Principles of Fluorescence Spectroscopy

316 QUENCHING OF FLUORESCENCE
Figure 8.66. Fluorophore-quencher conjugates that display intramolecular
quenching. Revised and reprinted with permission from
[154–155]. Copyright © 1990, American Chemical Society.
quenched. It is now known that this quenching is due to
electron transfer. 157–158 The amino-acid residues responsible
for flavin quenching were identified using site-directed
mutagenesis. Figure 8.68 shows the intensity decays of
FMN free in solution and when bound to flavin reductase
from Escherichia coli. The lifetime of FMN bound to the
wild-type (WT) protein is much shorter than for FMN in
solution. This flavin reductase contains three tyrosine (Y)
residues at position 35, 72, and 116. Mutant proteins were
prepared that contained only two of these residues. The
flavin lifetime was still short for the mutants without tyr 72
or tyr 116, indicating that these tyrosines were not responsible
for quenching. The lifetime of FMN increased dramatically
when tyr 35 was removed, demonstrating that this
residue is responsible for quenching. The quenching mechanism
is electron transfer from try 35 to FMN. Tyr 35 is
4.5D away from FMN (upper panel). The other two
residues are further away from the flavin: 9.6D and 7.0D for
tyr 72 and tyr 116, respectively. Electron-transfer reactions
Figure 8.67. Intramolecular quenching of a DBO-labeled oligomer by
a terminal guanine residue. Revised and reprinted with permission
from [156]. Copyright © 2004, American Chemical Society.
only occur over short distances, which explains the lack of
quenching by tyr 72 and tyr 116.
8.17.3. Sensors Based on Intramolecular
PET Quenching
Intramolecular photoinduced electron transfer (PET) often
results in quenching. The phenomenon of PET has been
widely used to develop fluorescent sensors. 159–163 In a typical
PET sensor an aromatic fluorophore such as anthracene
is covalently linked to an aliphatic amine, typically by a
short methylene chain. If the amine is not protonated it
quenches the fluorophore. Protonation of the amine
decreases its ability to donate an electron, so the fluorescence
intensity increases.
Figure 8.69 shows the structure of a typical PET sensor.
163 The anthracene group is quenched by the amino
groups. Addition of pyrophosphate (PPi) resulted in an
increase in fluorescence because of protonation of some of
the linked amino groups. The increased fluorescence may
have also been due in part to less motion of the side chain
during the excited-state lifetime, and thus less quenching.