Principles of Fluorescence Spectroscopy

598 TIME-RESOLVED PROTEIN FLUORESCENCE
Figure 17.41. Decay-associated spectra and time-resolved emission
spectra from myelin basic protein at 30°C, 289-nm excitation. Revised
from [119].
residues, multiple conformations, and spectral relaxation all
affect the intensity decays.
17.8. PHOSPHORESCENCE OF PROTEINS
Advanced Topic
Phosphorescence is usually observed at low temperature
and/or in the complete absence of quenchers. 122–124 These
conditions are needed because phosphorescence lifetimes
are typically long—milliseconds to seconds—and thus
quenched by low concentrations of oxygen or impurities.
Low temperatures are also needed to decrease the rates of
non-radiative decay to be comparable with the phosphorescence
emission rates. Otherwise, the quantum yield of
phosphorescence will be very small.
Figure 17.42. Lifetime distributions for tryptophan octyl ester (TOE)
in dodecylmaltoside (DM) micelles, 20°C. Revised from [121].
Figure 17.43 shows the fluorescence and phosphorescence
of tryptophan in a glass at low temperatures. 125 Phosphorescence
occurs at longer wavelengths than fluorescence,
and phosphorescence spectra are typically more
structured. The phosphorescence is shown as a separate
spectrum from the fluorescence. A separate phosphorescence
spectrum is only observed if detection of the phosphorescence
is gated to remove the fast decay fluorescence.
More typical spectra are shown in Figure 17.44 for NATA
in a glass-forming solvent at various temperatures. 126 At
low temperature (100 to 190EK) the phosphorescence
appears on the long-wavelength side of the fluorescence
spectrum. Without gated detection both emissions are
observed. The wavelength resolution of the phosphorescence
is lower in Figure 17.44 than in Figure 17.43. The