Photochemical behavior and Na+,K+-ATPase sensitivity of voltage-sensitive styrylpyridinium fluorescent membrane probes

Photochemistry and Photobiology
Steve AmorosoRonald J Clarke

Abstract

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.

References

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Citations

Aug 25, 2010·Journal of Bioenergetics and Biomembranes·Steve AmorosoRosanne Quinnell
Sep 18, 2014·Biophysical Journal·Laura J MaresRonald J Clarke
Jan 25, 2017·Biochimica Et Biophysica Acta. Biomembranes·Alvaro GarciaRonald J Clarke
Aug 6, 2019·Frontiers in Cellular Neuroscience·Bernd Kuhn, Christopher J Roome
Apr 22, 2008·The Journal of Physical Chemistry. B·T H Nguyen Pham, Ronald J Clarke

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Methods Mentioned

BETA
light jump
NMR

Software Mentioned

ANNINE

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