An Isothermal Titration Calorimetry Study: Complex Binding Isotherms obtained from the Interaction between Phytate and Tannins with Proteins

In a single Isothermal Titration Calorimetry (ITC) experiment, the Gibbs free energy (∆G), the enthalpy (∆H), entropy (∆S), specific heat capacity (ΔCp) and stoichiometry of binding (n) can be identified. Non-sigmoidal binding isotherms are particularly difficult to interpret, yet these are routinely found in ITC studies. ITC binding studies at 25 °C between tannins and bovine serum albumin (BSA) produced biphasic binding isotherms that were suggested to be indicators of positive cooperative binding. However, reducing the temperature to 10 °C produced monophasic binding isotherms, which raised questions regarding the validity of these binding isotherms being classified as positively cooperative binding. The biphasic binding isotherm had two separate temperature dependences, with the initial stage producing a positive ΔCp and the second stage a negative one. Subsequently, this binding isotherm was suggested to essentially be a composite of two crosslinking events. This idea of two different crosslinking events rather than positive cooperativity was supported by an ITC study on a phytate-lysozyme interaction. The ITC study showed that n became lower with decreasing temperature suggesting a crosslinking process became more extensive. Consequently, phytate may be less able to impair protein bioavailability at lower temperatures. Continuous Isothermal Titration Calorimetry (cITC) is a technique based on a continuous slow injection of titrant. The cITC binding isotherms obtained from the lysozyme-phytate interaction indicated that positive binding cooperativity was not occurring. To determine how phytate can be transported around the blood, an ITC study was performed using phytate and BSA in phosphate buffered saline pH 7.4. No binding was detected meaning that BSA does not transport phytate around the blood. However, in 10 mM ammonium acetate at pH 4 binding between BSA and phytate occurred. Conclusively, this reinforces earlier work that binding assays should be done in physiological conditions otherwise findings could be spurious.