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

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1200; p. 173
• Main Authors: Hori, Kanji, Hirayama, Makoto
• Format: Journal Article
• Language: English
• Published: United States 2014
• Subjects: Amination; Centrifugation - methods; Chromatography, High Pressure Liquid - methods; Fluorescent Dyes - chemistry; Fluorometry; Kinetics; Lectins - metabolism; Oligosaccharides - chemistry; Oligosaccharides - isolation & purification; Oligosaccharides - metabolism; Protein Binding; Substrate Specificity; Temperature; Ultrafiltration - methods
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-1292-6_16

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.