This paper investigates the use of isothermal titration calorimetry (ITC) as a tool for studying molecular systems in which weaker secondary interactions are present in addition to a dominant primary interaction. Such systems are challenging since the signal pertaining to the stronger primary interaction tends to overshadow the signal from the secondary interaction. The methodology presented here enables a complete and precise thermodynamic characterization of both the primary and the weaker secondary interaction, exemplified by the binding of beta-cyclodextrin to the primary and secondary binding sites of the bile salt glycodeoxycholate. Global regression analysis of calorimetric experiments at various concentrations and temperatures provide a precise determination of Delta H, Delta G degrees, and Delta C-p for both binding sites in glycodeoxycholate (K1 = 5.67 +/- 0.05 x 10(3) M-1, K2 = 0.31 +/- 0.02 x 10(3) M-1). The results are validated by a C-13 NMR titration and negative controls with a bile salt with no secondary binding site (glycocholate) (K = 2.96 +/- 0.01 x 10(3) M-1). The method proved useful for detailed analysis of ITC data and may strengthen its use as a tool for studying molecular systems by advanced binding models.
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