Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 243
of the increased dipole moment of the excited state. If the
biomolecule is rigid, there will be no relaxation, as was
seen for 4-AP in propanol at –132EC (Figure 7.7). If the
biomolecule is flexible, then the TRES should relax on a
timescale characteristic of the macromolecule. Timedependent
spectral shifts have been observed for probes
bound to proteins, 11–21 membranes, 22–33 micelles, 34–39 and
polymers, 40–41 and has recently been reviewed. 42 Spectral
relaxation of intrinsic tryptophan fluorescence in proteins
will be described in Chapter 17.
7.3.1. Spectral Relaxation of
Labeled Apomyoglobin
One example of TRES of a protein is provided by labeled
myoglobin. Myoglobin is a muscle protein that binds oxygen
from the blood and releases oxygen as needed to the
muscles. Myoglobin thus acts as an oxygen reservoir. In
myoglobin the oxygen molecule is bound to the heme
group, which is near the center of the protein. The heme
group can be removed from the protein, leaving a
hydrophobic pocket that is known to bind a number of fluorophores.
43–44 Myoglobin without the heme group is called
apomyoglobin. The dynamics of myoglobin are of interest
because myoglobin cannot bind and release oxygen without
undergoing structural fluctuations to allow diffusion of oxygen
through the protein. If the protein is flexible on the
nanosecond timescale for oxygen penetration, then it seems
likely the protein can be flexible during the nanosecond
decay times of bound fluorophores.
In the previous chapter we described Prodan and its
derivatives as being highly sensitive to solvent polarity. The
dynamics of the heme binding site was studied using the
probe Danca, which is an analogue of Prodan (Figure 7.9).
The carboxy cyclohexyl side chain serves to increase the
affinity of Danca for apomyoglobin, and to ensure it binds
to the protein in a single orientation. A single mode of binding
simplifies interpretation of the data by providing a
homogeneous probe population. Excitation of Danca results
in the instantaneous creation of a new dipole within the
apomyoglobin molecule. If myoglobin is flexible on the ns
timescale, one expects time-dependent shifts in its emission
spectrum as the protein rearranges around the new dipole
moment.
Intensity decays were measured at various wavelengths
across the emission spectrum (Figure 7.9). The amplitudes
of these decays were adjusted according to eq. 7.3. The
decays are somewhat faster at shorter emission wave-
Figure 7.9. Deconvolved fluorescence decays of Danca-apomyoglobin
complex from 400 to 528 nm at 298°K. The area under each trace
has been scaled to the steady-state intensity at that wavelength. Note
the intensity decays show a rise time at longer wavelengths. Revised
and reprinted with permission from [17]. Copyright © 1992,
American Chemical Society.
lengths. Importantly, there is evidence of a rise time at
longer wavelengths, which is characteristic of an excitedstate
process. A rise time can only be observed if the emission
is not directly excited, but rather forms from a previously
excited state. In this case the initially excited state
does not contribute at 496 and 528 nm, so that the decays at
these wavelengths show an initial rise in intensity. The
emission at these wavelengths is due to relaxation of the initially
excited state.
The time-dependent decays were used to construct the
time-resolved emission spectra (Figure 7.10). These spectra
shift progressively to longer wavelength at longer times.
Even at the earliest times (20 ps) the TRES are well shifted
from the steady-state spectrum observed at 77EK. At this
low temperature solvent relaxation does not occur. As was
described in Chapter 6 (Figure 6.28), Prodan-like molecules
can emit from locally excited (LE) and internal chargetransfer
(ICT) states. The short-wavelength emission at
77EK is probably due to the LE state. Hence, the emission
of DANCA-apomyoglobin is from the ICT state, which has
undergone nearly complete charge separation.
The TRES can be used to calculate the rates of spectral
relaxation. These data are usually presented as the average
energy of the emission versus time (Figure 7.11). Alternatively,
one can calculate the time-dependent change in the
emission maximum. In the case of Danca-apomyoglobin