Principles of Fluorescence Spectroscopy

304 QUENCHING OF FLUORESCENCE
Figure 8.42. Stern-Volmer plots for the iodide quenching of E. coli tet
repressor (wild type, WT) and its mutants (W75F and W43F). Revised
from [108].
quenching of the wild-type protein is intermediate between
the two single-tryptophan mutants. These results are consistent
with those obtained from the quenching-resolved emission
spectra. While the same information is available from
the mutant proteins, the use of quenching provided the
resolved spectra using only the wild-type protein.
It is valuable to notice a difference in the method of
data analysis for the modified Stern-Volmer plots (Section
Figure 8.43. Emission spectra of a four α-helix bundle in water in the
presence of halothane. Each peptide chain contains one tryptophan
residue. Revised from [114].
8.8.1) and for the quenching-resolved emission spectra. In
a modified Stern-Volmer plot one assumes that a fraction of
the fluorescence is totally inaccessible to quenchers. This
may not be completely true because one component can be
more weakly quenched, but still quenched to some extent.
If possible, it is preferable to analyze the Stern-Volmer plots
by nonlinear least squares, when the f i and K i values are
variable. This approach allows each component to contribute
to the data according to its fractional accessibility,
instead of forcing one to be an inaccessible fraction. Of
course, such an analysis is more complex, and the data may
not be adequate to recover the values of f i and K i at each
wavelength.
8.13. QUENCHING AND ASSOCIATION
REACTIONS
8.13.1. Quenching Due to Specific Binding
Interactions
In the preceding sections we considered quenchers that
were in solution with the macromolecule but did not display
any specific interactions. Such interactions can occur, and
often appear to be of static quenching. 110–115 One example is
provided by a synthetic peptide which spontaneously forms
a four α-helical bundle in aqueous solution (Figure 8.43).
The bundle consists of two peptide chains. Each peptide
chain contains two α-helical regions and a single tryptophan
residue. 114 The general anesthetics are nonpolar and
were expected to bind to the central nonpolar region of the
four helix bundles.
The effects of halothane on the emission intensity of
the four-helix bundle are shown in Figure 8.43. The emission
is strongly quenched by even low concentrations of
halothane. Quenching of the trp residues was also examined
in the presence of 50% trifluoroethanol (TFE). Halothane
quenching is much less efficient in this solvent (Figure
8.44). TFE is known to disrupt hydrophobic interactions but
to enhance helix formation in peptides. In 50% TFE the
peptide is expected to exist as two separate α-helical peptides
that are not bound to each other. These results show
that the trp residues are buried in a nonpolar region in the
four-helix bundle and become exposed to the solvent phase
when the two peptides dissociate.
How can one determine whether the quenching seen
for the helix bundle in water is due to halothane binding, or
to collisional quenching? One method is to calculate the
apparent bimolecular quenching constant (k q app). Assume
the decay time of trp residues in the bundle is near 5 ns. The