Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 895
A1.8. Figure 1.40 shows that the donor intensity increases
and the acceptor intensity decreases upon addition
of cAMP and PKI. These spectral changes
indicate a decrease in RET. The donor and acceptor
must move further apart when the protein binds
cAMP or PKI. According to publications 43 the C
and R subunits remain associated in the presence of
cAMP, but change this relative conformation (Figure
1.43). PKI was said to dissociate the subunit.
Figure 1.43. Effect of cAMP and protein kinase inhibitor (PKI) on the
structure and association of cAMP-dependent protein kinase. The
holoenzyme consists of two catalytic and two regulatory subunits.
CHAPTER 2
A2.1. The true optical density is 10. Because of stray light
the lowest percent transmission (%T) you can measure
is 0.01%. The %T of the rhodamine solution is much
less than 0.01%. In fact I/I 0 = 10 –10 and %T = 10 –8 %.
Hence your instrument will report an optical density
of 4.0. The calculated concentration of rhodamine B
would be 4 x 10 –5 M, 2.5-fold less than the true concentration.
A2.2. The concentrations of solutions are 10 –5 and 10 –7
M, respectively. A 1% error in %T means the OD
can be
OD log I 0
I
(2.13)
Hence the concentration can be from 0.97 x 10 –5 to
1.03 x 10 –5 M.
For the more dilute solution, the 1% error results
in a large error in the concentration:
I0 OD log
I
1
log
0.51
1.00
log
1.00
or log 1
0.49
or log 1.0
0.98
(2.14)
CHAPTER 3
The measured OD ranges from 0 to 0.009, so the calculated
concentration ranges from 0 to 2.9 x 10 –7 M.
This shows that it is difficult to determine the concentration
from low optical densities. In contrast, it is
easy to obtain emission spectra with optical densities
near 0.003.
A3.1. Binding of the protein to membranes or nucleic acids
could be detected by several types of measurements.
The most obvious experiment would be to look for
changes in the intrinsic tryptophan fluorescence upon
mixing with lipid bilayers or nucleic acids. In the case
of membranes one might expect the tryptophan emission
to shift to shorter wavelengths due to shielding of
the indole moiety from water. The blue shift of the
emission is also likely to be accompanied by an
increase in the tryptophan emission intensity. In the
case of nucleic acids, tryptophan residues are typically
quenched when bound to DNA, so that a decrease
in the emission intensity is expected.
Anisotropy measurements of the tryptophan
emission could also be used to detect binding. In
this case it is difficult to predict the direction of the
changes. In general one expects binding to result in
a longer correlation time and higher anisotropy (see
eq. 1.10), and an increase of the tryptophan
anisotropy is likely upon binding to proteins. However,
the anisotropy increase may be smaller than
expected if the tryptophan lifetime increases on
binding to the membranes (eq. 1.10).
In the case of protein binding to nucleic acids, it
is difficult to predict the anisotropy change. The
tryptophan residues would now be in two states,
free (F) and bound (B), and the anisotropy given by
r = r F f F + r B f B , (3.2)
where f F and f B represent the fraction of the total fluorescence
from the protein in each state. If the protein
is completely quenched on binding to DNA, then the
anisotropy will not change because f B = 0. If the protein
is partially quenched the anisotropy will probably
increase, but less than expected due to the small contribution
of the DNA-bound protein by the total fluorescence.