Principles of Fluorescence Spectroscopy

290 QUENCHING OF FLUORESCENCE
relative contribution of scattered light always increases with
quenching.
It is also important to consider the absorption spectra
of the quenchers. Iodide and acrylamide absorb light below
290 nm. The inner filter effect due to absorption can
decrease the apparent fluorescence intensity, and thereby
distort the quenching data. 38–39 Regardless of the quencher
being used, it is important to determine if the inner filter
effects are significant. If inner filter effects are present the
observed fluorescence intensities must be corrected. The
lifetime measurements are mostly independent of inner filter
effects because the lifetime measurements are relatively
independent of total intensity.
When using iodide or other ionic quenchers it is important
to maintain a constant ionic strength. This is usually
accomplished by addition of KCl. When using iodide it is
also necessary to add a reducing agent such as Na 2 S 2 O 3 .
Otherwise, I 2 is formed, which is reactive and can partition
into the nonpolar regions of proteins and membranes.
8.9. APPLICATIONS OF QUENCHING TO
PROTEINS
8.9.1. Fractional Accessibility of Tryptophan
Residues in Endonuclease III
There have been numerous publications on determining the
fraction of protein fluorescence accessible to
quenchers. 40–47 In Section 13.5 we show that proteins in the
native state often display a fraction of the emission that is
not accessible to water-soluble quenchers, and that denaturation
of the proteins usually results in accessibility of all
the tryptophan residues to quenchers. The possibility of
buried and exposed residues in an apurinic single protein is
illustrated by endonuclease III. Endo III is a DNA repair
enzyme that displays both N-glycosylase and apurinic/
apyrimidinic lyase activities. The structure of Endo III
shows two domains, with the DNA binding site in the cleft
region. Endo III contains two tryptophan residues. 42 Trp
132 is exposed to the solvent, and trp 178 is buried in one
of the domains (Figure 8.15). Hence one expects these
residues to be differently accessible to water-soluble
quenchers.
The Stern-Volmer plot for iodide quenching of Endo III
shows clear downward curvature (Figure 8.16, top). The
modified iodide Stern-Volmer plot for Endo III shows clear
evidence for a shielded fraction (bottom). Extrapolation to
high iodide concentrations yields an intercept near 2, indicating
that only half of the emission can be quenched by
Figure 8.15. Structure of Endo III showing the exposed residue trp
132 and the buried residue trp 178 near the iron-sulfur cluster.
Figure 8.16. Stern-Volmer plot and modified Stern-Volmer plot for
iodide quenching of Endo III showing evidence for two types of tryptophan
residues. The inaccessible fraction is f a = 0.47. Revised and
reprinted with permission from [42]. Copyright $©$ 1995, American
Chemical Society.