Titratable Histidine Research Articles

The Proton-binding Behavior of Human Hemoglobin and Its Subunits in Their Native State

Abstract The titration of the histidyl and lysyl residues has been investigated in oxy- and deoxyhemoglobin, in apohemoglobin, and in the isolated polypeptide chains with sulfhydryl groups blocked by p-hydroxymercuribenzoate (αpmb and βpmb). The number of titratable histidyl residues was found to be 5 or 6 per chain in hemoglobin and probably 7 in apohemoglobin and in the isolated αpmb and βpmb chains. This indicates that the number of titratable histidines per heme increased by 1 or 2 units per heme when the heme was removed or when the αpmb and βpmb chains were separated. The protonation of these new exposed residues was irrelevant to the helical structure of hemoglobin. The pKint of the lysine residues appeared to vary in the different proteins, being 10.6 in hemoglobin, 10.2 in apohemoglobin, 10.5 in the βpmb chains, and 10.3 in the αpmb chains. The difference shown between hemoglobin and apohemoglobin could indicate that the ionization of some groups in this class is sensitive to conformational changes. The difference in protons bound between apohemoglobin and oxyhemoglobin is consistent with the presence of at least 1 more histidyl residue in apohemoglobin and with a difference in the pK of some e-NH2 group. The difference in protons bound by ferrihemoglobin and oxyhemoglobin proved to be essentially accounted for by the ionization of the water molecule coordinated with the trivalent iron atom.