The goal of this research is to determine the molecular mechanism and driving forces that regulate communication between the oxygen binding sites of human hemoglobin. The overall oxygen binding properties of hemoglobin are well-documented, however, the molecular basis of positive cooperativity, which is crucial to physiological efficacy, is not understood. The primary functional units within the hemoglobin tetramer have only recently been identified as its alphabeta dimer components, as shown by the distribution of structural energetics among intermediate states. The order of oxygen binding, as well as its release, follows specific combinatorial rules, binding preferentially to both sites on one dimer within the tetramer, then binding to the sites on the remaining dimer. This functional asymmetry within the hemoglobin tetramer is also observed in initial studies on hybrid tetramers, where one alphabeta dimer is normal but the other dimer contains a single amino acid in the cross-dimer interface. The effect of the modification on the cooperative free energy of oxygen binding is felt only on the modified dimer, while the normal dimer is not significantly affected. Dr. Ackers plans to extend these studies for the 02 intermediates using recombinant hemoglobins with designed sets of altered residue sites. Subunit interactions of the recombinant systems will be studied by techniques of kinetics, analytical gel chromatography, cryogenic isoelectric focusing, as well as by direct 02 binding. As the Hb regulatory system is an important prototype for a large family of cooperative multi-site regulatory assemblies, the methods developed by this program should have wide applicability. Deeper understanding of human Hb mechanisms is also of current interest to the potential design of red cell substitute oxygen carriers and hemoglobin-based drug delivery systems.
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