title: Determination of binding constants between one protein and multiple carbohydrates by affinity chromatography on a microchip.
abstract: Development of rapid, reliable and high throughput methods for evaluating the interactions between different carbohydrates and a same protein is critical to carbohydrate drug development. In this study, we develop a novel strategy based on an affinity chromatography for quickly determining the binding constants of different carbohydrates to a same protein. The core of our method is the inversely proportional relationship between the binding constant and a new termed parameter, critical elution concentration (CMC). CMC is defined as the lowest concentration of displacing reagent, a series of carbohydrates herein, at which the protein specifically bond to the affinity column can be eluted off as an intact peak by the carbohydrate solution in a certain time. The interactions between a series of sulfate polysaccharides and granulocyte colony-stimulating factor (G-CSF) are selected as model. Through a 200 μm long heparin affinity column microfabricated inside a channel of 50 μm width and 20 μm height, the binding constant of each G-CSF-polysaccharide binding pair can be obtained within 1h, around one sixth of time needed by traditional capillary electrophoresis based method.
authors.affiliation: Jiangsu Normal University, School of Chemistry and Engineering, Xuzhou 221116, China.
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Liu
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.forename: Aiye
authors.initials: A
authors.lastname: Liang
authors.forename: Yuling
authors.initials: Y
authors.lastname: Li
authors.forename: Hongwei
authors.initials: H
authors.lastname: Gai
authors.forename: Bingcheng
authors.initials: B
authors.lastname: Lin
mesh_terms: D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry / Q000378:metabolism*; D006493:Heparin; D008956:Models, Chemical; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D011134:Polysaccharides; D016179:Granulocyte Colony-Stimulating Factor; D006493:Heparin