31P NMR Study of the Kinetics of Binding of myo‐Inositol Hexakisphosphate to Human Hemoglobin

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Abstract
The association and dissociation kinetics of the complexes of myo-inositol hexakisphosphate (P6-instol) 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 105 M−1 and 2 × 109 M−1. 31P NMR spectra of P6-instol 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 P6-instol NMR time scale. This observation cannot be reconciled with a single-step binding mechanism of P6-insitol to hemogloin. Analysis of the spectra revealed the occurrence of additional binding of P6-insitol to both Hb and HbCO. This binding was also observed in pH-stat experiments performed at low ionic strength. 31P NMR exoeriments carried out with hemoglobin of which the α-chain N termini were carbamylated, strongly suggest that these termini constitute the additional binding site for P6-insitol 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-insitol 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.