Binding of divalent metal ions to 1–25 β-caseinophosphopeptide: An isothermal titration calorimetry study

To better understand the mechanism of metal ion transport through the gastrointestinal tract to their absorption sites, isothermal titration calorimetry (ITC) was used to investigate the binding of dicationic metals to β-CN(1–25)4P, a β-casein tetraphosphorylated peptide. ITC technology was found suitable for studying weak bonds between metal ions and phosphopeptides and provided a direct means of thermodynamic and stoichiometric characterisation of complex formation. Thus, one mole of β-CN(1–25)4P binds two moles of Ca2+, Mg2+ or Zn2+ under experimental conditions close to those of the ileum (pH 8, 37 °C), with rather low binding affinity constants (K = 4900–11,200 M−1). These low affinities should facilitate the release of metal ions during intestinal absorption. By contrast, Cu2+ did not bind to β-CN(1–25)4P at pH 8, despite its reported significant affinity towards β-casein and the 1–25 peptide at near-neutral pH.