THERMODYNAMICS OF BOVINE SPLEEN GALECTIN-1 BINDING TO DISACCHARIDES -CORRELATION WITH STRUCTURE AND ITS EFFECT ON OLIGOMERIZATION AT THE DENATURATION TEMPERATURE

Isothermal titration calorimetry (ITC) measurements of the binding 1-b eta-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, methy l beta-lactoside, lactulose, 4-O-beta-D-galactopyranosyl-D-mannopyrano side, 3-O-beta-D-galactopyranosyl-D-arabinose, 2'-O-methyllactose, lac to-N-biose, N-acetyllactosamine, and thiodigalactopyranoside. The site binding enthalpies, Delta H-b, 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 constant s range from 4.86 +/- 0.78 x 10(3) M-1 for methyl beta-lactoside at 29 7.8 K to 6.54 +/- 0.97 x 10(4) M-1 for N-acetyllactosamine at 281.3 K. The binding reactions are enthalpically driven, exhibit enthalpy-entr opy compensation, and, with the exception of N-acetyllactosamine, foll ow a van't Hoff dependence of the binding constant on temperature. The number of contacts at distances <4.0 Angstrom between the disaccharid e and galectin was determined from the energy-minimized conformation o f the complex derived from the X-ray crystallographic structure of the galectin-N-acetyllactosamine complex determined by Liao et al. [Liao, D. I., Kapadia, G., Ahmed, H., Vasta, G. R., and Herzberg, O. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 1428-1432]. The binding enthalpies c alculated from changes in the solvent-accessible surface areas of the galectin binding site upon binding of the disaccharide were in close a greement with the experimental values for lactose, lactulose, lacto-N- biose, and N-acetyllactosamine, ail of which exhibit binding enthalpie s >-36 kJ mol(-1). Differential scanning calorimetry measurements on s olutions of galectin and its disaccharide complexes show that the gale ctin dimer does not dissociate upon denaturation in contrast to the le gume lectins. At the denaturation temperature, the galectin in the abs ence of sugar exists as a tetramer, and the extent of this association is substantially reduced in the presence of a disaccharide.