Fluorescence quenching and time-resolved fluorescence studies on peanut agglutinin

Abstract

Peanut agglutinin (PNA) has elicited much interest due to its specific recognition of the tumor associated disaccharide, 2-acetamido-2-deoxy-(3-O-β-D-galatopyranosyl)-D-galactopyranoside (Galβ13GalNAc; T-antigen). In this study, fluorescence quenching and time-resolved fluorescence measurements have been carried out to investigate the accessibility and environment of the tryptophan residues in this protein and to study the effect of saccharide binding. The emission λ(max) of native PNA is at 323 nm and is unaffected by binding of the specific ligand, lactose, but shifts to 362 nm upon denaturation with 6 M guanidinium hydrochloride, clearly indicating that the Trp residues which are buried in the hydrophobic interior of the lectin get exposed to the aqueous environment upon unfolding. At a quencher concentration of 0.5 M, the extent of quenching observed for the native lectin with acrylamide and iodide was 25% and 5%, respectively. In the presence of lactose the quenching by I- is unaffected while that by acrylamide is decreased to 22%, indicating that diffusion of this neutral quencher into the protein matrix is decreased by ligand binding. In time-resolved fluorescence experiments, the decay curves could be fitted to bi-exponential patterns with lifetimes of 0.72 and 4.37 ns for native PNA, 0.76 and 4.46 ns in presence of 0.1 M lactose, and 1.73 and 4.18 ns upon denaturation.