The dependence of ligand binding constant on pH. (a) Comparison of TFMSA (filled symbols) and EZA (open symbols) binding pH profiles. Curves show the fits according to Eq. (1) using parameters listed in the Tables 2 and 3. Datapoints show K
s obtained by ITC in phosphate (diamonds) and TRIS (triangles) buffers and by TSA (squares). (b) The dissection of the Gibbs free energies of EZA binding to hCA XIII as a function of pH. Datapoints are calculated from panel (a). Solid bent line shows the fit according to Eq. (1). Solid straight line shows the position of the intrinsic Gibbs free energy of binding which is independent of pH. Dashed and dotted lines show the contributions of the fractions of deprotonated EZA and protonated CA, respectively. (c) The observed and intrinsic Gibbs free energies of TFMSA binding to hCA XIII as a function of pH. Solid bent line is the fit according to Eq. (1) recalculated to Gibbs free energies. Straight line shows the intrinsic Gibbs free energy of TFMSA binding, independent of pH.