Misconceptions over Förster resonance energy transfer between proteins and ANS/bis-ANS: Direct excitation dominates dye fluorescence.
Our aim was to disprove the widespread misconception that Förster resonance energy transfer (FRET) is the only explanation for observing fluorescence from ANS (8-anilino-1-naphthalenesulfonic acid) and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid, dipotassium salt) following excitation at 280nm in the presence of protein. From ultraviolet (UV) absorption spectra and fluorescence emission spectra of bis-ANS and ANS in buffer and ethanol, direct excitation at 280nm was found to be the dominant mechanism for the resulting dye fluorescence. Furthermore, Tyr/Trp quenching studies were performed for solutions of N-acetyl-l-tryptophanamide, heat-stressed immunoglobulin G (IgG), and bovine serum albumin (BSA) by monitoring changes in steady state fluorescence spectra and time-resolved fluorescence decays as a function of dye concentration. Stronger quenching of the intrinsic BSA and IgG fluorescence in steady state than in time-resolved fluorescence by bis-ANS and ANS pointed toward static quenching being the dominant mechanism in addition to dynamic quenching and/or FRET. In conclusion, one should consider the role of direct excitation of ANS and bis-ANS at 280nm to ensure a proper interpretation of fluorescence signals resulting from dye-protein interactions. When ANS or bis-ANS is to be used for protein characterization, we recommend selectively exciting the dyes at the higher absorption wavelength maximum (370 or 385nm, respectively).
Hawe
Andrea
A
Division of Drug Delivery Technology, Leiden/Amsterdam Center for Drug Research, Leiden University, 2300 RA Leiden, The Netherlands. ahawe@chem.leidenuniv.nl
Poole
Robert
R
Jiskoot
Wim
W
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet
C515594:8-anilino-1-naphthalenesulfonic acid; D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D011506:Proteins; D012710:Serum Albumin, Bovine; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate)