Isothermal titration calorimetry (ITC) is a standard technique to determine the affinity between a protein and a small-molecule compound. The heat absorbed or released upon the protein-ligand binding is measured in ITC. In this study, we used the technique to verify the Kd values obtained by FTSA and SFA. The Kd of compounds 2, 3, 4, 8, 9, 12, 13 and 16 binding to CA II were measured by ITC. The experiments were performed using the iTC200 instrument (Malvern Instruments Ltd, Malvern, UK). Protein concentration in the cell was 10–20 μM and the compound concentration in the titration syringe was 100–200 μM. The 50 mM sodium phosphate buffer with 50 mM NaCl, pH 7.5, was used both for the protein and compound solutions. Experiment consisted of 19–20 injections of 2 μL (with the first smaller injection of 0.4 μL) every 180 s at a reference power of 5.0 μcal/s (20.9 μJ/s) and the stirring speed of 750 rpm, temperature 25°C. All ITC experiments were repeated at least twice. The data analysis was performed using Microcal ITC module of the Origin 7.0 software using the single-site binding model.

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