Analyzing ITC Data for the Enthalpy of Binding Metal Ions to Ligands

Isothermal titration calorimetry (ITC) has become increasingly popular because it is the only method that directly measures the enthalpy change for a reaction. When analysis is done properly and accurate thermodynamic values are obtained, ITC results can be compared to thermodynamic values collected by other methods and can be extrapolated to different conditions. However, incorrect assumptions are sometimes made in metal binding data analysis. To illustrate the importance of data analysis for enthalpy changes, copper (II) was titrated into ethylenediaminetetraacetic acid (EDTA) in three different buffers, allowing for accurate quantification of a metal-buffer interaction (ΔHMB). Introduction The enthalpy, ΔHITC, reported from the fit of titration curve represents the overall heat in the reaction cell. In order to account for all of the processes during a reaction it is recommended to analyze the data and account for each separate chemical event. Analysis may seem overwhelming at first, but it can be broken down into specific incremental events that may occur simultaneously in the reaction cell. Several papers have been published that have reviewed how to analyze ITC data and quantify protons released during a binding event in the ITC (1,2). However, when metal binding reactions are studied, an additional component in ITC data analysis is required due to other heat generating events occurring in the reaction cell. Quantification of the additional buffer-metal chemistry and its added complexity has been previously incorporated into data analysis, yet how these values were calculated is not always easily understood or straightforward (3,4). It is the goal of this paper to show the experimentalist how ΔHMB can be quantified and incorporated in data analysis. One of the major heat producing events in a reaction can be related to the displacement of protons from a binding pocket or binding ligand. Proton coupled equilibria, can originate from protonation of buffer and needs to be understood and quantified as a large portion of ΔHITC. The buffer protonation event can work in favor to the experimentalist as large amounts of heat are