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Preliminary characterization of bovine beta‐lactoglobulin after its conjugation to polyethylene glycol
Author(s) -
Nijs Michelle,
Azarkan Mohamed,
Smolders Nicole,
Brygier Jeanne,
Vincentelli Jean,
Petiaude Vries Ghislaine M.,
Duchateau Jean,
Looze Yvan
Publication year - 1997
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(19970405)54:1<40::aid-bit5>3.0.co;2-y
Subject(s) - pegylation , chemistry , polyethylene glycol , cysteine , lysine , peg ratio , amino acid , beta lactoglobulin , antigenicity , stereochemistry , whey protein , chromatography , organic chemistry , biochemistry , enzyme , finance , antigen , biology , economics , genetics
The major component of the whey fraction of bovine milk, β‐lactoglobulin (βLG), 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 βLG. 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 βLG 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 βLG. The antigenicity of the fully pegylated adduct was examined through antigenic competition towards native βLG. The pegylated protein exhibited less than 1/100 of the native βLG 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 βLG‐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 βLG is discussed. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 40–49, 1997.