This proposal is concerned with a study of: the kinetics and mechanisms of the binding of oxygen and CO to various hemoglobins, the assembly reactions of Hb, and the functional properties of intermediates in the assembly process. In the assembly reactions, we are interested in determining at what stages and to what extent co-operativity in ligand binding appears. These reactions are followed in laser light-scattering, fluorescence anisotropy stopped-flow, tandem flow, and flow-flash experiments. From the light-scattering and fluorescence measurements, relative populations of intermediates can be determined. Tandem flow studies allow us to trap the transient intermediates and determine their functional properties. Detailed stopped-flow studies of ligation are directed toward unravelling the early phases of ligation and conformational changes in T-state human hemoglobin. Laser photolysis will probe details of geminal and bimolecular CO recombination in hemo-globins which are at the two extremes of known CO reactivity. In one hemoglobin, n-sec studies can be carried out on both the T and R states. These studies will provide information on energy barriers for ligation on the heme cavity and for protein channels which may be involved in the bimolecular reactions. Recent synthetic advances allow us to propose experiments directed toward elucidating the roles of the alpha and beta chains in the Root effect, the most extreme of Bohr effects. This is to be accomplished by synthesizing both the modified hemoglobin and various blocked or valency-hybrids, permitting ligation to the single reactive chain to be followed in both R and T states. The simplest of co-operative Hbs, one that is dimeric, without a Bohr effect or detectable alpha-beta heterogeneity, will be studied in an attempt to provide a description of the most elementary co-operative unit. In general, these studies are all directed toward improving and enlarging our understanding of protein-protein and protein-ligand interactions; in particular, the study is of subunit interactions and the binding of oxygen and Co to diverse hemoglobins.
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