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 (KbH or b eta(4)) disease have higher proportions of HbH and more severe tissue hypoxia than patients with the deletion genotype. Because these patien ts' red blood cells (RBCs) contain mainly two Hb species, HbH and HbA, the high proportion of HbA can be exploited by lowering its oxygen af finity; this would probably increase oxygen delivery to the RBCs and i mprove the patients' clinical phenotype. Allosteric effecters that ind uce a low-affinity Hb may be useful in this regard. We investigated th e 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 ct-chains of deoxy-Hb, decreasing hemoglobin oxygen affinity. The blood used was obtained hem a patient with HbH disease (alpha(TSaudi) homozygote) wh ose HbH level was 33.5% as measured by high-performance liquid chromat ography. Oxygen binding studies were performed in RBCs and purified he molysates. RBCs incubated in the presence of 500 mu M RSR-4 nilino)-ca rbonyl]methyl]phenoxy]-2-methylpropionic acid) in standard conditions (pH 7.4, 0.14 M NaCl, 37 degrees C) displayed an increase in their P-5 0 value from 14.5 to 35.2 mm Hg. Oxygen binding studies in purified st ripped hemolysates (pH 7.2, 0.1 M NaCl, 25 degrees C) showed that addi tion of both 500 mu M RSR-4 and 1 mM of 2,3 diphosphoglycerate (DPG) l ed to an Ii-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 d ecrease, respectively. In both cases, the oxygen equilibrium curves (O ECs) were biphasic due to the presence of the noncooperative, high-oxy gen-affinity HbH (beta(4)) component. After addition of RSR-4 the lowe r part of the OEC (corresponding to HbH) was not shifted compared with the upper part (corresponding to HbA). These results were confirmed b y kinetic studies of CO recombination. Both experiments demonstrated t hat RSR-4 does not affect beta(4) Hb. Our findings provide an experime ntal model for lowering; the oxygen affinity of HbA in HbH-containing cells and suggest that the oxygen delivery capability of the latter wo uld be thereby improved.