Synthesized allosteric effectors of the hemoglobin molecule: a possible mechanism for improved erythrocyte oxygen release capability in hemoglobinopathy H disease.

Patients with the nondeletion genotype of hemoglobinopathy H (HbH or beta4) disease have higher proportions of HbH and more severe tissue hypoxia than patients with the deletion genotype. Because these patients' red blood cells (RBCs) contain mainly two Hb species, HbH and HbA, the high proportion of HbA can be exploited by lowering its oxygen affinity; this would probably increase oxygen delivery to the RBCs and improve the patients' clinical phenotype. Allosteric effectors that induce a low-affinity Hb may be useful in this regard. We investigated the effect of a bezafibrate derivative, RSR-4, on the oxygen affinity of RBCs and purified hemolysates containing HbA and HbH. This allosteric effector crosses RBC membranes and binds reversibly to the alpha-chains of deoxy-Hb, decreasing hemoglobin oxygen affinity. The blood used was obtained from a patient with HbH disease (alphaTSaudi homozygote) whose HbH level was 33.5% as measured by high-performance liquid chromatography. Oxygen binding studies were performed in RBCs and purified hemolysates. RBCs incubated in the presence of 500 microM RSR-4 (2-[[[(3,5-dichloroanilino)-carbonyl]methyl]phenoxy]-2-methylpropi onic acid) in standard conditions (pH 7.4, 0.14 M NaCl, 37 degrees C) displayed an increase in their P50 value from 14.5 to 35.2 mm Hg. Oxygen binding studies in purified stripped hemolysates (pH 7.2, 0.1 M NaCl, 25 degrees C) showed that addition of both 500 microM RSR-4 and 1 mM of 2,3 diphosphoglycerate (DPG) led to an 11-fold decrease in oxygen affinity, whereas the addition of the natural effector DPG or RSR-4 alone produced a 2.7- and 5.7-fold decrease, respectively. In both cases, the oxygen equilibrium curves (OECs) were biphasic due to the presence of the noncooperative, high-oxygen-affinity HbH (beta4) component. After addition of RSR-4, the lower part of the OEC (corresponding to HbH) was not shifted compared with the upper part (corresponding to HbA). These results were confirmed by kinetic studies of CO recombination. Both experiments demonstrated that RSR-4 does not affect beta4 Hb. Our findings provide an experimental model for lowering the oxygen affinity of HbA in HbH-containing cells and suggest that the oxygen delivery capability of the latter would be thereby improved.