Determining the affinity of anti-mitotic compounds binding to colchicine binding site of tubulin by affinity probe capillary electrophoresis

The colchicine binding site of tubulin is often used to screen the anti-mitotic compounds, which are widely used as anti-cancer therapies. In the present work, an affinity probe capillary electrophoresis (APCE) method was developed for determining the affinity of anti-mitotic compounds. To this end, a fluorescently labeled affinity probe, 5-carboxyfluorescein-colchicine (F-colchicine), was prepared for the affinity competition experiment. The probe can form a stable complex with tubulin with the binding stoichiometry of 0.75, and the dissociation constant K-d of the complex was determined as 5.7 x 10(-5) mol/L. In the affinity competition experiment, F-colchicine was incubated with tubulin and the test compound in the solution. The F-colchicine-tubulin complexes and free F-colchicine were quickly separated by CE and the concentration of free F-colchicine was accurately determined with the laser induced fluorescence detection. The affinity constant of the tested compound can be measured with the affinity competition binding curve. The enantiomers of the anti-mitotic compound were evaluated by using the method. The binding affinity of the enantiomers displayed an enantioselective manner. Compared to other affinity binding assay methods, our method is more straightforward, more accurate, and more cost-effective.