title: Preliminary characterization of bovine beta-lactoglobulin after its conjugation to polyethylene glycol.
abstract: The major component of the whey fraction of bovine milk, beta-lactoglobulin (betaLG), has been transformed by grafting polyethylene glycol chains either on the thiol group (free and after reduction of the S-S bridges) of the cysteine residues, or on the amino group of the lysine residues and/or of the N-terminal amino acid. Acylation of the protein was achieved at a controlled pH of 7.0 using increasing ratios of activated PEG to betaLG. Transformation of the dimeric form into the monomer occurred at least for the fully pegylated adduct. The number of polymer chains fixed per mole of protein was determined by dosage of the free amino functions still present after reaction. The incidence of pegylation on the secondary structure of betaLG was evaluated using the Fourier Transform Infrared Spectroscopy (FTIR). Denaturation studies with guanidinium hydrochloride (Gu-HCl) by means of spectrofluorimetric measurements, showed an identical behavior of native as well as of pegylated betaLG.The antigenicity of the fully pegylated adduct was examined through antigenic competition towards native betaLG. The pegylated protein exhibited less than 1/100 of the native betaLG inhibition capacity, that could moreover never be complete. This is thus demonstrating the loss of accessibility for at least multiple conformational epitopes through pegylation procedure.Spectrofluorimetric measurements showed that betaLG-N-PEG(7) was still able to bind retinol while no effect on the intrinsic fluorescence could be detected by adding palmitic acid. Whether this last ligand binds or not to pegylated betaLG is discussed.
authors.affiliation: Protein Chemistry Unit, Faculty of Medicine, University of Brussels, 808 Route de Lennik, B-1070 Bruxelles, Belgium.
authors.forename: M
authors.initials: M
authors.lastname: Nijs
authors.forename: M
authors.initials: M
authors.lastname: Azarkan
authors.forename: N
authors.initials: N
authors.lastname: Smolders
authors.forename: J
authors.initials: J
authors.lastname: Brygier
authors.forename: J
authors.initials: J
authors.lastname: Vincentelli
authors.forename: G M
authors.initials: GM
authors.lastname: Petiau-de Vries
authors.forename: J
authors.initials: J
authors.lastname: Duchateau
authors.forename: Y
authors.initials: Y
authors.lastname: Looze