3.4. Isothermal Titration Calorimetry (ITC)

The interactions between the molecules SeDG and unsym(Se) with Gal-1 and Gal-3^CRD were performed by isothermal titration calorimetry (ITC) experiments [31]. The analyses were carried out on an ITC200 calorimeter (MicroCal/GE Healthcare, Boston, MA, USA) at 298 K in 20 mM NaP, 150 mM NaCl pH 7.5 buffer. SeDG and unsym(Se) (500 μM) were dissolved in the same protein buffer and were titrated into a solution of Gal-1 or Gal-3^CRD (20 μM). A solution of 39.4 μL of the ligands was titrated in aliquots of 1.5 μL into a cell containing 270 μL of protein. Injections were performed every 150 s, for a total of 27 injections (0.4 μL for the first injection), with a 1000 rpm stir speed. Control experiments were carried out by performing identical injections of SeDG and unsym(Se) into the cell containing buffer without protein. The binding stoichiometry, enthalpy, and equilibrium association constants were determined by fitting the corrected data to a one-set-of-site-binding model with MicroCal analysis software (GE Healthcare) and are summarized in Table 1.