Transposing sequences between fetal and adult hemoglobins indicates which subunits and regulatory molecule interfaces are functionally related

To correlate amino acid sequence changes with hemoglobin function we are ca rrying out a detailed recombinant analysis of the adult hemoglobin/fetal he moglobin (HbA/HbF) systems. The important physiological differences between these two tetramers lie at unspecified sites in the 39 sequence substituti ons of the 146 amino acids in their beta and gamma chains. In this paper, s ignificant differences in the tetramer-dimer dissociation constants (referr ed to as tetramer "strength" or "stability") of adult (HbA) and fetal (HbF) hemoglobin tetramers have been used to probe the relationship between the allosteric, sliding interface and the effects of the allosteric regulator, 2,3-DPG, in promoting oxygen release. The single amino acid difference at t he allosteric interfaces of these two hemoglobins, Glu-43(beta) --> Asp-43( gamma), which is not near the DPG binding site, leads to a significantly lo wer DPG response, approaching that of HbF. The results are inconsistent wit h the long-held idea that the replacement of His-143(beta) in HbA to Ser-14 3(gamma) in HbF is solely responsible for the lowered DPG response in HbF. On the other hand, the Val-1(beta) --> Gly-1(gamma) replacement near the DP G binding site has no effect on the DPG response. The replacement of His-11 6(beta) by the hydrophobic Ile-116(gamma) at the rigid alpha(1)beta(1) inte rface has a marginal yet detectable effect on the allosteric alpha(1)beta(2 ) interface. The results, overall, are interpreted using a model involving electrostatic coupling between certain side chains and extend the concept o f a long-range relationship between some distant regions of the tetramer th at are likely mediated through the central cavity.