Fluorescence based ligand-binding studies of ß-lactoglobulin

Päivi Tammela, M.Sc. & Heikki Vuorela, Prof., Ph.D, Faculty of Pharmacy, University of Helsinki, Finland

Abstract

This application note describes the advantage of Thermo’s Varioskan in ligand binding studies of ß-lactoglobulin. The experiments are based on tryptophan fluorescence quenching in consequence of ligand-binding. The method can be easily applied to other respective protein-ligand studies as well.

The protein ß-lactoglobulin (ß-LG) is the major whey protein in bovine milk. It has been recognized that ß-LG may play a role in the transport of retinol and fatty acids (Sawyer et al. 1998). ß-LG usually exists as a dimer in which the molecular weight of the subunit is about 18000 Da (Papitz et al. 1986). The ß-LG molecule consists of an anti-parallel ß-sheet, formed by 9 strands wrapped around to form a flattened cone or calyx. The core of the ß-LG is an eight-stranded, antiparallel ß-barrel. The extra ß-strand forms the dimer interface (Sawyer & Kantopidis 2000). There is also an α-helix lying on the surface. ß-LG binds ligands of a diverse nature. ß-LG can bind hydrophobic ligands as retinol, retinoic acid, long-chain fatty acids and aromatic compounds. The retinol binding site is inside the ß-barrel (Naryan & Berliner, 1997).

There are many analyses which can be appropriate if the ligand binds to ß-LG. The most commonly used methods are based on fluorescence. ß-LG contains two tryptophan residues, Trp19 and Trp61 (Cho et al. 1994). The binding of retinoids and palmitate to ß-LG causes a quenching of the fluorescence of Trp19, which is present at the bottom of calyx (Wang et al. 1997).