Thermodynamics of Bovine Spleen Galectin-1 Binding to Disaccharides: Correlation with Structure and Its Effect on Oligomerization at the Denaturation Temperature † Frederick P. Schwarz,* ,‡ Hafiz Ahmed, § Mario A. Bianchet, | L. Mario Amzel, | and Gerardo R. Vasta § Center for AdVanced Research in Biotechnology and National Institute of Standards and Technology, 9600 Gudelsky DriVe, RockVille, Maryland 20850, Center of Marine Biotechnology, UniVersity of Maryland Biotechnology Institute, Columbus Center, 701 East Pratt Street, Baltimore, Maryland 21202, and Department of Biophysics and Biophysical Chemistry, The Johns Hopkins UniVersity School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205 ReceiVed July 8, 1997; ReVised Manuscript ReceiVed October 15, 1997 ABSTRACT: Isothermal titration calorimetry (ITC) measurements of the binding 1- 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 -lactoside, lactulose, 4-O--D-galactopyranosyl-D-mannopyranoside, 3-O--D-galactopyranosyl-D-arabinose, 2′-O-methyllactose, lacto-N-biose, N-acetyllactosamine, and thiodigalactopyranoside. The site binding enthalpies, ∆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 constants range from 4.86 ( 0.78 × 10 3 M -1 for methyl -lactoside at 297.8 K to 6.54 ( 0.97 × 10 4 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 <4.0 Å between the disaccharide and galectin was determined from the energy- minimized conformation of 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 calculated from changes in the solvent-accessible surface areas of the galectin binding site upon binding of the disaccharide were in close agreement with the experimental values for lactose, lactulose, lacto-N- biose, and N-acetyllactosamine, all of which exhibit binding enthalpies >-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. Lectins bind to carbohydrates such as those on the surfaces of plant and animal cells with a high degree of specificity and effect processes within and without the cell. Although, over 5000 lectins have been isolated from biological systems, their precise biological functions are not yet fully understood. Information on the thermodynamics of their carbohydrate binding properties would help elucidate their biological roles. Previous investigations of the carbohydrate binding thermo- dynamics of plant lectins have shown that these reactions are enthalpically driven with little increase in the heat capacity change and they exhibit enthalpy-entropy com- pensation (1-3). Comparisons of the thermodynamics of binding of various carbohydrate derivatives to the X-ray crystallographic structure of a few of the carbohydrate-lectin complexes have successfully identified the structural deter- minants of the binding reactions (4). Recently, thermody- namic binding studies have been extended to lectins isolated from animal cells, specifically the galectins, to determine their thermodynamic binding properties. In particular, Ram- kumar et al. (5) have shown that the carbohydrate binding reactions of the 14 kDa sheep spleen galectin are also enthalpically driven with little increase in the heat capacity change and they exhibit enthalpy-entropy compensation. If the structure of the carbohydrate-galectin complex were known, then comparison of the thermodynamics of the different carbohydrate derivatives would lead to the identi- fication of the structural determinants such as the intermo- lecular interactions and changes in the solvent-accessible surface area responsible for the specificity of the binding thermodynamics. This knowledge is important in elucidating the relationship between structure and function in lectins, as well as the specific biological role of the galectins. † Supported by Grant 03-4-38512 from the Collaborative Research Program, University of Maryland Biotechnology Institute, to F.P.S. and G.R.V., Grant 95-31 from the Lucille P. Markey Trust Fund, and Grant MCB-94-06649 from the National Science Foundation to G.R.V. * To whom correspondence should be sent. ‡ Center for Advanced Research in Biotechnology and National Institute of Standards and Technology. § University of Maryland Biotechnology Institute. | The Johns Hopkins University School of Medicine. 5867 Biochemistry 1998, 37, 5867-5877 S0006-2960(97)01647-4 CCC: $15.00 © 1998 American Chemical Society Published on Web 04/14/1998