Principles of Fluorescence Spectroscopy

416 ADVANCED ANISOTROPY CONCEPTS
Figure 12.5. Anisotropy decay of rhodamine B in water and with
0.04, 0.08, and 0.27% Ludox (from bottom to top). For the highest silica
concentration α 1 (H 2 O) = 0.70 and α 2 (silica) = 0.30 (eq. 12.8).
Revised from [18].
time, which also displayed restricted motion. Associated
anisotropy decays have also been observed for labeled
oligonucleotides when bound to proteins 14–15 and for rhodamine
and proteins in lipid–protein systems. 16–17
A dramatic example of an associated anisotropy decay
is shown in Figure 12.5. These anisotropy decays were
observed for rhodamine B (RhB) in the presence of Ludox,
which is colloidal silica. In the absence of silica the RhB
anisotropy decays rapidly to zero. In the presence of
increasing amounts of silica (bottom to top) the anisotropy
initially decreases and then increases to nearly the timezero
value. 18 The initial rate of anisotropy decay is similar
to that observed without silica. This suggests that in all
samples, with and without silica, the anisotropy for times
less than 1 ns is due to RhB that is not bound to silica. It
seems logical to conclude that the anisotropy at long times
is due to RhB bound to the silica particles. Since the longtime
anisotropy is close to the time-zero anisotropy, the correlation
time of RhB bound to silica must be long.
Associated anisotropy decays cannot always be distinguished
from non-associated decays. 1–3 For instance, if the
lifetimes of RhB were the same when in water and when
bound to silica, the observed anisotropy decay would be a
multi-exponential but the anisotropy would not increase at
Figure 12.6. Klenow fragment of E. coli DNA polymerase with a bound DNA oligomer. The dansyl-labeled base (D) is located at the 10th residue
from the 5' end of the 17-mer. Revised from [19].