Principles of Fluorescence Spectroscopy

298 QUENCHING OF FLUORESCENCE
σ are found by least-squares or other filtering procedures.
Depth-dependent quenching was used for the analysis
shown in Figure 8.31.
8.10.5. Boundary Lipid Quenching
Advanced Topic
Quenching in membranes can also be used to study boundary
lipids, which are the lipid molecules surrounding a fluorophore
or a membrane-bound protein. 80–86 Suppose a protein
is surrounded by a discrete number of lipid molecules,
and that the tryptophan fluorescence is accessible to
quenchers in the membrane phase. Then the number of
boundary lipid molecules can be estimated from
F Fmin (1 Q)
F0 Fmin n
(8.35)
where F 0 is the intensity in the absence of quencher and
F min is the intensity when the probe is in pure quencher
lipid. F is the intensity at a given mole fraction of quencher
lipid.
This model was tested using small probes in membranes,
and for Ca 2+ -ATPase, which is a large membranebound
protein containing 11 to 13 tryptophan residues. For
tryptophan octyl ester, the intensity decreased according to
n = 6, indicating that each tryptophan was surrounded by 6
lipid molecules (Figure 8.32). For the Ca 2+ -ATPase, the
intensity decreased with n = 2. This does not indicate that
only two lipid molecules surrounded this protein, but that
only two lipid molecules are in contact with tryptophan
residues in the Ca 2+ -ATPase. Similar results of two boundary
quenchers were found for the Ca 2+ -ATPase using 1,2bis(9,10-dibromooleoyl)phosphatidylcholine.
82
8.10.6. Effect of Lipid–Water Partitioning on
Quenching
In the preceding examples of quenching in membranes the
quenchers were not soluble in water. Hence, the quencher
concentrations in the membrane were known from the
amount of added quencher. However, there are many
instances where the quencher partitions into the membranes,
but some fraction of the quencher remains in the
aqueous phase. Consequently, the quencher concentration
in the membrane is not simply determined by the amount of
Figure 8.32. Quenching of tryptophan octyl ester (") and the
Ca 2+ -ATPase (!) by a spin labeled (7,6)PC in egg PC vesicles.
(7,6)-PC in a phosphatidylcholine in which the spin label is
located on the 8th carbon atom chain of the 2-position fatty acyl
group. The structure of the nitroxide spin label is shown in Figure
8.27. The solid lines are for the indicated values of n (eq. 8.35).
The dashed lines show the theoretical curves for n = 10 or 2 in
(eq. 8.35). Revised and reprinted with permission from [80].
Copyright © 1981, American Chemical Society.
quencher added, but also by the total lipid concentration in
the sample. In these cases it is necessary to determine the
lipid–water partition coefficient in order to interpret the
observed quenching.
Consider a quencher that distributes between the membrane
and aqueous phases. At non-saturating concentrations
of quencher the concentrations in the water (w) and membrane
(m) phases are related by the partition coefficient
P Q m / Q w
(8.36)
The total (T) concentration of quencher added ([Q] T ) partitions
between the water and membrane phases according to
Q TV T Q mV m Q wV w
(8.37)
where V m and V w represent the volume of the membrane
and water phases, respectively. By defining
α m V m/V T
(8.38)