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J. Biol. Chem., Vol. 265, Issue 23, 13533-13539, 08, 1990
RI MacDonald
Self- or concentration quenching of octadecylrhodamine B (C18-Rh)
fluorescence increases linearly in egg phosphatidylcholine (PC) vesicles
but exponentially in vesicles composed of egg PC:cholesterol, 1:1, as the
probe concentration is raised to 10 mol%. Cholesterol- dependent
enhancement of self-quenching also occurs when N-(lissamine-
rhodamine-B-sulfonyl)dioleoylphosphatidylethanolamine is substituted for
C18-Rh and resembles that in dipalmitoylphosphatidylcholine vesicles below,
as opposed to above, the phase transition. These effects are not due to
changes in dimer:monomer absorbance. Stern- Volmer plots indicate a
dependence of quenching on nonfluorescent dimers both in the presence and
absence of cholesterol. Decreases in fluorescence lifetimes with increasing
probe concentration parallel decreases in residual fluorescence of C18-Rh
with increasing probe concentration in PC and PC + cholesterol membranes,
respectively. Decreases in the steady-state polarization of C18-Rh
fluorescence as its concentration is raised to 10 mol% indicate energy
transfer with emission between probe molecules in PC and to a lesser extent
in PC + cholesterol membranes. The calculated R0 for 50% efficiency of
energy transfer from excited state probe to monomer was 55-58 A and to
dimer was 27 A. Since lateral diffusion of C18-Rh is probably too slow to
permit collisional quenching during the lifetime of the probe, even if
C18-Rh were concentrated in a separate phase, C18-Rh self-quenching appears
to be due mainly to energy transfer without emission to nonfluorescent
dimers.
Characteristics of self-quenching of the fluorescence of lipid- conjugated rhodamine in membranes
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208.
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