(3aS,4S,6aR)-N-[2-[[(3',6'-Dihydroxy-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthen]-5-yl)carbonyl]amino]ethyl]hexahydro-2-oxo-1H-thieno[3,4-d]imidazole-4-pentanamide

Biotin-4-Fluorescein is used to quantitate biotin binding sites. Biotin-4-Fluorescein is a reagent that binds to avidin more readily than conventional fluorescein biotin. This bifunctional biotin-fluorescein conjugate demonstrates better binding and stronger fluorescence than biotin fluorescein. It has similar avidin-binding properties in terms of high affinity, fast association, and non-cooperative binding to avidin and streptavidin tetramers. These exceptional properties are attributed to the small size/length of the new ligand since all larger/longer biotin derivatives are known for their mutual steric hindrance and anti-cooperative binding in 4:1 complexes with avidin and streptavidin tetramers. Specific binding of this biotin-fluorescein conjugate towards avidin and streptavidin is accompanied by 84-88% quenching of ligand fluorescence. It is used for the quantitation of biotin-binding sites. Both the fluorescence and absorbance of biotin-4-fluorescein are quenched upon binding to one of the four biotin-binding sites of streptavidin, avidin, or the streptavidin conjugates of fluorescent dyes or enzymes. As a result, when a known concentration of biotin-4-fluorescein is added to a known amount of streptavidin, one can estimate the number of biotin-binding sites.