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.
D002263:Carboxyhemoglobin / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D010833:Phytic Acid / Q000097:blood*; D011485:Protein Binding
D006454:Hemoglobins; D010833:Phytic Acid; D002263:Carboxyhemoglobin