Complexometric titrations of protein-bound metal ions: a method for determining binding constants

A method for quantifying the affinity of proteins for specific metal ions has been developed. Both the stoichiometry and the binding constants of the protein-bound metal ion can be determined by titrating protein-bound metal ions with complexometric reagents and observing electrochemically the change in free metal ion concentration. The technique is limited to cases where the affinity of the macromolecule for the metal ion is less than or similar to the affinity of the complexometric reagent for the metal ion. The method has been employed successfully in the study of both Cu(II) and Ag(I) binding to the apoprotein of bovine cuprozinc superoxide dismutase.