Principles of Fluorescence Spectroscopy

296 QUENCHING OF FLUORESCENCE
Figure 8.27. Structures of a bromo-PC (top), 9,10-dibromostearic
acid (middle), and a nitroxide labeled fatty acid (bottom), which act as
localized quenchers in membranes.
Figure 8.28. Structures of the fluorenyl fatty acids. Revised from
[62].
of the fatty acid (Figure 8.27, middle), so one expects maximal
quenching for those fluorenyl groups located as deep
as the bromine atoms. The quenching data reveal larger
amounts of quenching when the fluorenyl groups are placed
more deeply in the bilayer by a longer methylene chain
between the fluorenyl and carboxyl groups (Figure 8.29).
Hence, the fluorenyl probes are located as expected from
Figure 8.29. Quenching of fluorenyl fatty acids (Figure 8.28) by 9,10dibromostearic
acid in erythrocyte ghost membranes. Revised and
reprinted with permission from [62]. Copyright © 1992, American
Chemical Society.
their structure. For these fluorenyl fatty acids a continuing
alkyl chain beyond the fluorenyl group was important for
probe localization.
Another example of using localized quenchers is provided
by studies of pyrene-labeled lipids. 63 A number of
pyrene PCs were used that had a different number of methylene
groups (n) between the glycerol backbone and the
pyrene. This localizes the pyrene groups at different depths
in the bilayers (Figure 8.30). Three different bromo-PCs
with the bromides at position 6 and 7, 9 and 10, or 11 and
12 were used as quenchers. The apparent Stern-Volmer
quenching constants were measured for each pyrene PC and
each quencher (Figure 8.31). The largest quenching constant
was observed for the smallest number of methylene
groups in the pyrene PC (n) when using Br 6,7 PC, in which
the bromides are located just beneath the membrane surface.
For larger values of n, maximum quenching was
observed for Br 11,12 PC, where the bromides are located
more deeply in the membrane. In this case the fluorophores
were localized in bilayer as would be predicted from their
structure. Bromo-PCs are known to quench the fluorescence
of membrane-bound proteins, 64–66 and have been
used to study lateral phase separations in membranes. 67–68
8.10.4. Parallax and Depth-Dependent Quenching
in Membranes Advanced Topic
The use of quenching to determine the location of fluorophores
in membranes has been formalized using two similar
methods: parallax quenching 69–75 and depth-dependent
quenching. 76–78 The basic idea is to compare the amount of
quenching observed for quenchers that are located at two