Principles of Fluorescence Spectroscopy

534 PROTEIN FLUORESCENCE
Figure 16.6. Absorption spectra of indole in cyclohexane
with increasing amounts of ethanol. Data courtesy of Dr. Ignacy Gryczynski.
As the ethanol concentration is increased the vibrational
structure at 288 nm decreases. Similar results have been
observed for indole derivatives. 38–39 These spectral changes
can be understood as a red shift of the 1 L a absorption spectrum
due to interaction of indole with the polar solvent.
This energy shift is probably due to both hydrogen bonding
interactions with the imino group and the general effects of
solvent polarity. The 1 L b state is less sensitive to solvent,
and its absorption is less affected by polar solvent.
The properties of the 1 L a and 1 L b states explain the
complex emission spectra displayed by indole (Figure
16.5). In a completely nonpolar solvent the structured 1 L b
state can be the lowest energy state, resulting in structured
emission. The presence of polar solvent decreases the energy
level of the 1 L a state, so that its unstructured emission
dominates. At higher ethanol concentrations these specific
solvent interactions are saturated, and indole displays a
polarity-dependent red shift consistent with general solvent
effects. Very few proteins display a structured emission,
which suggests few tryptophan residues are in completely
nonpolar environments.
16.1.3. Excited-State Ionization of Tyrosine
Tyrosine displays a simple anisotropy spectrum, but it is
important to recognize the possibility of excited-state ionization.
Excited-state ionization occurs because the pKA of
the phenolic hydroxyl group decreases from 10.3 in the
Figure 16.7. Normalized emission spectra of tyrosine at pH 7 and in
0.01 M NaOH (pH 12). Modified from [16].
ground state to about 4 in the excited state. Ionization can
occur even at neutral pH, particularly if the solvent contains
proton acceptors such as acetate. Tyrosinate emission is
most easily observed at high pH, where the phenolic OH
group is ionized in the ground state (Figure 16.7). In 0.01
M NaOH (pH 12) the emission of tyrosine is centered near
345 nm. 6,40 The emission from tyrosinate can be mistaken
for tryptophan. The decay time of tyrosinate at pH 11 has
been reported to be 30 ps. 41
In Figure 16.7 the tyrosine hydroxyl group is ionized in
the ground state. Tyrosinate emission can also be observed
at neutral pH, particularly in the presence of a base that can
interact with the excited state. One example is shown in
Figure 16.8, which shows the emission spectra of tyrosine
at the same pH, but with increasing concentrations of
acetate buffer. The emission intensity decreases with
increased acetate concentrations. This decrease occurs
because the weakly basic acetate group can remove the phenolic
proton, which has a pK a of 4.2 to 5.3 in the first singlet
state. 40–43 If the tyrosinate form does not emit, the
acetate behaves like a collisional quencher, and the extent
of excited-state ionization and quenching depends on the
acetate concentration. Tyrosine can also form ground-state
complexes with weak bases such as phosphate. 44–45
Because of its low quantum yield the emission from
tyrosinate is not easily seen in Figure 16.8. This emission is