Suitable experimental conditions are required to characterize interferon-alpha2b synthetic peptides.
The binding and antiproliferative activities of synthetic peptides 29-35 and 122-139 of interferon-alpha2b, both of which contain a cysteine residue in their sequences, were studied in the presence or absence of a dissociation medium containing mainly urea, dithiothreitol and 2-mercaptoethanol. Although interferon-alpha2b peptides either did not modify or slightly increased 125I-labelled interferon-alpha2b specific binding to WISH cell-membrane receptors in the absence of dissociation medium, significant binding inhibition was obtained when both peptides were assayed in dissociation medium. Furthermore, also in the presence of dissociating agents, the two fragments inhibited cell growth in a concentration-dependent manner, the 122-139 sequence being more effective than the 29-35 sequence. No additive effect on interferon binding and cell proliferation was observed when both peptides were added simultaneously. Results obtained after submitting peptide 122-139 to gel filtration or PAGE under different experimental conditions showed the presence of dimers and/or noncovalent aggregates arising from intermolecular disulfide bridges or hydrophobic interactions. Thus, our results indicated that peptide effects on 125I-labelled interferon-alpha2b binding and WISH cell proliferation were clearly manifested when the amount of monomeric species increased, showing that suitable experimental conditions should be used to study peptide behavior. The ability of both peptides to effectively trigger an interferon-specific biological action, such as cell growth inhibition, strongly suggested that 29-35 and 122-139 interferon-alpha2b fragments constitute the conformational epitope or mimotope that interacts with the cytokine-specific receptor.
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Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina.
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D001665:Binding Sites; D002455:Cell Division; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel / Q000379:methods; D003545:Cysteine / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry* / Q000302:isolation & purification; D008623:Mercaptoethanol / Q000494:pharmacology; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D011485:Protein Binding; D011994:Recombinant Proteins; D014508:Urea / Q000494:pharmacology
D000939:Epitopes; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D010455:Peptides; D011994:Recombinant Proteins; D008623:Mercaptoethanol; D014508:Urea; D003545:Cysteine; D004229:Dithiothreitol