Isothermal Titration Calorimetry (ITC) is a commonly used technique to determine the stoichiometry, affinity, and enthalpy of binding reactions in solution [1,2]. In many cases, conclusions are made on the basis of titrations performed in one buffer system. In this article, we will demonstrate a potential problem that may lead to incorrect conclusions about the energetics of a given reaction based on data acquired using a single buffer system, which originates from changes in pKas of titratable groups upon complex formation. In a binding reaction, pKas of titratable group on the protein and the ligand may shift upon formation of the complex. As a result, protons are either released or taken up by these groups and the buffer responds to such changes either by releasing or taking up protons to maintain pH. Therefore, the observed enthalpy (ΔHobs), includes contributions from the intrinsic enthalpy of binding (ΔHint) and the heat of ionization of the buffer (ΔHion). ΔHint can be determined based on the following equation and using different buffers with different ΔHion. While the y-intercept yields ΔHint, the slope of the line yields the net protonation, Δn. ΔHobs=ΔHint+Δn (ΔHion) It is important to note, that the ΔHint, determined in this manner, will still include contributions from the heat of ionization of the titratable groups in the protein–ligand complex. Furthermore, it is also a common practice to include a certain level of salt (50-200 mM) to eliminate the heat of nonspecific binding of the buffer to the protein. The importance of determining the intrinsic enthalpy of a reaction can be seen in the experimental data shown in Figure 1. Titration of the aminoglycoside phosphotransferase(3′)-IIIa (APH) with the aminoglycoside antibiotic netilmicin yielded an endothermic reaction when the titration was performed in Tris-HCl pH 7.4 (Figure 1, right panel), while the same titration in PIPES buffer at the same pH yielded an exothermic reaction (Figure 1, left panel). Tris-HCl, ACES, and PIPES buffers have ΔHion of 11.7, 7.5, and 2.7 kcal/mol respectively [3]. Analysis of these data (Table 1) as described above clearly shows that this is an exothermic reaction with the ΔHint=-7.7 ± 0.8 kcal/mol.

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