Development of a Fluorescence Titration Method to Measure the Association Constants of Small Molecule Fluorophores with Cyclodextrins and Fluorophore Specific Antibodies
Meyers, Jessica R.
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In this study, a protocol was developed to measure the association constant (Ks) of a fluorophore and a non-fluorescent binding protein using fluorescence titration techniques. A spectrofluorimeter was used to measure the fluorescence intensity of a solution during the titration of two binding partners, and by using non-linear regression techniques, the Ks was measured. One of the binding partners must be fluorescent and result in a change in its emission spectrum upon binding to a macromolecular binding host. The fluorophores 8-anilino-l-naphthalenesulfonate (ANS) and dimethylaminonaphthalene sulfonyl (dansyl) amide were used as small molecule ligands, and P-cyclodextrin (BCD), hydroxypropyl-P-cyclodextrin (HPBCD), and an antibody against dansyl (anti-DAN) were used as macromolecular binding hosts. A method for measuring and calculating Ks was developed for four pairs of binding partners: ANS:BCD, dansyl:BCD, ANS:HPBCD, and dansyl:anti-DAN. This method involves the titration of one binding partner into the other at specified concentration ranges, and a non-linear regression equation for calculating the Ks. This protocol can be used for measuring the association constant of these fluorescent ligands and any other macromolecular binding host.