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 Ca(2+), Mg(2+) or Zn(2+) under experimental conditions close to those of the ileum (pH 8, 37°C), with rather low binding affinity constants (K=4900-11,200M(-1)). These low affinities should facilitate the release of metal ions during intestinal absorption. By contrast, Cu(2+) 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.
Keywords: Caseinophosphopeptide; ITC; Metal chelation; Mineral bioavailability; β-Casein.
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