Confirmation of a unique intra-dimer cooperativity in the human hemoglobinalpha(1)beta(1) half-oxygenated intermediate supports the symmetry rule model of allosteric regulation

The contribution of the alpha(1)beta(1) half-oxygenated tetramer [alpha bet a:alpha O(2)beta O-2] (species 21) to human hemoglobin cooperativity was ev aluated using cryogenic isoelectric focusing, The cooperative free energy o f binding, reflecting O-2-driven protein structure changes, was measured as (21)Delta G(c) = 5.1 +/- 0.3 kcal for the Zn/FeO2 analog. For the Fe/FeCN analog, (21)Delta G(c) was estimated as 4.0 kcal after correction for a CN ligand rearrangement artifact, demonstrating that ligand rearrangement does not invalidate previous conclusions regarding this species. In the context of the entire Db cooperativity cascade, which includes eight intermediate species, the 21 tetramer is highly abundant relative to the other doubly-li gated species, providing strong support for the previously determined conse nsus partition function of O-2 binding and for the Symmetry Rule model of h emoglobin cooperativity (Ackers et al,, Science 1992;255:54-63). Cooperativ ity of normal human hemoglobin is shown to depend on site-configuration, an d not solely the number of O-2 bound, nor the occupancy of alpha vs. beta s ubunits, Verification of a unique contribution from the alpha(1)beta(1) dou bly-oxygenated species to the equilibrium O-2 binding curve strongly reinfo rces the Symmetry Rule interpretation that the alpha(1)beta(1) dimer acts b oth as a structural and functional element in cooperative O-2 binding. (C) 2000 Wiley-Liss, Inc.