A proposed solution for the determination of the ionization constants of sets of ionizing groups in proteins

A mixture of acids with known p K ′ and in a known concentration was dissolved to simulate the ionic character of hemoglobin. The titration curves of the mixture were obtained in water and 3.6 M KCl at 20°C. These curves were subjected to analysis by a reiterative curve‐fitting procedure to determine if one could evaluate both the group ionization constants and the numbers of groups. This approach was successful in that the calculated parameters were within the experimental error encountered in obtaining these constants on each of the model compounds. However, analysis for the electrostatic interaction parameter w indicated that there was a possible effect on the ionization of formic acid, pyridine, imidazole, and the α‐amino group of glycylglycine in water and on imidazole and the α‐amino group of glycylglycine in 3.6 M KCl.