Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 435
Figure 12.35. Absorption spectra of perylene along the long (z) and
short (y) axes. Revised from [83].
tron density. Absorption spectra in stretched films have
been used to determine the direction of transition moments
in a number of fluorophores of biochemical interest, including
adenine, 87 2-aminopurine, 88 Yt-base 89 , DAPI, 90 Hoechst
33342, 85 and polynuclease aromatic hydrocarbons. 91
12.9.1. Anisotropy of Planar Fluorophores with
High Symmetry
The fundamental anisotropy can be determined in part by
the symmetry of the molecule. This is illustrated by triphenylene,
which has threefold symmetry and a fundamental
Figure 12.36. Polarized absorption spectra of 9-aminoacridinium in a
stretched PVA film. Revised from [86].
Figure 12.37. Structure and symmetry axes of triphenylene.
anisotropy of r 0 = 0.1 (Figure 10.37). 92 This anisotropy
value is found whenever the transition moments are randomized
in a plane. The anisotropy value of 0.1 can be calculated
from the additivity law of anisotropy (eq. 10.6). Following
excitation along one of the axes the emission is randomized
among the three identical axes. Since the axes are
identical, a third of the emission originates from each transition,
resulting in r 0 = 0.10 (Problem 12.3).
12.10. LIFETIME-RESOLVED ANISOTROPIES
In Sections 10.5 and 10.6 we saw how the Perrin equation
could be used to estimate the apparent volume of macromolecule.
Examination of eq. 10.45 suggests an alternative
method of estimating the rotational correlation time. Suppose
the lifetime (τ) of the probe could be decreased by collisional
quenching. Then a plot of 1/r versus τ would have
a slope of (r 0 /θ) –1 , and thus allow measurement of the correlation
time. These measurements are called lifetimeresolved
anisotropies. 93–98 The first suggestion of lifetimeresolved
anisotropies appeared early in the literature as a
means to study polymers 98–99 as well as proteins. 100
There are advantages to the use of lifetime-resolved
measurements. The lifetime can typically be decreased with
only a modest change in solution conditions. This is particularly
true for oxygen quenching since oxygen diffuses rapidly
and is an efficient quencher. The use of oxygen quenching
is demonstrated in Figure 12.38, which shows anisotropy
values of a peptide hormone when the single-tryptophan
residue was quenched to various lifetimes. 101 The proteins
were human luteinizing hormone (hLH) and its β subunit
(βhLH). The intact hormone has a molecular weight of
28 kD, and the β subunit is 14 kD. The apparent correlation
times were 6.0 and 4.9 ns, respectively. The calculated correlation
times are 10.1 and 5.2 ns, respectively. The meas-