REVERSIBLE PHOTOBLEACHING OF FLUORESCEIN CONJUGATES IN AIR-SATURATED VISCOUS SOLUTIONS - SINGLET AND TRIPLET-STATE QUENCHING BY TRYPTOPHAN

Fluorescence recovery after photobleaching (FRAP) measurements on air- saturated aqueous solutions of fluorescein made viscous with glycerol or sucrose revealed a rapid component of fluorescence recovery with ex ponential time constants of 30-120 mu s at viscosities of 15-300 cP. T he rapid recovery process was not related to fluorophore translational diffusion and was insensitive to fluorophore concentration and the ad ditive used to increase solution viscosity, At constant viscosity, the rate of reversible photobleaching recovery increased 2.5-fold in an O -2- vs N-2-saturated solution. The relative efficiency of reversible-t o-irreversible photobleaching decreased with increasing photobleaching time and/or beam intensity. Reversible photobleaching was also detect ed for conjugates of fluorescein with dextrans and proteins in viscous media. In screening triplet state quenchers that might influence the reversible recovery, it was found that tryptophan enhanced the rate of reversible photobleaching recovery (two-fold increase at 8 mM) and qu enched the fluorescein singlet state (Stern-Volmer constant, 12 M(-1)) . Analysis of fluorescein lifetimes and photobleaching parameters for a series of fluorescein-labeled proteins with different numbers of try ptophans were also carried out, The results provide evidence for an ox ygen-dependent, reversible photobleaching mechanism for the fluorescei n chromophore involving triplet state relaxation. The identification o f reversible fluorescein photobleaching has important implications for FRAP measurements of rapid solute diffusion in biological systems.