Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 547
Figure 16.30. Collisional quenching of buried (W 1 ) and surface accessible (W 2 ) tryptophan residues in proteins.
single-tryptophan azurin Pae. The spectrum of the
quenched tryptophan residue can be seen from the difference
spectrum, and is characteristic of an exposed residue
in a partially hydrophobic environment. In this favorable
case, one residue is quenched and the other is not, providing
resolution of the two emission spectra.
16.6.1. Effect of Emission Maximum on Quenching
Water-soluble quenchers, including iodide and acrylamide,
do not readily penetrate the hydrophobic regions of proteins.
There is a strong correlation between the emission
maximum and quenching constant. 101–102 Blue-shifted tryptophan
residues are mostly inaccessible to quenching by
acrylamide, and red-shifted residues are nearly as accessible
as tryptophan in water. This correlation can be seen in
the acrylamide quenching of several proteins (Figure
16.32). The emission of azurin is essentially unchanged in
up to 0.8 M acrylamide. In contrast, the exposed tryptophan
residue in adrenocorticotropin hormone (ACTH) is almost
completely quenched at 0.4 M acrylamide (Figure 16.32,
left panel).
Quenching data are typically presented as Stern-
Volmer plots, which are shown for several single-tryptophan
proteins (Figure 16.32, right panel). In these plots the
larger slopes indicate larger amounts of quenching and can
be used to calculate the bimolecular quenching constant
(k q ). The buried single-tryptophan residue in azurin Pae is
not affected by acrylamide. In contrast, the fully exposed
residue in ACTH is easily quenched by acrylamide. ACTH
is quenched nearly as effectively as NATA. A plot of the
bimolecular quenching constant (k q ) for acrylamide versus
emission maximum for a group of single-tryptophan proteins
is shown in Figure 16.33. These data show that k q