HEMOGLOBIN AND MYOGLOBIN KINETIC STUDIES

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.