Photoacoustic determination of fluorescent quantum yields of protein probes

Pulsed-laser photoacoustic methods may be used to determine the fluorescent quantum yields of fluorophores in solution. Of interest to biological spectroscopists are the fluorescent quantum yields of probes bound covalently or noncovalently to proteins. Previous studies (J.R. Small et al., Fluorescence Detection III, E.R. Menzel, ed., SPIE Proceedings 1054, pp. 26-35, 1989) have been extended to examine some common protein probes and their fluorescent quantum yields. Examples include the probes Prodan [6-propionyl-2-(dimethylamino)naphthalene] and Acrylodan [6-acryloyl-2-(dimethylamino)naphthalene] in a variety of protein and solvent environments. We have found that, at the simplest level, the pulsed-laser photoacoustic technique gives us excellent results for the fluorescent quantum yields of fluorophores free in solution, but interestingly anomalous results for the fluorophores bound to proteins. The source of the anomalous protein results has not yet been determined, but several possibilities are discussed.