentration at pH 7.0, 37 degrees C, using H64Y/V68F apomyoglobin as a hemin acceptor reagent. Hemin dissociation rates were also measured for native isolated alpha and beta chains and for recombinant hemoglobin tetramers stabilized by alpha subunit fusion. The rate constant for hemin dissociation from beta subunits in native hemoglobin increases from 1.5 h-1 in tetramers at high protein concentration to 15 h-1 in dimers at low concentrations. The rate of hemin dissociation from alpha subunits in native hemoglobin is significantly smaller (0.3-0.6 h-1) and shows little dependence on protein concentration. Recombinant hemoglobins containing a fused di-alpha subunit remain tetrameric under all concentrations and show rates of hemin loss similar to those observed for wild-type and native hemoglobin at high protein concentration. Rates of hemin dissociation from monomeric alpha and beta chains are much greater, 12 and 40 h-1, respectively, at pH 7, 37 degrees C. Aggregation of monomers to form alpha1beta1 dimers greatly stabilizes bound hemin in alpha chains, decreasing its rate of hemin loss approximately 20-fold. In contrast, dimer formation has little stabilizing effect on hemin binding to beta subunits. A significant reduction in the rate of hemin loss from beta subunits does occur after formation of the alpha1beta2 interface in tetrameric hemoglobin. These results suggest that native human hemoglobin may have evolved to lose heme rapidly after red cell lysis, allowing the prosthetic group to be removed by serum albumin and apohemopexin.