RESUMEN
The development of a biosensor based on surface plasmon resonance is described for the detection of carbohydrate-binding proteins in solution on a Biacore 2000 instrument, using immobilized glycopeptides as ligands. Their selection was based on previous screenings of solid-phase glycopeptide libraries with Ricinus communis agglutinin (RCA(120)) and human adhesion/growth-regulatory galectin-1 (h-Gal-1). Glycopeptides were immobilized on Au sensor chips functionalized with mixed self-assembled monolayers of different ratios of 11-mercapto-1-undecanol and 11-mercaptoundecanoic acid, and of 3-mercapto-1-propanol and 11-mercaptoundecanoic acid. The biosensors were optimized for the detection of RCA(120), and a detection limit of 0.13 nM was obtained. Subsequent experiments with h-Gal-1 indicated a detection limit of at least 0.9 nM for this lectin. Additionally, the effect of interfering proteins on the sensitivity of the optimized biosensor was investigated.
Asunto(s)
Técnicas Biosensibles/métodos , Glicopéptidos/química , Resonancia por Plasmón de Superficie/métodos , Técnicas Biosensibles/instrumentación , Proteínas Portadoras/análisis , Galectina 1/análisis , Lectinas de Plantas/análisis , Sensibilidad y EspecificidadRESUMEN
Two combinatorial glycopeptide libraries were synthesized on solid support via the "split-and-mix" method combined with the ladder synthesis strategy. The O-glycopeptide library contained Gal(beta1-O)Thr, whereas the S-,N-glycopeptide library contained both Gal(beta1-S)Cys and Gal(beta1-N)Asn. In this model study, the two libraries were screened against the fluorescently labeled lectin Ricinus communis agglutinin (RCA120). The screening results showed that both O- and S- or S-,N-glycopeptides were recognized by the lectin with similar amino acid recognition patterns. Surface plasmon resonance interaction studies demonstrated that both the selected S- or S-,N-glycopeptide hits and the O-glycopeptides bound to the lectin with a similar affinity. Structure 19, containing two glycosylated cysteine residues, bound to the receptor with the highest affinity (KA = 3.81 x 10(4) M(-1)), which is comparable to N-acetyllactosamine. Competition assays, in which some selected glycopeptides and methyl beta-d-galactopyranoside competed for the binding site of immobilized RCA120, showed that the glycopeptide-lectin interaction was carbohydrate-specific. Incubation of the O- and S-,N-glycopeptides with beta-galactosidase demonstrated the complete stability of S-,N-glycopeptides toward enzymatic degradation, whereas O-glycopeptides were not completely stable.