Binding of [1-13C]galactose-labeled N-acetyllactosamine to Erythrina cristagalli agglutinin as studied by 13C-NMR.
The equilibrium binding kinetics of enzymatically prepared N-acetyllactosamine to the lectin from Erythrina cristagalli have been investigated by 13C-NMR spectroscopy. Under the experimental conditions used, NMR signals in the spectrum, corresponding to both the free and bound disaccharide species, were observed for the first time. This has permitted the simultaneous determinations of the equilibrium binding constant and the number of binding sites per lectin molecule. At the relatively high lectin concentrations used (0.3-0.87 mM), the association constants determined at 31 degrees C (approximately 6 X 10(3) M-1) are typically lower then those obtained by other methods employing much lower lectin concentrations. Extrapolation of the experimentally observed values to infinite dilution gave a better fit of the data (Ka approximately 1.4 X 10(4) M-1) with the binding constant determined by other methods (K approximately 1.1 X 10(4) M-1). The sugar residence time on the lectin (approximately 0.2 s) was determined directly from the signal's line-width using total line-shape analysis. Similar NMR experiments may permit an analysis of the interaction of the lectin with glycoproteins and cells labelled with 13C-enriched galactose residues. Moreover, information on lectin-galactose interactions at the binding site may be obtained by using galactose labeled at various carbons.
D000606:Amino Sugars / Q000378:metabolism*; D002247:Carbon Isotopes; D005690:Galactose; D007553:Isotope Labeling; D007700:Kinetics; D037102:Lectins / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D037121:Plant Lectins; D011975:Receptors, Mitogen / Q000032:analysis*
D000606:Amino Sugars; D002247:Carbon Isotopes; D037102:Lectins; D037121:Plant Lectins; D011975:Receptors, Mitogen; C045629:erythrina lectin; C000458:N-acetyllactosamine; D005690:Galactose