Localization of pyridoxal-5-phosphate covalently bound to the human hemoglobin molecule. A spectrofluorimetric study

Human apohemoglobin tryptophan residues were localized in the regions of th e protein globule with restricted mobility. By the method of dynamic quench ing of phosphopyridoxyl chromophore fluorescence, the heterogeneity of pyri doxal-5-phosphate molecules covalently bound to the human hemoglobin molecu les was determined from the accessibility to solvent. The first four pyrodo xal-5-phosphate molecules are localized in the hydrophobic regions of the h emoglobin molecule; at the same time, they have a high mobility. One of the se molecules is situated at the site inaccessible to the solvent, which coi ncides with the anion-binding center of the oxyhemoglobin molecule. The nex t pyridoxal-5-phosphate molecules modify the surface amino groups of the pr otein. In the apohemoglobin molecule, the pyridoxal-5-phosphate binding sit es are more exposed to the solvent, as compared to hemoglobin. In the hemog lobin molecule modified by pyridoxal-5-phosphate, an effective electron eli citation energy transfer from tryptophan residues to phosphopyridoxyl chrom ophores occurs. The effective distances between tryptophanyls of single sub units of hemoglobin and the covalently bound pyridoxal-5-phosphate molecule were estimated to be 19 Angstrom for the alpha-subunit and 17 Angstrom for the beta-subunit.