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Photochemical Behavior and Na + ,K + ‐ATPase Sensitivity of Voltage‐sensitive Styrylpyridinium Fluorescent Membrane Probes
Author(s) -
Amoroso Steve,
Agen Vanessa V.,
StarkePeterkovic Thomas,
McLeod Malcolm D.,
Apell HansJürgen,
Sebban Pierre,
Clarke Ronald J.
Publication year - 2006
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 - 10.1562/2005-06-08-ra-569
Subject(s) - fluorescence , chemistry , excited state , photochemistry , membrane , photodissociation , ion , excitation , absorption (acoustics) , analytical chemistry (journal) , materials science , atomic physics , optics , biochemistry , physics , electrical engineering , organic chemistry , composite material , engineering , chromatography
RH421 is a widely used voltage‐sensitive fluorescent membrane probe. Its exposure to continuous illumination with 577 nm light from an Hg lamp leads, however, to an increase in its steady‐state fluorescence level when bound to lipid membranes. The increase occurs on the second time scale at typical light intensities and was found to be due to a single‐photon excited‐state isomerization. Modifications to the dye structure are, therefore, necessary to increase photochemical stability and allow wider application of such dyes in kinetic studies of ion‐transporting membrane proteins. The related probe ANNINE 5, which has a rigid polycyclic structure, shows no observable photochemical reaction when bound to DMPC vesicles on irradiation with 436 nm light. The voltage sensitivity of ANNINE 5 was tested with the use of Na + ,K + ‐ATPase membrane fragments. As long as ANNINE 5 is excited on the far red edge of its visible absorption band, it shows a similar sensitivity to RH421 in detecting charge‐translocating reactions triggered by ATP phosphorylation. Unfortunately the wavelengths necessary for ANNINE 5 excitation are in a region where the Hg lamps routinely used in stopped‐flow apparatus have no significant lines available for excitation.