Determination of galectin-4N-ligand interactions by TFS and ITC. Galectin-4N (5 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 figure show buffer control titrations. Ligand concentrations vs changes in fluorescence intensity were plotted and analysed using One site- Specific binding model to obtain the binding affinities to LacNAc (B), lactose (D) and galactose (F). Heat changes for titration of galectin-4N (100 µM) with 20 mM LacNAc (G), 30 mM lactose (H) and 200 mM galactose (I) were measured by ITC (upper panels) with data fit to One set of sites interaction model (bottom panels). Note the very small heat change by galactose which indicates very little, if any, binding taking place (I). 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-4N binding to LacNAc and lactose.