A fluorometric assay for the biotin-avidin interaction based on displacement of the fluorescent probe 2-anilinonaphthalene-6-sulfonic acid

Abstract

Avidin and biotin can be sensitively and accurately quantitated using the fluorescent probe 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS). In the presence of avidin, the fluorescence of 2,6-ANS is blue shifted with a large increase in quantum yield. Biotin binding causes complete displacement of the bound fluorophore with concomitant quenching of the fluorescence. The fluorometric monitoring of the displacement of 2,6-ANS can be used as a facile method of measuring the biotin-avidin interaction. 2,6-ANS displacement gives the same stoichiometry as the method using 4′-hydroxyazobenzene-2-carboxylic acid. Our initial studies of an affinity-purified avidin revealed that, of the four binding sites on the avidin tetramer, a mean of three remain available for biotin (or dye) binding; this finding highlights a caveat concerning the use of affinity-purified oligomeric-binding proteins with multiple sites. As compared with previous fluorescence methods, the use of 2,6-ANS gives high sensitivity without the necessity of preparing and purifying a covalent avidin conjugate. In addition, the present method: is potentially more sensitive than those based on optical absorbance; uses a probe that has increased stability and a larger Stokes shift compared with fluorescein; is not subject to protein interference; and gives accurate results over a wide range of 2,6-ANS and avidin concentrations.