PEROXIDASE-LABELING OF HUMAN SERUM TRANSFERRIN BY CONJUGATION TO OLIGOSACCHARIDE MOIETIES

The generation of reactive aldehydes on the carbohydrate moieties of t he human serum transferrin was performed by a derivatization procedure based on the mild oxidation with sodium periodate and subsequent reac tion with peroxidase hydrazide. The synthesized conjugate was compared to that obtained by modification of the amino acid side chains of tra nsferrin. The conjugate reaction mixture assayed by SDS-PAGE consisted , besides unreacted compounds, of three main bands, corresponding to a molar ratio transferrin:peroxidase of 1:1, 1:2, 1:3. After blotting, these bands were identified by either anti-peroxidase and anti-transfe rrin antibodies on nitrocellulose membrane. ELISA detection method sho wed that the conjugate via oligosaccharide moieties (glycans) was stil l recognized not only by the anti-transferrin antibodies but also by t he specific cellular receptor, while the conjugate via amino acids fai led to display this latter ability. The different behaviour can be pro bably due to a significant damage of the protein structure or, possibl y, to the peroxidase binding at sites recognized by the receptor. The results reported here indicate that the conjugation procedure through glycans leads to stable and selected transferrin-conjugates fully exhi biting their biological activity. (C) 1998 Published by Elsevier Scien ce BN. All rights reserved.