Determination of galectin-3-ligand interactions by TFS and ITC. Galectin-3 (10 µM) florescence intensity with increased concentrations of LacNAc (A) lactose (C) and galactose (E) were recorded by fluorescence spectroscopy. Numbers indicate the maximum fluorescence wavelength and corresponding fluorescence intensity for the first (no ligand/buffer titration, black) and last (red) titration traces. Inserts in each panel show buffer control titrations. Ligand concentrations vs changes in fluorescence intensity at single wavelength were plotted and analysed using One site- Specific binding model to obtain the galectin-3 binding affinities to LacNAc (B) lactose (D) and galactose (F). Heat changes for titration of galectin-3 (100 µM) with 2 mM LacNAc (G), 4 mM lactose (H) and 20 mM galactose (I) were recorded by ITC (upper panels) with data fit to One set of sites interaction model (bottom panels). Data were plotted as means ± SEM from three independent experiments for each assessment. Panel (J) shows thermodynamic profiles with enthalpy (∆H), entropy (∆S) and Gibbs free energy (∆G) changes associated with galectin-3 binding to LacNAc, lactose and galactose.