Principles of Fluorescence Spectroscopy

402 TIME-DEPENDENT ANISOTROPY DECAYS
Figure 11.24. Stern-Volmer plot for the quenching of t-COPA by the
spin probes 5NS and 16NS in unilamellar vesicles of DMPC at 17°C
(gel phase) and 30°C (fluid phase). The concentration refers to the
quencher concentration in the membrane phase. Revised and reprinted
from [85], from the Biophysical Society.
in the intensity decay has a lifetime of about 20 ns. This
means that the anisotropy decay of t-COPA can be measured
to longer times than DPH, which has a typically lifetime
in membranes near 9 ns. The time-zero anisotropy is
near 0.385, making it a useful anisotropy probe. The
anisotropy decays of t-COPA are multi-exponential in solvents
and in lipid bilayers. In solvents the anisotropy decays
to zero (not shown). In membranes t-COPA behaves like a
highly hindered rotor. Below the phase transition the
Figure 11.25. Time-resolved fluorescence anisotropy of t-COPA in
unilamellar vesicles of DMPC in the gel (16°C) and fluid (31°C) phases.
Revised and reprinted from [85], from the Biophysical Society.
anisotropy of t-COPA displays a high r 4 value near 0.31.
Above the phase transition the value of r 4 decreases to 0.07
(Figure 11.25). In total, this polyene probe behaves similarly
to DPH in model membranes.
11.8. ANISOTROPY DECAYS OF NUCLEIC ACIDS
Studies of DNA by fluorescence can be traced to the use of
dyes to stain chromatin for fluorescence microscopy. The
use of time-resolved fluorescence for DNA dynamics originated
with the measurement of anisotropy decays of ethidium
bromide (EB) bound to DNA. 86–89 These early studies
showed the anisotropy at long times did not decay to zero
(Figure 11.26), which is similar to that found for DPH in
membranes. Initially the results were interpreted in terms of
the angle through which the EB could rotate within the
DNA helix. The anisotropy values in Figure 11.26 are lower
than expected for ethidium bromide. This is because the
experiments were performed with natural or unpolarized
light for the excitation. When the excitation source is unpolarized,
the emission is still polarized but the anisotropy
values are half those observed with polarized excitation.
Since these early studies there has been theoretical
progress in the use of fluorescence to study DNA dynam-
Figure 11.26. Anisotropy decay of ethidium bromide (EB) bound to
calf thymus DNA. The excitation was with polarized or natural light.
Revised from [86].