Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 305
Figure 8.44. Tryptophan fluorescence intensities of the four α-helix
bundle in the presence of halothane. Revised from [114].
data in Figure 8.43 indicate that the trp residues are about
50% quenched at 142 µM halothane. This corresponds to a
Stern-Volmer quenching constant of K SV = 7042 M –1 , which
is two to three orders of magnitude larger than possible for
diffusion-limited quenching in water. These results show
that hydrophobic interactions of halothane result in the
appearance of static quenching of the trp residues. These
interactions result in an increase in the local concentration
of halothane near the trp residues, as shown in the molecular
dynamics snapshot of this system (Figure 8.45).
At a more microscopic level there may still be a
dynamic component to the quenching. There is no reason
to expect the formation of ground-state complexes
between the trp and halothane. Hence the quenching may
be dynamic but due to diffusion motions of halothane with-
Figure 8.45. Molecular dynamic snapshot of the four α-helix bundle
with one bound halothane molecule. The two peptides are shown in
light and dark green, the tryptophans are red and the methionines are
yellow-orange. The halothane molecule contains the multicolor
spheres. Reprinted from [114]. Courtesy of Dr. Jonas Johansson from
the University of Pennsylvania, USA.
Figure 8.46. Quenching of HSA by caffeine. Revised from [113].
in the hydrophobic region of the bundle. There is not
always a clear distinction between static and dynamic
quenching. Actual and apparent complex formation may
be distinguished from the absorption spectra, which is only
expected to change for actual formation of ground-state
complexes.
Another example of quenching due to a specific binding
interaction is shown for binding of caffeine to HSA, an
experiment most of us start each morning (Figure 8.46).
The Stern-Volmer plots show a value of K SV = 7150 M –1 .
This value is obviously too large to be due to collisional
quenching, especially for a lifetime near 5 ns. The apparent
value of k q app is 1.4 x 10 12 , over 100-fold larger than the
maximum diffusion limited rate. Hence, the caffeine must
be bound to the HSA. Caffeine is an electron-deficient molecule,
and may form ground-state complexes with indole.
This possibility could be tested by examination of the
absorption spectra of HSA in the absence and presence of
caffeine. If ground-state association with indole occurs,
then the trp absorption spectrum is expected to change.
Another indicator of complex formation is the temperature
dependence of the Stern-Volmer plots. For diffusive
quenching one expects more quenching at higher temperatures.
In the case of HSA and caffeine there is less quenching
at higher temperatures (Figure 8.46), which suggests the
complex is less stable at higher temperatures.
8.14. SENSING APPLICATIONS OF QUENCHING
Fluorescence quenching has been used for sensing of a
wide variety of analytes including oxygen, NO, and heavy
metals. Fluorescence sensing is discussed in more detail in
Chapter 19. The use of collisional quenching for sensing is
illustrated by chloride-sensitive fluorophores.