Calorimetric studies of ternary complexes of Ni(II) and Cu(II) nitrilotriacetic acid and N-acetyloligohistidines

The thermodynamics of Cu²⁺- and Ni²⁺-NTA binding to polypeptides containing 2-6 histidine residues in neutral buffered aqueous solutions were characterized by isothermal titration calorimetry (ITC). The apparent dissociation constants of ternary complexes of N-acetylhexahistidine-Ni²⁺-NTA and N-acetylhexahistidine-Cu²⁺-NTA were 69.5 μM and 27.1 μM, respectively. Decreased number of histidine residues in the oligohistidine fragment caused gradual decrease in the stability of the mixed metal complexes and a change in the binding stoichiometry from ∼1:1 for N-acetylhexahistidine and N-acetylpentahistidine to ∼0.5:1 for N-acetyltetrahistidine and ∼0.3:1 for N-acetyltrihistidine, N-acetyldihistidine and N-acetylhistidine ligands. In all cases, the binding interactions are shown to be enthalpically favored. Other metal cations formed less stable mixed metal complexes with stability constants in the order of Cu²⁺ > Ni²⁺ > Mg²⁺, Co²⁺, Zn²⁺ >> Mn²⁺, Ca²⁺. The significance of the data is discussed in terms of important practical applications.