This contribution illustrates the advantages of some chromophoric and fluorophoric carbohydrate derivatives such asp-nitrophenyl (pNO2Phe) or 4-methylumbelliferyl (MeUmb) glycosides andN-dansylgalactosamine in studies of the binding equilibrium and kinetics with some plant lectins. The methods used involve continuous titrations of changes in ligand or protein absorption and ligand fluorescence, including substitution titrations as well as stopped-flow, temperature-jump or pressure-jump relaxation kinetics.
When monitored by temperature-jump relaxation, binding of MeUmbαGal to the bloodgroup A specific lectin GSAI-A4 fromGriffonia simplicifolia is a simple bimolecular association with parametersk+= 9.4 × 104 M-1 s-1 andk-1 = 5.3 s-1 at 23°C, but binding to the GSAI-B4 lectin is biphasic.
The complementarity of the peanut agglutinin binding site with Galβ1 → 3GalNAc that occurs in manyO-glycoproteins follows from enthalpic considerations and also from the value of the dissociation-rate parameterk-1 = 0.24 s-1 of the MeUmbβGalβl → 3GalNAc.lectin complex. This value, obtained by stopped-flow kinetics is 100 times smaller than for other mono-and disaccharides investigated. The binding mechanism is simple and the derivatisation of Galβ1 → 3GalNAc does not affect the affinity to a considerable degree.
The binding preference of tetravalentsoybean agglutinin for MeαGalNAc over MeαGal by a factor of 25 is mainly of enthalpic origin with an additional 7 kJ mol-1; the NAc group causes perturbation of a tryptophanyl residue, evidenced by protein difference absorption spectrometry. In the glycosides, a large aglycon likeβpNO2 Phe orβMeUmb hardly affects the affinity of SBA but a largeN-dansyl group increases the affinity by a factor 20 as compared to GalNAc. The 10-fold increase in carbohydrate-specificN-dansylgalactosamine fluorescence, together with a very favourable entropic contribution point at the presence of a hydrophobic region in the vicinity of the carbohydrate-binding site. The dissociation-rate parameter of the MeUmbβGalNAc SBA complex is slower than for any reported monosaccharide-lectin complex: 0.4 s-1.
The divalent lectin fromErythrina cristagalli preferentially binds the Galβ1 → 4GlcNAc structure that occurs in manyN-glycoproteins. The combining site was mapped thermodynamically with carbohydrates ranging from mono-to pentasaccharides as derived fromN-glycoproteins. Here, N-dansylgalactosamine was used as a fluorescent indicator ligand in substitution titrations. When Galβ1 → 4GlcNAc was linkedα1 → 2 orα1 → 6 to Man, the binding enthalpy and entropy remained practically constant. Application of stopped flow kinetics and pressure-jump relaxation withN-dansylgalactosamine gave mono-exponential signal changes with a concentration dependence corresponding tok+ = 4.8 x 104 M-1 s-1k- = 0.4 to 0.66 s-1 and a change in reaction volume of+7ml/mol.
Volume 44 | Issue 5
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