title: beta-Lactoglobulin binding properties during its folding changes studied by fluorescence spectroscopy.
abstract: The milk protein, beta-lactoglobulin (BLG) exhibits structural and binding properties which vary widely, depending on the medium. These properties of BLG are reflected in fluorescence intensities, steady-state anisotropies and phase lifetimes of BLG tryptophan residues and of retinol and diphenyl hexatriene (DPH) bound to BLG, as functions of pH, ethanol concentration and protein modifications (22% ethylated, 90% methylated and 85% acetylated BLGs). Tryptophan quenching experiments show that retinol and DPH bind to BLG in 1:1 molar ratios with apparent dissociation constants around 10(-7) - 10(-8) M. The strength of retinol binding is pH-dependent in the range 3-8, whereas that of DPH binding is not. Two different binding sites for these two ligands coexist on the protein. Modified BLGs exhibit higher affinities for DPH than the unmodified protein. At all pH values investigated, the fluorescence emission at 480 nm of retinol/BLG mixtures and retinol, DPH and tryptophan anisotropies and lifetimes change dramatically with midpoint at 27% ethanol for the first parameter and 35% for the others, suggesting simultaneous 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.
authors.affiliation: L.E.I.M.A.-Institut National de la Recherche Agronomique, Nantes, France.
authors.forename: E
authors.initials: E
authors.lastname: Dufour
authors.forename: C
authors.initials: C
authors.lastname: Genot
authors.forename: T
authors.initials: T
authors.lastname: Haertlé
mesh_terms: D004161:Diphenylhexatriene / Q000737:chemistry; D000431:Ethanol; D005454:Fluorescence Polarization; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000737:chemistry*; D017510:Protein Folding; D012996:Solutions; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000032:analysis / Q000737:chemistry; D014801:Vitamin A / Q000737:chemistry
chemical_list: D007782:Lactoglobulins; D012996:Solutions; D014801:Vitamin A; D004161:Diphenylhexatriene; D000431:Ethanol; D014364:Tryptophan