Measurement of monovalent and polyvalent carbohydrate-lectin binding by back-scattering interferometry

Abstract

Carbohydrate-protein binding is important to many areas of biochemistry. Here, backscattering interferometry (BSI) has been shown 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 that were generated within several minutes and were highly reproducible in multiple wash/measure cycles provided adsorption coefficients that showed mannose to bind to concanavalin A (conA) with 3.7 times greater affinity than glucose consistent with literature values. Galactose was observed to bind selectively and with similar affinity to the lectin BS-1. The avidities of polyvalent sugar-coated virus particles for immobilized conA were much higher than monovalent glycans, with increases of 60-200 fold per glycan when arrayed on the exterior surface of cowpea mosaic virus or bacteriophage Qbeta. Sugar-functionalized PAMAM dendrimers showed size-dependent adsorption, which was 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 is sensitive to the number of binding events, rather than changes in mass. The operational simplicity and generality of BSI, along with the near-native conditions under which the target binding proteins are immobilized, make BSI an attractive method for the quantitative characterization of the binding functions of lectins and other proteins.

Figure 1

Functional preparation of immobilized lectins in for BSI measurements.

Figure 2

Measurement of monovalent carbohydrate binding to immobilized biotin-conA. (A) Comparison of sugars on immobilized commercial biotin-conA. (B) Comparison of mannose binding to different sources of biotinylated conA (deposited at the same concentration): red squares = commercially available material; blue diamonds = conA biotinylated in house; green triangles = an equimolar mixture of commercial conA-biotin and biotinylated BSA. (C) Comparison of sugars on immobilized biotin-BS-1. Error bars on all plots are derived from three independent experiments using different chips, showing a high degree of reproducibility.

Figure 3

Polymer virus- and dendrimer-carbohydrate adducts.

Figure 4

Measurement of polyvalent virus-carbohydrate binding to immobilized biotin-conA.

Figure 5

Binding of mixed mannose-galactose particles to immobilized conA and BS-1 lectins. (A, B) BSI measurements of the binding of the indicated CPMV-(sugar)₁₉₂ particles to immobilized conA and BS-1. (C, D) Plots of values of 1/K_ads derived from A and B, in terms of the overall concentrations of the indicated glycans presented on the virus surface (black) and the concentrations of the virus particles (blue). (E, F) BSI measurements of the binding of the indicated G4 and G6 dendrimer-(sugar)_n particles to immobilized conA. (G, H) Plots of 1/K_ads derived from E and F. In all cases, 1/K_ads values were calculated from each curve independently, ignoring the relative differences in signal magnitudes between curves. See the text for further discussion.

Figure 6

(A and B) BSI measurement of the treatment of CPMV-mannose particle 11 bound to an extravidin-conA channel with increasing concentrations of (A) soluble conA and (B) mannose. (C) Cartoon representation of experiments measuring binding and competition with free sugar and receptor. For particles bearing different numbers of sugars, saturation is reached with approximately the same number of virions bound, and yet the signal intensities in the two cases are markedly different.