Measurement of Mono- and Polyvalent Carbohydrate-Lectin Binding by Back-Scattering Interferometry

• Published in: Analytical chemistry (Washington) Vol. 81; no. 12; pp. 4889 - 4897
• Main Authors: Kussrow, Amanda, Kaltgrad, Eiton, Wolfenden, Mark L., Cloninger, Mary J., Finn, M.G., Bornhop, Darryl J.
• Format: Journal Article
• Language: English
• Published: 15.06.2009
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac900569c

Carbohydrate-protein binding is important to many areas of biochemistry. Back-scattering interferometry (BSI) is shown here to be a convenient and sensitive method for obtaining quantitative information about the strengths and selectivities of such interactions. The surfaces of glass microfluidic channels were covalently modified with extravidin, to which biotinylated lectins were subsequently attached by incubation and washing. The binding of unmodified carbohydrates to the resulting avidin-immobilized lectins was monitored by BSI. Dose-response curves, generated within several minutes and highly reproducible in multiple wash/measure cycles, provided adsorption coefficients that showed mannose to bind to concanavalin A with 3.7 times greater affinity than glucose, in line with literature values. Galactose was found to bind selectively and with similar affinity to the lectin BS-1. The avidities of polyvalent sugar-coated virus particles for immobilized conA were far higher than monovalent glycans, with increases of 60–200 fold per glycan when arrayed on the exterior surface of cowpea mosaic virus or bacteriophage Qβ. Sugar-functionalized PAMAM dendrimers showed size-dependent adsorption consistent with the expected density of lectins on the surface. The sensitivity of BSI matches or exceeds that of surface plasmon resonance and quartz crystal microbalance techniques, and differs in its sensitivity to the number of binding events rather than changes in mass. Its operational simplicity, generality, and the near-native conditions under which the target binding proteins are immobilized make it an attractive method for the quantitative characterization of the binding functions of lectins and other proteins.