KINETIC SPECTROSCOPY OF ERYTHROSIN PHOSPHORESCENCE AND DELAYED FLUORESCENCE IN AQUEOUS-SOLUTION AT ROOM-TEMPERATURE

The photophysics and polarization of the phosphorescence and delayed f luorescence of erythrosin in conditions compatible with the current bi ological applications of the dye (aqueous buffers at pH 7.4 at ambient temperatures) and in ethanol have been studied as a function of dye c oncentration (10(-7)-10(-5) M) and temperature (245-333 K), The emissi on decay is strictly single exponential and the detailed kinetic analy sis of all the rate processes connected with the emitting T-1 state sh owed that (1) the lowering of the emission lifetime at the higher temp eratures is due to a very efficient self-quenching process, (2) the ba ck intersystem crossing rate T-1 curved right arrow S-1 is temperature dependent (Delta E-TS approximate to 7 kcal mol(-1)) but the T-1 curv ed right arrow S-0 is not (E-a < 0.1 kcal mol(-1)) and (3) both inters ystem crossing processes are very sensitive to solvent polarity, which accounts for the solvent dependence of the phosphorescence yield and lifetime. The high value of the phosphorescence anisotropy (r(0) = 0.2 5 +/- 0.006) is independent of the excitation and emission wavelengths , and its evolution in time accurately reflects the rotational restric tions in solid solutions. The relevance of these findings to studies w ith protein-dye conjugates is also outlined to facilitate the design a nd interpretation of phosphorescence depolarization experiments that p robe the mu s-ms dynamics of biomolecules and supramolecular systems.