title: Centrifugal ultrafiltration-HPLC method for interaction analysis between lectins and sugars.
abstract: 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.
authors.affiliation: Graduate School of Biosphere Science, Hiroshima University, Kagamiyama 1-4-4, Higashi-Hiroshima, 739-8528, Japan, kanhori@hiroshima-u.ac.jp.
authors.forename: Kanji
authors.initials: K
authors.lastname: Hori
authors.forename: Makoto
authors.initials: M
authors.lastname: Hirayama
mesh_terms: D000586:Amination; D002498:Centrifugation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D005470:Fluorometry; D007700:Kinetics; D037102:Lectins / Q000378:metabolism*; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D011485:Protein Binding; D013379:Substrate Specificity; D013696:Temperature; D014462:Ultrafiltration / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D037102:Lectins; D009844:Oligosaccharides