H + titration studies of human hemoglobin

Abstract

The normal and differential titration curves of human Hb and HbO 2, of the isolated α- and β-chains, of apohemoglobin and of HbO 2 with the SH groups blocked by iodoacetamide, were determined and analysed. 1. 1. From the differential titration curves of the α-chains and of Hb and HbO 2 it can be concluded that all carboxyl groups and all Lys and Arg residues are titratable in these molecules. 2. 2. Seven His residues are found to be titratable in the α- and 6 in the β-chains. The titration curve of the α-chains could be calculated with only one p K value for the His residues, whereas p K values varying from 5.6 to 8 were necessary to obtain a good fit for the β-chains. 3. 3. Assuming that the acid Bohr groups are carboxyl groups 20 His residues appeared to be titratable in Hb and HbO 2, which means that probably 6 His residues become titratable upon dissociation of hemoglobin into its subunits. 4. 4. In HbO 2 the p K of the β 93 SH groups was found to be 9.9. 5. 5. By blocking the reactive SH groups with iodoacetamide, 2 His residues become titratable which are masked in HbO 2. 6. 6. On removing the heme from hemoglobin probably 8 His residues become unmasked. Assuming that in globin all 6 SH groups are titratable, their p K is found near 9.