Abstract
The values of pKa for β-146 and β-2 histidines in human hemoglobin were determined by the mass spectrometric method using the hydrogen-deuterium exchange reaction. The effects of ligand binding and allosteric interaction on these pKa values were investigated.
The pKa values of β-146 histidine in the oxy- and deoxy-forms of the stripped hemoglobin were little affected by addition of inositol hexaphosphate (7.0 in the oxy- and 8.2 in the deoxy-form). Blocking of the reactive thiol group at β-93 cysteine decreased the pKa values of the amino acid remarkably in both the oxy- and deoxy-forms, whereas the difference in pKa between the oxy- and deoxy-forms (d pKa) remained unchanged. Values of the pKa in the tetramer of
the p-chain were found to be 7.4 in both the oxy- and deoxy-forms. The pKa value in methemoglobin was found to be 7.0.
Values of pKa of β-2 histidine on human hemoglobin were measured mass-spectrometrically after isolation of histidine as the methyl thiohydantoin derivative from deuterated hemoglobin by stepwise Edman degradation. From these measurements a pKa value of 6.9 for this histidine in both oxy- and deoxy-hemoglobin was found in the absence of inositol hexaphos-phate. The pKa value of the histidine in oxyhemoglobin was little affected by inositol hexaphosphate, while the value in deoxyhemoglobin was shifted to a higher value of 7.7 by inositol hexaphosphate.
