Research under this program has yielded the first determination of cooperativity contributions by all eight configurationally-distinct O2 bound intermediates of human hemoglobin at physiological pH. Results showed that the site-specific distribution of O2 affinities through the 16-step binding cascade does not conform to either of the traditional allosteric models ("concerted" MWC or "sequential" KNF). However, the experimentally-resolved distribution was fully consistent with the Symmetry Rule model (Ackers et. al (1992) Science 255, 54) which incorporates both sequential and concerted structural transitions according to site-specific rules. In the next phase, these studies will be extended to learn how the O2-linked proton release (i.e. the physiologically important Bohr effect) is modulated at all sixteen reaction steps of the binding cascade. To accomplish this objective, the ten combinatorial forms of human hemoglobin having 0 -> 4 O2 bound will be investigated at a series of pH values (7.0 - 9.5) to evaluate their site-specific contributions to the classical Bohr effect. Experimental methods developed previously under this program for hybrid tetramer studies will be employed (i.e., stopped-flow and haptoglobin kinetics, isoelectric focusing, dimer hybridization, analytical gel chromatography and direct O2 binding). The resulting databases will be analyzed using thermodynamic linkage theory to yield the 16 site-specific Bohr proton contributions of the cascade.
The significance of this research lies both in its methodological innovation for experimentally dissecting the functional rules of a complex macromolecular system at the site-specific level and in the importance of human hemoglobin itself, as a prototypic model for numerous other allosteric proteins. While the importance of understanding complex multi-subunit protein systems is widely recognized, as a result of the explosive growth of genomic and proteomic databases and the numerous discoveries from cell biology and the exciting goals of protein design, there is much ongoing interest and debate over the molecular mechanisms of allosterically-regulated proteins, including that of human hemoglobin.
