Abstract
The binding of Artocarpus integrifolia lectin (jacalin) to 4-methylumbelliferyl (Meumb)-glycosides, Gal alpha Meumb, Gal beta Meumb, GalNAc alpha Meumb, GalNAc beta-Meumb, and Gal beta 3GalNAc beta Meumb was examined by extrinsic fluorescence quenching titration and stopped flow spectrofluorimetry. The binding was characterized by 100% quenching of fluorescence of Meumb-glycosides. Their association constants range from 2.0 x 10(4) to 1.58 x 10(6) M-1 at 15 degrees C. Entropic contribution is the major stabilizing force for avid binding of Meumb-glycosides indicating the existence of a hydrophobic site that is complementary to their methylumbelliferyl group. The second order association rate constants for interaction of these sugars with lectin at 15 degrees C vary from 8.8 x 10(5) to 3.24 x 10(6) M-1 S-1, at pH 7.2. The first order dissociation rate constants range from 2.30 to 43.0 S-1 at 15 degrees C. Despite the differences in their association rate constants, the overall values of association constants for these saccharides are determined by their dissociation rate constants. The second order rate constant for the association of Meumb-glycosides follows a pattern consistent with the magnitude of the activation energies involved therin. Activation parameters for association of all ligands illustrate that the origin of the barrier between binding of jacalin to Meumb-glycosides is entropic, and the enthalpic contribution is small. A correlation between these parameters and the structure of the ligands on the association rates underscores the importance of steric factors in determining protein saccharide recognitions.
Highlights
From the Molecular Biophysics Unit,Indian Institute of Science, Bangalore 560 012, India and the YRosewell Park Memorial Institute, New York, New York 10018
Meumb-glycosidesindicating the existencoef a hydrophobic site that is complementary to their methylumbelliferyl group
In order to further probe the role of bulkier substituents at the anomeric position, we have studied the interaction between 4-methylumbelliferyl glycosides of galactose, N-acetylgalactosamine and the T-antigenic dissacharide and jacalin
Summary
The concentration of fluorescent sugars was determined using the molar extinction coefficient (e) value of1.36 X lo M" cm" a t 318 nm [11]. M" (S.E.;n = 4) at 28.2'C and the values of the intercepts at theabscissa, n,equal to 3.6 k 0.18 (S.E.;n = 4), 4.0 f 0.25 (S.E.; n = 4), and 3.8 f 0.22 (S.E.;n = 4), respectively, for jacalin with M,of 66,000, indicating that the lectin is tetrameric in nature. Meumb-glycosides fluorescence,the KOvalues for their binding were calculated by the method of Chipman et al [14] by plotting which allows averaging of over 10 or more values. The values for other Meumb-glycoside range from 2.0 x IO4 (GalpMeumb) ciating the complex with a 10 mM solution of an inhibitory sugar, to 1.58 X lo6M-' (GalNAcaMeumb) at 15 "C. The value of KO for each of the ligands decreases with increasing temperature,asillustrated byVan't Hoff plots
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