Development of a fluoroscence titration method to measure the association constants of small molecule fluorophores with cyclodextrins and fluorophore specific antibodies

CHED 296

Jessica Rachel Meyers, k03jm01@kzoo.edu, Department of Chemistry, Kalamazoo College, 1200 Academy St, Kalamazoo, MI 49006
A protocol was developed to measure the association constant (KS) of a small molecule fluorophore and a non-fluorescent binding partner. Fluorescence intensity data was collected during titrations of the fluorescent ligands and association hosts, and were fit using non-linear regression analysis to determine the KS of each ligand/binding host pair. The fluorophores 8-anilino-1-naphthalenesulfonate (ANS) and dimethylaminonaphthalene sulfonyl (dansyl) amide were used as small molecule ligands because they exhibit enhanced fluorescence and a hypsochromic shift in their emission spectra when bound to a macromolecular host. The binding hosts used in the development of this protocol were â-cyclodextrin (BCD), hydroxypropyl-â-cyclodextrin (HPBCD), and an antibody raised against the dansyl fluorophore (anti-DAN). The resulting protocol can be used to determine the KS between ANS or dansyl amide and water soluble macromolecular hosts.