Determination of binding constants between one protein and multiple carbohydrates by affinity chromatography on a...

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.