Principles of Fluorescence Spectroscopy

294 QUENCHING OF FLUORESCENCE
Figure 8.22. Oxygen quenching of 2-methylanthracene (2-MA) at
30.6°C, in DMPC (T c = 24°C) and DPPC (T c = 37°C) bilayers.
of cholesterol where the amount of quenching decreases
(Figure 8.23, !).
In order to calculate the bimolecular quenching constants
it is necessary to know the oxygen concentration in
the membranes. Unfortunately, precise values are not
known, particularly for membranes with different lipid
compositions. The oxygen solubility in membranes is usually
taken as equal to that in nonpolar solvents, and thus
Figure 8.23. Oxygen quenching of pyrene dodecanoic acid in erythrocyte
ghost membranes with various cholesterol/protein ratios: ", control,
no added cholesterol; Chol/Protein ratios: O, 0.19; , 0.35; ,
0.65; △, 0.71; !, 0.83. Revised from [59].
approximately fourfold larger than in water. Assuming a
lifetime of PDA near 120 ns, one can use the data in Figure
8.23 to calculate k q = 0.22 x 10 10 M –1 s –1 . The permeability
(P) of a membrane can be approximated by P = k q D/∆x,
where D is the oxygen diffusion coefficient and ∆x is the
membrane thickness. Given that the diffusion coefficient of
oxygen is about 1/5 of that in water, and the lipid–water
partition coefficient is near 5, this equation suggests that
biological membranes do not pose a significant diffusive
barrier to oxygen.
8.10.2. Localization of Membrane-Bound
Tryptophan Residues by Quenching
Collisional quenching is a short-range interaction, so that
the extent of quenching can be used to indicate the amount
of molecular contact between the fluorophore and
quencher. This concept has been used to study the location
of tryptophan residues in membrane-spanning peptides. 61 A
series of peptides were synthesized that contained a single
tryptophan residue. These peptides were roughly of the
form K q L k WL m K m , where K is a charged amino acid lysine,
L is a nonpolar amino acid leucine, and W is tryptophan.
The number of nonpolar residues on each side of the tryptophan
was varied to position the trp at various distances
from the center of the DOPC bilayers. Figure 8.24 shows
acrylamide quenching of the peptides when bound to
DOPC vesicles. The extent of quenching depends strongly
Figure 8.24. Acrylamide quenching of tryptophan residues in a membrane-spanning
peptide. Revised and reprinted with permission from
[61]. Copyright © 2003, American Chemical Society.