Macro and micro enhancers of the 8-anilino-1-naphthalenesulfonate (ANS) fluorescence. Is ANS indeed a hydrophobic probe?

A study on the absorption and fluorescence properties of the 8-anilino-1-naphthalenesulfonate (ANS) fluorescent probe was performed in order to (i) verify the validity of its classification as hydrophobic probe and (ii) to assess the reliability of the interpretation of the ANS fluorescence enhancement upon protein binding as the evidence for the existence of hydrophobic binding sites on the protein molecules. We observed an enhancement of the ANS fluorescence in hydrophilic media: DMSO, polyethylene glycol (PEG400) and glycerol to the values characteristic of ANS complexes with globular proteins, and all ANS fluorescence characteristics (except anisotropy) in PEG400 and in complex with bovine serum albumin are identical. We observed an increase in the ANS fluorescence with a nonzero anisotropy in an aqueous medium in the presence of an amphiphilic cetyltrimethylammonium cation as a result of the formation of the 1:1 complex with ANS. Water molecules quench the fluorescence of ANS. The enhancement of the ANS fluorescence in aqueous media in the presence of fluorescence enhancers is accounted for by their blocking the access of water molecules to the region close to the excited ANS molecule, which is critical for the fluorescence.