Equilibrium aspects of the binding of myo-inositol hexakisphosphate to human hemoglobin as studied by 31P NMR and pH-stat techniques.
The interaction of myo-inositol hexakisphosphate (P6-inositol) with human hemoglobin has been 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/deoxyhemoglobin and P6-inositol/carboxyhemoglobin 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 affinities of P6-inositol towards deoxyhemoglobin (Hb) and carboxyhemoglobin (HbCO) have been calculated as a function of pH. The shape of the proton absorption curves was found to be strongly dependent on the ligation state of the hemoglobin molecule. The pH dependence of the 31P NMR spectra of P6-inositol bound to Hb or HbCO provides a monitor for the proton-binding behaviour of the phosphate groups of P6-inositol when present in the central cavity of the protein. It appears that this behaviour is only slightly dependent on the ligation state of the hemoglobin molecule. The NMR spectral data were interpreted in terms of a model which takes into account the electrostatic interaction between the phosphate groups within the P6-inositol molecule as well as 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 behaviour 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.
D002263:Carboxyhemoglobin / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010833:Phytic Acid / Q000097:blood*; D011485:Protein Binding
D006454:Hemoglobins; D010833:Phytic Acid; D002263:Carboxyhemoglobin