BETA-LACTOGLOBULIN BINDING-PROPERTIES DURING ITS FOLDING CHANGES STUDIED BY FLUORESCENCE SPECTROSCOPY

The milk protein, beta-lactoglobulin (BLG) exhibits structural and bin ding properties which vary widely, depending on the medium. These prop erties of BLG are reflected in fluorescence intensities, steady-state anisotropies and phase lifetimes of BLG tryptophan residues and of ret inol and diphenyl hexatriene (DPH) bound to BLG, as functions of pH, e thanol concentration and protein modifications (22% ethylated, 90% met hylated and 85% acetylated BLGs). Tryptophan quenching experiments sho w that retinol and DPH bind to BLG in 1:1 molar ratios with apparent d issociation constants around 10(-7)-10(-8)M. The strength of retinol b inding is pH-dependent in the range 3-8, whereas that of DPH binding i s not. Two different binding sites for these two ligands coexist on th e protein. Modified BLGs exhibit higher affinities for DPH than the un modified protein. At all pH values investigated, the fluorescence emis sion at 480 nm of retinol/BLG mixtures and retinol, DPH and tryptophan anisotropies and lifetimes change dramatically with midpoint at 27% e thanol for the first parameter and 35% for the others, suggesting simu ltaneous beta-strand to alpha-helix transition and the dissociation of BLG complexes at 35% ethanol. An intermediate state, possibly 'molten globular', occurs around 20% ethanol, as deduced from anisotropy and lifetime measurements.