Binding of zinc caseinophosphopeptides as assessed by isothermal titration calorimetry

Introduction: Caseinophosphopeptides (CPPs) are the phosphorylated peptides derived from β-casein (β-CPP) or αs-casein (αs-CPP). These molecules are known to be biologically active in that they exert a positive effect on the absorption and metabolism of calcium and other minerals (iron, zinc) (Bouhallab & Bouglé, 2004). In the present study, we report on the ability of CPPs to bind zinc using isothermal titration calorimetry. Methods: The pools of CPPs were obtained by simulated gastrointestinal digestion from α-casein and β-casein and selective precipitation with CaCl2, and identified by HPLC-ESI-MS/MS according the method of García-Nebot et al.(2010). For the ITC assays, the CPP preparations and zinc solutions (ZnCl2) were prepared with buffer HEPES (pH 7), MES (pH 6), or with deionized water (pH 5). Isothermal titration measurements were carried out on a VP-ITC Microcalorimeter (Microcal). Results and discussion: Both samples (β-CPP and αs-CPP) interact with zinc and the interactions were found to be endothermic. The experimental results obtained with the two CPP preparations were better fitted using a model involving four binding sites. The shape of the curve indicates that all binding sites belong to the same family. The process was enthalpically unfavourable (interval 4x103-1.5x104 cal/mol) and entropically favourable (interval 30-65 cal/mol) for three of the four binding sites. The association constants of Zn+2 to the different sites of CPPs ranged between ≈103 M-1 and ≈1.5x105 M-1. The affinity constants were decreased to about 103 M-1 by decreasing the pH value underlying theinvolvement of electrostatic forces in the formed complexes. Conclusion: The association constants obtained reflect the relative homogeneity and low affinity of the binding, fact that could allow a progressive release of zincin the intestinal lumen and facilitating thus its absorption