PRELIMINARY CHARACTERIZATION OF BOVINE BETA-LACTOGLOBULIN AFTER ITS CONJUGATION TO POLYETHYLENE-GLYCOL

The major component of the whey fraction of bovine milk, beta-lactoglo bulin (beta LG), 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 protei n was achieved at a controlled pH of 7.0 using increasing ratios of ac tivated PEG to beta LG. Transformation of the dimeric form into the mo nomer occurred at least for the fully pegylated adduct. The number of polymer chains fixed per mole of protein was determined by dosage of t he free amino functions still present after reaction. The incidence of pegylation on the secondary structure of beta LG was evaluated using the Fourier Transform Infrared Spectroscopy (FTIR). Denaturation studi es with guanidinium hydrochloride (Gu-HCl) by means of spectrofluorime tric measurements, showed an identical behavior of native as well as o f pegylated beta LG. The antigenicity of the fully pegylated adduct wa s examined through antigenic competition towards native beta LG. The p egylated protein exhibited less than 1/100 of the native beta PLG inhi bition capacity, that could moreover never be complete. This is thus d emonstrating the loss of accessibility for at least multiple conformat ional epitopes through pegylation procedure. Spectrofluorimetric measu rements showed that beta LG-N-PEG(7) was still able to bind retinol wh ile no effect on the intrinsic fluorescence could be detected by addin g palmitic acid. Whether this last ligand binds or not to pegylated be ta LG is discussed. (C) 1997 John Wiley & Sons, Inc.