title: Photochemical behavior and Na+,K+-ATPase sensitivity of voltage-sensitive styrylpyridinium fluorescent membrane probes.
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.
authors.affiliation: School of Chemistry, University of Sydney, Sydney, Australia.
authors.forename: Steve
authors.initials: S
authors.lastname: Amoroso
authors.forename: Vanessa V
authors.initials: VV
authors.lastname: Agon
authors.forename: Thomas
authors.initials: T
authors.lastname: Starke-Peterkovic
authors.forename: Malcolm D
authors.initials: MD
authors.lastname: McLeod
authors.forename: Hans-Jürgen
authors.initials: HJ
authors.lastname: Apell
authors.forename: Pierre
authors.initials: P
authors.lastname: Sebban
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Clarke