Surface plasmon resonance study of protein-carbohydrate interactions using biotinylated sialosides

Lectins are carbohydrate binding proteins found in plants, animals, and microorganisms. They serve as important models for understanding protein-carbohydrate interactions at the molecular level. We report here the fabrication of a novel sensing interface of biotinylated sialosides to probe lectin-carbohydrate interactions using surface plasmon resonance spectroscopy (SPR). The attachment of carbohydrates to the surface using biotin-NeutrAvidin interactions and the implementation of an inert hydrophilic hexaethylene glycol spacer (HEG) between the biotin and the carbohydrate result in a well-defined interface, enabling desired orientational flexibility and enhanced access of binding partners. The specificity and sensitivity of lectin binding were characterized using Sambucus nigra agglutinin (SNA) and other lectins including Maackia amurensis lectin (MAL), concanavalin A (Con A), and wheat germ agglutinin (WGA). The results indicate that alpha 2,6-linked sialosides exhibit high binding affinity to SNA, while alteration in sialyl linkage and terminal sialic acid structure compromises the affinity by a varied degree. Quantitative analysis yields an equilibrium dissociation constant (K-D) of 777 +/- 93 nM for SNA binding to Neu5Ac alpha 2,6-LHEB. Transient SPR kinetics confirms the KD value from the equilibrium binding studies. A linear relationship was obtained in the 10-100 mu g/mL range with limit of detection of similar to 50 nM. Weak interactions with MAL, Con A, and WGA were also quantified. The control experiment with bovine serum albumin indicates that nonspecific interaction on this surface is insignificant over the concentration range studied. Multiple experiments can be performed on the same substrate using a glycine stripping buffer, which selectively regenerates the surface without damaging the sialoside or the biotin-NeutrAvidin interface. This surface design retains a high degree of native affinity for the carbohydrate motifs, allowing distinction of sialyl linkages and investigation pertaining to the effect of functional group on binding efficiency. It could be easily modified to identify and quantify binding patterns of any low-affinity biologically relevant systems, opening new avenues for probing carbohydrate-protein interactions in real time.