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A PHOTOCHEMICAL TECHNIQUE TO ENHANCE SENSITIVITY OF DETECTION OF FLUORESCENCE RESONANCE ENERGY TRANSFER
Author(s) -
Mekler Vladimir M.
Publication year - 1994
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1994.tb09665.x
Subject(s) - förster resonance energy transfer , photobleaching , photochemistry , chemistry , acceptor , fluorescence , acridine orange , reaction rate constant , kinetics , resonance fluorescence , energy transfer , analytical chemistry (journal) , optics , chemical physics , organic chemistry , apoptosis , biochemistry , physics , quantum mechanics , condensed matter physics
A photochemical kinetic method of measuring small values of efficiency of fluorescence resonance energy transfer (FRET) between special probes is proposed. The FRET efficiency (ω) is determined from kinetics of the photochemical reaction of the energy acceptor sensitized by FRET from the energy donor. The choice of an appropriate donor‐acceptor pair permits the minimization of background reactions. Application of the method is demonstrated by the detection of FRET from 2,5‐ bis (5‐ tert ‐butyl‐2‐benzoxasolyl)thiophen (BBOT) to acridine orange (AO) in phospholipid vesicles. Photobleaching of AO in the presence of CBr 4 was applied as a photochemical reaction of the acceptor. The reaction was monitored by steady‐state fluorescence measurements. The FRET measurements were carried out by the proposed technique when the probe/lipid ratio and ω were as small as 1.1 × 10 −5 M/M and 0.0017, respectively. Under these conditions, the rate constant of AO photobleaching was increased by 26% as compared with that of the reference sample without BBOT. The results suggest that applications of the technique may be useful in the study of the membrane topography.