31P NMR study of the kinetics of binding of myo-inositol hexakisphosphate to human hemoglobin. Observation of fast-exchange kinetics in high-affinity systems.

The association and dissociation kinetics of the complexes of myo-inositol hexakisphosphate (P6-inositol) with deoxyhemoglobin (Hb) and carboxyhemoglobin (HbCO) have been investigated by 31P NMR between pH 6.8 and pH 5.5. These complexes represent high-affinity systems with binding constants varying between 10(5) M-1 and 2 X 10(9) M-1. 31P NMR spectra of P6-inositol were recorded in the presence of hemoglobin as a function of the P6-inositol/hemoglobin molar ratio. It appeared that the exchange of the polyphosphate molecule between the solution and the central cavity binding site is fast on the NMR time scale. This observation cannot be reconciled with a single-step binding mechanism of P6-inositol to hemoglobin. Analysis of the spectra revealed the occurrence of additional binding of P6-inositol to both Hb and HbCO. This binding was also observed in pH-state experiments performed at low ionic strength. 31P NMR experiments carried out with hemoglobin of which the alpha-chain N termini were carbamylated, strongly suggest that these termini constitute the additional binding site for P6-inositol. A model is proposed which accounts for the enhancement of exchange kinetics in these high-affinity systems. In this model a rapid migration is assumed for P6-inositol between the central cavity binding site and an entry/leaving site on the hemoglobin molecule. Based on this model 31P NMR linewidths and chemical shift patterns for this three-site exchange problem were calculated.

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.