Fabrication of Carbohydrate Surfaces by Using Nonderivatised Oligosaccharides, and their Application to Measuring the Assembly of Sugar-Protein Complexes

摘要

Surface-based tools, such as microarrays and optical biosensors, are being increasingly applied to the analysis of carbohydrate-protein interactions. A key to these developments is the presentation of the carbohydrate to the protein target. Dual polarisation interferometry (DPI) is a surface-based technique that permits the real-time measurement of the changes in thickness, refractive index and mass of adsorbates 100 nm thick or less on the surface of a functionalised waveguide. DPI has been used to design and characterise a surface on which the orientation and density of the immobilised carbohydrates is suitable for studying their interactions with proteins and where nonspecific binding is reduced to less than 5% of total binding. A thiol-functionalised surface was derivatised with a heterobifunctional crosslinker to yield a hydrazide surface. This was treated with oligosaccharides, derived from keratan sulfate (KS) chondroitin sulfate (CS) and heparin, that possess a reducing end. To block the unreacted hydrazide groups, the surface was treated with an aldehyde-functionalised PEG. The heparin DP-10 surfaces were then used to determine the performance of the immobilised DP-10 with respect to binding of two well-characterised proteins, lactoferrin (Lf) and fibroblast growth factor-2. The results show that Lf could adopt two different orientations, at high protein loadings the protein layer thickness corresponded to an "end-on" orientation of Lf, whilst rinsing with buffer saw the Lf molecules adopt a "side-on" configuration. In the case of FGF-2, a single monolayer of protein bound to DP-10 was observed. These results demonstrate that the new surface can be used to resolve key questions relating to the binding of proteins to carbohydrates, including, when used in DPI, the resolution of the geometry of complexes, an area that is frequently controversial.