Equilibrium Oxygen Binding to Human Hemoglobin Cross-linked Between the α Chains by Bis(3,5-dibromosalicyl) Fumarate

Abstract

Oxygen equilibrium curves of human hemoglobin Ao (HbAo) and human hemoglobin cross-linked between the α chains (α α Hb) by bis(3,5-dibromosalicyl) fumarate were measured as a function of pH and chloride or organic phosphate concentration. Compared to HbAo, the oxygen affinity of α α Hb was lower, cooperativity was maintained, although slightly reduced, and all heterotropic effects were diminished. The major effect of α α-cross-linking appears to be a reduction of the oxygen affinity of R-state hemoglobin under all conditions. However, while the oxygen affinity of T-state α α Hb was slightly reduced at physiologic chloride concentration and in the absence of organic phosphates, KT was the same for both hemoglobins in the presence of 2,3-diphosphoglycerate (or high salt) and higher for α α Hb in the presence of inositol hexaphosphate. The reduced O2 affinity arises from smaller binding constants for both T- and R-state α α Hb rather than through stabilization of the low affinity conformation. All four Adair constants could be determined for α α Hb under most conditions, but a3 could not be resolved for HbAo without constraining a4, suggesting that the cross-link stabilizes triply ligated intermediates of hemoglobin.