EVALUATION OF THE STOICHIOMETRY AND ENERGETICS OF CARBOHYDRATE-BINDING TO RICINUS-COMMUNIS AGGLUTININ - A CALORIMETRIC STUDY

High-sensitivity isothermal titration calorimetry has been used to inv estigate the thermodynamics of binding of Ricinus communis agglutinin to galactose, lactose and their derivatives in the temperature range 2 80.5-298 K. The present study unequivocally establishes the carbohydra te-binding stoichiometry of the tetrameric agglutinin from castor bean as two, i.e. the (As-sB)(2)-type tetramer of the agglutinin has two e quivalent sites that are noninteracting and independent. The site bind ing constants range from 2.2 x 10(3) M-1 at 282 K for galactose to 4.8 4 x 10(4) M-1 at 281 K for N-acetyl-lactosamine. The binding enthalpie s range from -21.9 kJ.mol(-1) at 293 K for 4-methylumbelliferyl-beta-g alactoside to -50.2 kJ.mol(-1) at 292.9 K for thiodigalactoside. The o bservation of limited entropy-enthalpy compensation for binding of the sugars to the lectin indicates that reorganization of water molecules plays an important role in binding. As the slope of the compensation plot is greater than unity, the reactions are largely enthalpically dr iven. These studies show that the stronger binding of N-acetyl-lactosa mine than lactose is due to a favourable interaction between the aceta mido group of the reducing-end N-acetylglucosamine of the former and t he corresponding loci in the agglutinin molecule. Preferential binding of methyl-beta-galactoside over methyl-alpha-galactoside also indicat es the apolar nature of the interaction with the methyl group of the f ormer sugar.