Centrifugal ultrafiltration-HPLC method for interaction analysis between lectins and sugars

The centrifugal ultrafiltration-HPLC method is a simple and rapid method for analyzing the binding interaction between lectins and sugars (oligosaccharides). In this method, a lectin is mixed with a fluorescent-labeled oligosaccharide in buffer and the unbound oligosaccharide recovered by centrifugal ultrafiltration is isolated and quantified by high-performance liquid chromatography. The binding activity is defined as a ratio (percentage) of the amount of bound oligosaccharide to that added, where the former is obtained by subtracting the amount of unbound oligosaccharide from the latter. The oligosaccharide-binding specificity of a lectin can be determined by comparing the binding activities with a variety of fluorescent-labeled oligosaccharides. The association constant and the optimum pH and temperature of the binding interaction between lectins and fluorescent-labeled oligosaccharides can be easily analyzed by this method.