Simultaneous addition of two ligands: A potential strategy for estimating divalent ion affinities in EF-hand proteins by isothermal titration calorimetry

Capable of providing a detailed thermodynamic picture of noncovalent association reactions, isothermal titration calorimetry (ITC) has become a popular method for studying protein–ligand interactions. We routinely employ the technique to study divalent ion-binding by two-site EF-hand proteins from the parvalbumin- and polcalcin lineages. The combination of high Ca2+ affinity and relatively low Mg2+ affinity, and the attendant complication of parameter correlation, conspire to make the simultaneous extraction of binding constants and –enthalpies for both ions challenging. Although global analysis of multiple ITC experiments can overcome these hurdles, our current experimental protocol includes upwards of 10 titrations – requiring a substantial investment in labor, machine time, and material. This paper explores the potential for using a smaller suite of experiments that includes simultaneous titrations with Ca2+ and Mg2+ at different ratios of the two ions. The results obtained for four proteins, differing substantially in their divalent ion-binding properties, suggest that the approach has merit. The Ca2+- and Mg2+-binding constants afforded by the streamlined analysis are in reasonable agreement with those obtained from the standard analysis protocol. Likewise, the abbreviated analysis provides comparable values for the Ca2+-binding enthalpies. However, the streamlined analysis can yield divergent values for the Mg2+-binding enthalpies – particularly those for lower affinity sites. This shortcoming can be remedied, in large measure, by including data from a direct Ca2+ titration in the presence of a high, fixed Mg2+ concentration.