Peroxidase-labelling of human serum transferrin by conjugation to oligosaccharide moieties.
The generation of reactive aldehydes on the carbohydrate moieties of the human serum transferrin was performed by a derivatization procedure based on the mild oxidation with sodium periodate and subsequent reaction with peroxidase hydrazide. The synthesized conjugate was compared to that obtained by modification of the amino acid side chains of transferrin. 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-transferrin antibodies on nitrocellulose membrane. ELISA detection method showed that the conjugate via oligosaccharide moieties (glycans) was still recognized not only by the anti-transferrin antibodies but also by the specific cellular receptor, while the conjugate via amino acids failed to display this latter ability. The different behaviour can be probably due to a significant damage of the protein structure or, possibly, 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 exhibiting their biological activity.
Department of Biomedical Sciences and Technologies, University of L'Aquila, Italy. dalessandro@aquila.infn.it
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D'Alessandro
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D'Andrea
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Oratore
D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007124:Immunoenzyme Techniques; D009844:Oligosaccharides; D009195:Peroxidase / Q000378:metabolism*; D011990:Receptors, Transferrin / Q000378:metabolism; D014168:Transferrin / Q000032:analysis* / Q000378:metabolism
D009844:Oligosaccharides; D011990:Receptors, Transferrin; D014168:Transferrin; D009195:Peroxidase