Principles of Fluorescence Spectroscopy

302 QUENCHING OF FLUORESCENCE
Figure 8.36. Steady-state emission spectra of (dashed) diphenylhexatriene
(DPH) and (solid) I-AEDANS in 1.2 mM SDS micelles at
23°C. The concentration of diphenylhexatriene was 2 µM, that of I-
AEDANS 170 µM. The excitation wavelength was 337 nm. Revised
from [103].
Figure 8.37. Stern-Volmer plots for acrylamide quenching of I-
AEDANS, DPH, or I-AEDANS and DPH, in SDS micelles. For the
mixture the solid line represents the fit with calculated parameters K 1
= 9.9 M –1 , K 2 = 0 M –1 , f 1 = 0.69, f 2 = 0.31. The lower panel shows the
residuals for these values. Revised from [103].
and the high solubility of acrylamide in water, DPH is
weakly quenched by acrylamide. In contrast, I-AEDANS is
strongly quenched. The extent of quenching for the mixture
is intermediate between that observed for each probe alone.
As expected for a mixture of fluorophores, the Stern-
Volmer plots curve downward due to the increasing fractional
contribution of the more weakly quenched species at
higher quencher concentrations.
Figure 8.38. Quenching resolved emission spectra of the DPH-I-
AEDANS mixture (bottom). The solid line shows the emission spectrum
of the mixture. The upper panel shows the wavelength-dependent
quenching constant, with average values of K 1 = 9.6 M –1 and K 2 =
0.47 M –1 . Revised from [103].
The curvature in the Stern-Volmer plots are used to
recover the values of K i (λ) and f i (λ) at each wavelength. In
this case the K i (λ) values were not used as global parameters,
so that K 1 (λ) and K 2 (λ) were obtained for each wavelength.
At wavelengths above 420 nm there were two values
of 0.47 and 9.6 M –1 , representing the quenching constants
of DPH and I-AEDANS, respectively. At the shortest wavelength
below 420 nm there is only one K i (λ) value because
only DPH emits. The recovered values of f i (λ) were used to
calculate the individual spectra from the mixture (Figure
8.38). In Chapters 4 and 5 we showed how the component
spectra for heterogeneous samples could be resolved using
the time-domain or the frequency-domain data. The use of
wavelength-dependent quenching provides similar results,
without the use of complex instrumentation. Of course, the
method depends on the probes being differently sensitive to
collisional quenching, which requires that the decay times
and/or accessibility to quenchers be different.
8.12.2. Quenching-Resolved Emission Spectra of
the E. Coli Tet Repressor
The tet repressor from E. coli is a DNA-binding protein that
controls the expression of genes that confer resistance to