Demonstration of inositol hexaphosphate induced changes in structure at ligand binding sites in carp hemoglobin carbonyl

Infrared and ¹³C NMR spectra for carbonyl ligands bound to hemoglobin from carp, Cyprinus carpio, at pH 6.0 and 25°C undergo marked shifts when inositol hexaphosphate (IHP) is added to the solution. The relative intensities of the two C-0 stretch bands at 1951 and 1968 cm^-1 due to two ligand site conformers are altered by IHP in the same manner as found upon lowering the pH in the absence of IHP. Both the lowering of pH and the binding of IHP enhance the 1968 cm^-1 band conformer, the conformer normally of much lower stability. ¹³C NMR spectra indicate the C0 sites for only one type of subunit, probably the β-subunit, are altered by IHP. These findings demonstrate directly that the observed IHP-induced shift from high affinity (R) to a low affinity (T) form is accompanied by a significant change in ligand binding site structure at two of the four subunits.