Principles of Fluorescence Spectroscopy

374 FLUORESCENCE ANISOTROPY
Figure 10.23. Effect of E. coli. helicase on a fluorescein-labeled DNA
oligomer. Revised from [56].
Figure 10.23 shows anisotropy measurements of a fluorescein-labeled
oligomer in the presence of helicase. The
anisotropy increases immediately upon addition of helicase
to the DNA, showing that the binding reaction occurs rapidly.
The anisotropy then remains constant because there is
no source of energy to disrupt the hydrogen bonded base
pairs. Upon addition of ATP the anisotropy drops rapidly to
a value lower than the starting value. The final anisotropy is
lower because the labeled DNA strand is no longer bound
to the complementary strand. The single-stranded oligomer
has a lower molecular weight and is more flexible than the
double-stranded oligomer. In this experiment the DNA
strands did not reassociate during the experiment. This helicase
will not rebind to DNA shorter than a 20-mer. These
results show that anisotropy measurements can be used to
follow the kinetics of biochemical reactions on a rapid
timescale.
10.7.4. Melittin Association Detected from
Homotransfer
In the preceding examples the binding reaction was detected
from the increase in anisotropy resulting from the larger
correlation time. Homo-resonance energy transfer can also
be used to detect binding interactions. This is shown for
melittin, which self-associates at high salt concentrations
(Chapter 16). Melittin was randomly labeled with fluorescein
isothiocyanate. 57 When the solution contained a small
fraction of labeled melittin (1 of 25) the anisotropy
increased at high salt concentrations (Figure 10.24). When
all the melittin molecules were labeled, their anisotropy
decreased markedly at high salt concentration. This
decrease may be unexpected because melittin forms a
tetramer in high salt solution, so its correlation time should
increase fourfold. The decrease in anisotropy can be understood
from the spectral properties of fluorescein (Chapter
3). The small Stokes shift and large overlap results in a
Förster distance (R 0 ) of 53 Å for homotransfer. The
anisotropy decrease in Figure 10.24 is due to homotransfer
between fluoresceins in the melittin tetramer.
10.8. ANISOTROPY OF MEMBRANES AND
MEMBRANE-BOUND PROTEINS
10.8.1. Membrane Microviscosity
Fluorescence anisotropy measurements can be used to
study biological membranes. These studies have their origin
Figure 10.24. Self-association of melittin labeled with fluorescein in
a 1:24 mixture with unlabeled melittin (") or for all melittin labeled
with fluorescein (!). Revised and reprinted with permission from
[57]. Copyright © 1995, Biophysical Society.