Isothermal Titration Calorimetry

The thermodynamics of a metal-peptide interaction tells about how the metal is binding, what the driving force is, and if other metals or peptides can effectively compete under biological conditions. There are many techniques that are used to determine the equilibrium binding constant, but only calorimetry accurately measures the enthalpy that is absorbed or released during binding. Isothermal titration calorimetry (ITC) is an attractive technique because it both measures the binding enthalpy and the equilibrium constant (Keq). Indeed, in one experiment the enthalpy and equilibrium constant are measured directly, and Gibbs free energy (ΔG°) and entropy (ΔS°) are determined from these experimental values, ΔG° = -RTln(Keq) = ΔH° – TΔS°.

The main components of a titration microcalorimeter are shown below. At its core is an adiabatic jacket that houses two cells of equal volume: the sample cell which typically holds the biomolecule and is where the reaction takes place; and the reference cell which is filled with water. Each of the cells have heaters that maintain constant temperature and thermocouples that provide feedback to the heaters to ensure both cells are at the same temperature. Inserted into the sample cell is the syringe that injects precise volumes of titrant, typically a metal solution, and stirs to ensure rapid mixing. As each aliquot of titrant is injected and a reaction occurs, the ITC must adjust the power supplied to the sample cell heaters to maintain the same experimental temperature as the reference cell.

If the binding event is exothermic, there will be a decrease of power to the sample cell and, conversely, if the binding event is endothermic there will be an increase of power. The power supplied to the sample cell is plotted versus time to produce the raw data consisting of injection peaks, which are integrated and normalized for the concentration of the titrant to produce a thermogram that is then fit to a non-linear least-squares mathematical model to obtain the stoichiometry at inflection (n), the apparent equilibrium constant (KITC) and the apparent binding enthalpy (ΔHITC). The parameters KITC and ΔHITC are a summation of all competing equilibria and a post hoc analysis must be performed (see Grossoehme, et al.) in order to determine the actual equilibrium constant and enthalpy for a metal-protein binding event (KMP and ΔHMP, respectively).