Concanavalin A-Polysaccharides binding affinity analysis using a quartz crystal microbalance

There is no comparative data available on the binding constants of Concanavalin A (Con A) and glycogen and Con A-mannan using quartz crystal microbalance (QCM), cost and time efficient system for biosensor analysis. It is hypothesized that a QCM can be used in its flow injection mode to monitor the binding affinity of polysaccharides to an immobilized lectin, Con A. The biosensor is prepared by immobilizing Con A on a 5 MHz gold crystal by carbodiimide crosslinking chemistry. The attachment efficiency is monitored by Fourier Transform Infrared Spectroscopy. Equilibrium association and dissociation constants describing Con A-polysaccharides interaction are determined in a saturation binding experiment, where increasing concentrations of polysaccharides are run on a Con A-immobilized gold crystal surface, and the frequency shifts recorded on the frequency counter. The molecular weights (MW) of glycogen from Oyster and mannan from Saccharomyces cerevisiae are determined by size exclusion chromatography. The MW for glycogen and mannan are 604±0.002 kDa and 54±0.002 kDa, respectively. The equilibrium association and dissociation constants for Con A-glycogen and Con A-mannan interactions are KA=3.93±0.7×106M−1/KD=0.25±0.06μM and KA=3.46±0.22×105M−1/KD=2.89±0.20μM (n=3), respectively. Their respective frequency and motional resistance shifts relationship (ΔF/ΔR) are 37.29±1.55 and 34.86±0.85 Hz/Ω (n=3), which support the validity of Sauerbrey׳s rigidity approximation. This work suggests that Con A-mannan complex could be potentially utilized for insulin delivery and the targeting of glucose-rich substances and glycoproteins when fast drug release is desired.