Principles of Fluorescence Spectroscopy

288 QUENCHING OF FLUORESCENCE
Figure 8.12. Quenching of etheno-ATP (ε-ATP, "), ε-ADP (∆), ε-AMP (G) and ε-Ad (∇) by thallium, iodide, and acrylamide in 10 mM phosphate
buffer, 0.1 M KCl, 20°C, pH 7.0. Revised from [33].
8.8. FRACTIONAL ACCESSIBILITY TO
QUENCHERS
Proteins usually contain several tryptophan residues that are
in distinct environments. Each residue can be differently
accessible to quencher. Hence one can expect complex
Stern-Volmer plots, and even spectral shifts due to selective
quenching of exposed versus buried tryptophan residues.
One example is quenching of lysozyme. This protein from
egg white has six tryptophan residues, several of which
are known to be near the action site. Lysozyme fluorescence
was observed with increasing concentrations of trifluoroacetamide
(TFA), which was found to be a collisional
quencher of tryptophan fluorescence. 36 The Stern-Volmer
plot curves downward towards the x-axis. As will be
described below, this is a characteristic feature of two fluorophore
populations, one of which is not accessible to
the quencher. In the case of lysozyme the emission spectrum
shifts progressively to shorter wavelengths with
increasing TFA concentrations (Figure 8.13, right). This
indicates that those tryptophan residues emitting at larger
wavelengths are quenched more readily than the shorter
wavelength tryptophans.
The emission spectrum of the quenched residues can
be calculated from taking the difference between the
unquenched and quenched emission spectra. This spectrum
shows that the quenched residues display an emission maxima
at 348 nm. The protected residues display an emission
Figure 8.13. Quenching of lysozyme by trifluoroacetamide (TFA).
Left: Stern-Volmer plot. Right: Emission spectra with increasing concentrations
of TFA. Also shown is the difference spectrum (diff),
0.0–0.77 M TFA. Revised and reprinted from [36]. Copyright © 1984,
with permission from Elsevier Science.
maxima at 333 nm. Similar results were obtained for
quenching of lysozyme by iodide. 37
8.8.1. Modified Stern-Volmer Plots
The differing accessiblities of tryptophan residues in proteins
has resulted in the frequent use of quenching to
resolve the accessible and inaccessible residues. 37 Suppose
there are two populations of fluorophores, one of which is
accessible (a) to quenchers and the other being inaccessible
or buried (b). In this case the Stern-Volmer plot will display