Equilibrium Aspects of the Binding of myo‐Inositol Hexakisphosphate to Human Hemoglobin as Studied by 31P NMR and pH‐Stat Techniques

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Abstract
The interaction of myo-inositol hexakisphosphate (P6-inositol) with human Hb was studied as a function of pH using pH-stat techniques and 31P NMR. With the pH-stat method the following data were obtained: the association constants for the P6-inositol/Hb and P6-inositol/HbCO complexes at alkaline and acid pH, respectively, and the proton absorption curves associated with the protein/phosphate interaction for both complexes from pH 5.5 to pH 9. From these data the affinities of P6-inositol towards Hb and HbCO were calculated as a function of pH. The shape of the proton absorption curves was strongly dependent on the ligation state of the Hb molecule. The pH dependence of the 31P NMR spectra of P6-inositol bound to Hb or HbCO provides a monitor for the proton-binding behavior is only slightly dependent on the ligation state of the Hb molecule. The NMR spectral data were interpreted in terms of a model which takes into account the electrostatic interaction between the phosphate groups and positively charged groups on the protein. To account for the discrepancy between the pH-stat and 31P NMR results, i.e., a strong dependence of the proton-absorption curves and a weak dependence of the proton-binding behavior of P6-inositol on the ligation state of the protein, respectively, it is proposed that a conformational change takes place in HbCO upon P6-inositol binding.