Effect of BSA binding on photophysical and photochemical properties of triarylmethane dyes

We have employed a combination of steady-state and time-resolved spectroscopic techniques to explore the effect of protein binding on the photophysical and photochemical properties of three triarylmethane dyes: ethyl violet, crystal violet, and malachite green. Our results indicate that the binding sites of bovine serum albumin (BSA) are very efficient in preventing fast nonradiative relaxation processes that occur via rotational motion of the aromatic rings of these triarylmethanes. As a result, remarkable enhancements in fluorescence quantum yield and lifetime, intersystem crossing efficiency, and photoreactivity are observed upon protein binding. The 532 nm laser-induced photobleaching of ethyl violet noncovalently bound to BSA yields leuco ethyl violet and 4,4'-bis(diethylamino)benzophenone as reaction products. The former was more prominent in nitrogen-purged samples and the latter in air-equilibrated samples. The time-resolved transient spectra of the ethyl violet complex show superimposed elements of the spectroscopic signatures of both ethyl violet triplet and the semireduced dye radical. Based on the nature of the reaction photoproducts and transient intermediates, the first step of the bleaching process is postulated to be an electron or hydrogen atom transfer from the protein to the dye moiety. An analogous reaction mechanism was observed for protein-bound crystal violet.