The proton-binding behavior of human hemoglobin and its subunits in their native state.

The titration of the histidyl and lysyl residues has been investigated in oxyand deoxyhemoglobin, in apohemoglobin, and in the isolated polypeptide chains with sulfhydryl groups blocked by p-hydroxymercuribenzoate (gMB and BP”“). 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 sMB and pPMB 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 aPMB and fiPMB 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 pPMB chains, and 10.3 in the gMB 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 difierence 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.