| Abstract
| - The complete binding cascade of human hemoglobin consists of eight partially ligatedintermediates and 16 binding constants. Each intermediate binding constant can be evaluated via dimer−tetramer assembly when ligand configurations within the tetramer are fixed through the use of hemesiteanalogs. The Zn/Fe analog, in which the nonbinding Zn2+ heme substitutes for deoxy Fe2+ heme, alsopermits direct measurement of O2 binding to the remaining Fe2+ hemesites within the symmetricallyligated Hb tetramers. Measurement of O2 binding over a range of Zn/Fe Hb concentrations to bothα-subunits (species 23) or to both β-subunits (species 24) shows noncooperative binding and incompletesaturation of the available Fe2+ hemesites. In contrast, the asymmetrically ligated Zn/FeO2 species 21, inwhich both oxygens are bound to one of the dimers within the tetramer, exhibits positive cooperativityand >90% ligation under atmospheric conditions. These properties are confirmed in the present study bymeasurement of the rate constant for tetramer dissociation to free dimer. The binding constants thus derivedfor these partially ligated intermediates are consistent with the stoichiometric constants measured fornative hemoglobin by standard O2 binding techniques, providing additional evidence that Zn2+-hemesubstitution provides an excellent deoxy hemoglobin analog. There is no evidence that Zn-substitutionstabilizes a low-affinity form of the tetramer, as previously suggested. These characterizations demonstratedistinct, nonadditive physical properties of the doubly ligated tetrameric species, yielding an asymmetricdistribution of cooperativity within the cascade of O2 binding by human hemoglobin.
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