| Polyvalent carbohydrate-protein interactions were investigated for the detection of proteins including biowarfare agents. A carbohydrate microarray was developed which differentiates cholera and tetanus toxins from other biowarfare agents. The monosaccharides, sialic acid and N-acetyl-galactosamine, bind cholera and tetanus toxins, showing density and concentration dependence. The array system determined that cholera toxin has association rate constants of 2.5 +/- 0.03 x 104 M-1s-1 to NeuAc and 1.8 +/- 0.00 x 105 M-1s -1 to anti-CT mAb, dissociation rate constants of 2.0 +/- 0.12 x 10-4 s-1 to NeuAc and 9.0 +/- 0.15 x 10-4 s-1 to anti-CT mAb, and equilibrium association constants of 1.3 +/- 0.10 x 108 M-1 to NeuAc and 2.1 +/- 0.03 x 108 M-1 to anti-CT mAb. The slower association and dissociation rates are consistent with the toxin's pentavalent binding to NeuAc, which results in higher avidity than is possible with the lower-valency antibody. A trisaccharide microarray, employing the trisaccharide, NeuAc-alpha2,3-GalNAc-beta1,4-Glc, synthesized in Chapter three, shows preliminary affinity for several biowarfare agents. Specific binding patterns were observed with the binding of toxins and carbohydrates.;A synthesis of the NeuAc-alpha2,3-GalNAc-beta1,4-Glc ligand was developed. Click chemistry successfully coupled the azidoalkyl-saccharides to a disulfide-containing linker. It is interesting that the copper(I)-catalyzed cycloaddition proceeds in the presence of disulfides and thiols.;The four-state rate equation and rate constants describing polyvalent binding at the single-molecule level were elucidated using an alpha-hemolysin ion-channel glycosylated with disaccharides specific (our 'Hit' ligand disaccharide, Gal-alpha1,3-GlcN(isovaleryl), which is specific for BP lectin) or promiscuous (the natural disaccharide receptor, Gal-beta1,3-GalNAc) for Bauhinia purpurea (BP) and Arachis hypogea (AH) lectins. The four-state model explains the complexity of bivalent binding, indicating two possible bivalent states exist for binding to the heptavalent ion-channel, both yielding different current changes. BP lectin binds to its promiscuous, natural disaccharide ligand at twice the rate to the Hit disaccharide ligand, which is specific for BP lectin, but dissociates more slowly from the Hit ligand. This may explain the higher affinity of BP lectin for the Hit ligand observed in on-bead screens. |
