If a molecule contains two groups that bind a common ligand, then to determine their group dissociation constants it is necessary (1) to assume that some property of the molecule is affected by the degree of ligation of only one of the groups, and (2) to determine how this property varies with the concentration of free ligand. When assignment of group dissociation constants made on this basis suggests that the degree of ligation of each group greatly affects the dissociation constant of the other, then the original assumption that the chosen property is completely unaffected by ligation of the second group is rendered doubtful. Dissociation constants of interacting groups can therefore not be reliably determined.
