Thermodynamics of Bovine Spleen Galectin-1 Binding to Disaccharides: Correlation with Structure and Its Effect on Oligomerization at the Denaturation Temperature

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Abstract
Isothermal titration calorimetry (ITC) measurements of the binding 1-β carbohydrate-substituted galactopyranoside derivatives to galectin-1 from bovine spleen, a dimer with one binding site per subunit, were performed at 283−285 and 298 K. The disaccharides were lactose, methyl β-lactoside, lactulose, 4-O-β-d-galactopyranosyl-d-mannopyranoside, 3-O-β-d-galactopyranosyl-d-arabinose, 2‘-O-methyllactose, lacto-N-biose, N-acetyllactosamine, and thiodigalactopyranoside. The site binding enthalpies, ΔHb, are the same at both temperatures and range from −42.2 ± 3.3 kJ mol-1 for thiodigalactopyranoside to −24.5 ± 0.5 kJ mol-1 for lacto-N-biose, and the site binding constants range from 4.86 ± 0.78 × 103 M-1 for methyl β-lactoside at 297.8 K to 6.54 ± 0.97 × 104 M-1 for N-acetyllactosamine at 281.3 K. The binding reactions are enthalpically driven, exhibit enthalpy−entropy compensation, and, with the exception of N-acetyllactosamine, follow a van't Hoff dependence of the binding constant on temperature. The number of contacts at distances −36 kJ mol-1. Differential scanning calorimetry measurements on solutions of galectin and its disaccharide complexes show that the galectin dimer does not dissociate upon denaturation in contrast to the legume lectins. At the denaturation temperature, the galectin in the absence of sugar exists as a tetramer, and the extent of this association is substantially reduced in the presence of a disaccharide.