The binding of EZA and TFMSA to hCA XIII by isothermal titration calorimetry (ITC). (a) Raw ITC curve of EZA binding to hCA XIII at pH 7.0, TRIS chloride buffer, at 25°C. (b) Integrated ITC curves of EZA binding to hCA XIII in sodium phosphate buffer, 25°C, at several pHs: 6.5 (■), 7.5 (◯), and 8.5 (▲). (c) Integrated ITC curves of EZA binding to hCA XIII in TRIS chloride (◊) and sodium phosphate (▲) buffer, pH 8.5, at 25°C. (d) Observed integral enthalpies of EZA (□) and TFMSA (◆) binding to hCA XIII as a function of buffer deprotonation enthalpy, pH 7.0, at 25°C in the following buffers (in the order of increasing enthalpy of deprotonation, shown in the brackets, in kJ/mol): Pi (5.1), PIPES (11.5), MES (15.5), HEPES (21.0), MOPS (22.2), Imidazole (36.6), and TRIS (47.6). (e) Integral observed enthalpies of EZA binding to hCA XIII as a function of temperature. The slope is equal to the observed heat capacity of binding.