Quaternary equilibrium analysis of the imidazole methemoglobin bound with inositol hexaphosphate

The visible and proton NMR spectral responses of imidazole methemoglobin by the binding of inositol hexaphosphate were examined in the 2-40 degrees C range. The magnitude of the +/- (inositol hexaphosphate) visible difference spectrum increased and the intensity of the 33 ppm NMR peak decreased with lowering of the temperature. The NMR results were quantitatively analyzed with a simple two-state allosteric model. The results show that the T conformer fraction is 0.6 at 20 degrees C and that the equilibrium shifts toward the T state at lower temperature. The large changes in delta H and delta S associated with the equilibrium suggest participation of numerous factors in the determination of the equilibrium position. The increase in the T conformer population of imidazole methemoglobin, which is pure low-spin, suggests that the appearance of the T state with decreasing temperature is not directly coupled to an increase in spin of the heme iron.