[6] Allosteric equilibrium measurements with hemoglobin valency hybrids

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This chapter discusses the allosteric equilibrium measurements with hemoglobin (Hb) valency hybrids samples.There is a need to correct for the influence of a slightly oxidized sample. Although the oxidation levels can usually be maintained below 5%, this level is worse than it seems when dealing with a tetrameric system. This problem may be aggravated in Hb mutants, natural or artificial, in which the rate of autoxidation is increased. Thus, an understanding of how ferric subunits affect their neighbors within an Hb tetramer is important in order to compare the ligand-binding parameters of different samples. The use of highly oxidized solutions provides another method to study the triply liganded form. With different ferric ligands and the use of external effectors– whether the two-state model is still operational at the triply liganded level can be determined. By comparing different ferric ligands with varying amounts of high- and low-spin ferric hemes, the coupling of the spin state and the allosteric equilibrium can be studied.

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