The paper reports the experimental results of bioluminescence quenching in the coupled enzyme system NADH:FMN-oxidoreductase-luciferase in the presence of xanthene dyes (fluorescein, eosin Y, erythrosin B) featured by the rate constants of intersystem crossing. From the spectral data and with the help of kinetic model the rate constants of energy transfer from emitter to dye were determined (2.9 × 1012–6.5 × 1013 M−1 s−1) and the emitter lifetime was estimated (1.2–2.7 ns). The calculated rate constants of energy transfer in a series of xanthene molecules are higher than the diffusion-controlled constants. The rate constants of energy transfer correlate with the probability of intersystem crossing of a dye. The combined analysis of emitter fluorescence quenching and acceptor fluorescence enhancement indicates that the significant bioluminescence intensity decrease in the presence of xanthene dyes is determined by both the quenching processes via the energy transfer and the inhibition of bioluminescent reaction.

The role of energy transfer in bioluminescence quenching by xanthene dyes

Modeling of kinetics of photoprocesses in polyatomic molecules based on four-level energy scheme is carried out. The
model includes the processes in dye under cw photoexcitation and takes into account a reverse intersystem crossing and
photochemical reaction from the upper triplet states. The analytical time-dependent relation for dye concentration of the
initial form was obtained taking into account the contrast of rates of various photoprocesses and using of quasistationary
approximation. It was shown that rate constant of photolysis correlates with probability of intersystem crossing of the
dyes. For the xanthene dyes in gelatin it was shown that simple experimental method of cw laser photolysis combined
with the kinetic model could be effective technique of study of upper excited states participation.

K. Tarbetskaya

Papers

Study of photoreduction kinetics of xanthene dyes with intersystem crossing from upper triplet states