Fluorescence-polarization studies on binding of 4-methylumbelliferyl β-D-galactopyranoside to Ricinus communis (castor-bean) agglutinin

Abstract

The binding of Ricinus communis (castor-bean) agglutinin 1 to saccharides was studied by equilibrium dialysis and fluorescence polarization by using the fluorescently labelled sugar 4-methylumbelliferyl β-D-galactopyranoside. No appreciable change in ligand fluorescence took place on its binding to Ricinus agglutinin 1; on the other hand, the fluorescence of 4-methylumbelliferyl β-D-galactopyranoside was considerably polarized on its binding to the lectin. The association constants obtained by Scatchard analysis of equilibrium-dialysis and fluorescence-polarization data do not differ much from each other, and at 25°C, K_a = 2.4 (±0.2) × 10⁴M^-1. These values agree reasonably well with that reported in the literature for Ricinus agglutinin 1. The number of binding sites obtained by the different experimental procedures is 1.94 ± 0.1 per molecule of 120000 daltons and is equal to the reported value of 2. The consistency in the values of K_a and number of binding sites indicate the absence of additional subsites on Ricinus agglutinin 1 for its specific sugars. In addition, the excellent agreement between the binding parameters obtained by equilibrium dialysis and fluoresence polarization indicate the potential of ligand-fluorescence-polarization measurements in the investigation of lectinsugar interactions.