Isothermal titration calorimetry measurements of Ni(II) and Cu(II) bindingto His, GlyGlyHis, HisGlyHis, and bovine serum albumin: A critical evaluation

The binding of Ni(II) and Cu(II) to histidine, to the tripeptides GlyGlyHis and HisGlyHis, and to the protein bovine serum albumin has been studied by isothermal titration calorimetry (ITC) to determine the experimental condi tions and data analysis necessary to reproduce literature values for the bi nding constants and thermodynamic parameters. From analysis of the ITC data , we find that there are two major considerations for the use of this metho d to accurately quantify metal ion interaction with biological macromolecul es. First, to determine true pH-independent binding constants, ITC data mus t be corrected for metal ion competition with protons by accounting for the experimental pH and pK(a) values of the metal-binding residues. Second, me tal interaction with the buffer (stability and enthalpy of formation of met al-buffer complex(es)) must be included in the analysis of the ITC data to determine the binding constants and the change in enthalpy. While it may be possible to use a buffer that forms only weak, and therefore negligible, c omplexes with the metal, a buffer that has a strong and well-characterized interaction has the benefit of suppressing metal ion hydrolysis and precipi tation, and of allowing the quantification of high-affinity metal-binding s ites on biological macromolecules. This study has also quantified the contr ibution of the N-terminal imidazole of HisGlyHis to the stability of the Cu (II) and Ni(II) complexes of this protein sequence and has provided new ins ight about Cu(II) binding to albumin.