Principles of Fluorescence Spectroscopy

248 DYNAMICS OF SOLVENT AND SPECTRAL RELAXATION
Figure 7.18. Time-resolved emission maxima and spectral half widths
for TNS-labeled egg lecithin. Redrawn from [48].
Examination of the time-dependent value of ∆ν(t) can
reveal the nature of the relaxation process. For Patman in
DPPC vesicles at 46EC there is an increase in half width at
intermediate times. This suggests that spectral relaxation
around Patman is best described as a two-state process.
Such behavior is not seen for all probes. For TNS-labeled
vesicles 26,48 the half-width remains constant during spectral
relaxation (Figure 7.18). This suggests that relaxation
around TNS is best described by a continuous process in
which an emission spectrum of constant shape slides to
longer wavelengths.
7.4.2. Spectral Relaxation of Membrane-Bound
Anthroyloxy Fatty Acids
Another example of spectral relaxation in membranes is the
anthroyloxy fatty acids, which have been extensively used
as membrane probes. One advantage of these probes is that
the fluorophore can be localized at the desired depth in the
membrane by its point of attachment to the fatty acid. The
localized anthroyloxy groups have been used to study the
dynamics of spectral relaxation at various depths in membranes.
The time-resolved emission spectra of these probes
are sensitive to the location of the probe in these membranes.
29 This is seen by the larger time-dependent shifts for
2-AS than for 16-AP (Figure 7.19). At first glance this difference
seems easy to interpret, with larger spectral shifts
for the probe located closer to the polar membrane-water
Figure 7.19. Time-resolved emission spectra of anthroyloxy fatty
acids (2-AS and 16-AP) in egg phosphatidylcholine vesicles. Revised
from [29].
interface. However, closer inspection reveals that the emission
spectra of 16-AP are more shifted to the red at early
times following excitation. This is evident from the timedependent
emission maxima (Figure 7.20). It seems that the
probes located deeper in the bilayer should display a more
blue-shifted emission. The reason for the larger red shift of
16-AP is not understood at this time. However, it is thought
that the anthroyloxy probes near the ends of acyl chains can
fold back to the lipid–water interface. The data in Figure
7.20 are consistent with the fluorophores in both 12-AS and
16-AP being localized near the lipid–water interface.
The rates of spectral relaxation for the anthroyloxy
probes also seem to be unusual. The fluidity of lipid bilayers
is thought to increase towards the center of the bilayer.
Examination of the time-dependent emission maxima (Figure
7.20) suggests that the relaxation becomes slower for
probes localized deeper in the membranes. This is seen
more clearly in the correlation functions (eq. 7.12, below),
which display the relaxation behavior on a normalized scale
(Figure 7.21). The apparent relaxation times increase from
2.7 to 5.9 ns as the anthroyloxy moiety is attached more distant
from the carboxy group. These results can be understood
by the membrane becoming more fluid near the cen-