Fluorescence polarization as a tool to study lectin-sugar interaction: an investigation of the binding of 4-methylumbelliferyl β-D-galactopyranoside to Abrus precatorious agglutinin

Abstract

Polarization of ligand fluorescence was used to study the binding of 4-methylumbelliferyl β-D-galactopyranoside (MeUmb-Galp) to Abrus precatorious agglutinin. The binding of the fluorescent sugar to the lectin led to considerable polarization of the MeUmb-Galp fluorescence, which was also quenched by about 30% on binding to the lectin. The binding of the fluorescent sugar was carbohydrate-specific, as evidenced by inhibition of both fluorescence polarization and quenching when lectin was preincubated with lactose. The association constant as determined by fluorescence polarization is 1.42×10⁴ M⁻¹ at 25°C and is in excellent agreement with those determined by fluorescence quenching (K_a=1.51×10⁴ M⁻¹) and equilibrium dialysis (K_a=1.62×10⁴ M⁻¹) at 25°C. The numbers of binding sites as determined by fluorescence polarization, quenching and equilibrium dialysis agree very well with one another, n being equal to 2.0±0.05. The consistency between the association constant value determined by fluorescence polarization, quenching and equilibrium dialysis shows the validity of this approach to study lectin-sugar interaction.