Principles of Fluorescence Spectroscopy

896 ANSWERS TO PROBLEMS
Energy transfer can also be used to detect protein
binding. Neither DNA nor model membranes possess
chromophores that can serve as acceptors for
the tryptophan fluorescence. Hence it is necessary
to add extrinsic probes. Suitable acceptors would
be probes that absorb near 350 nm, the emission
maximum of most proteins. Numerous membranes
and nucleic-acid probes absorb near 350 nm. The
membranes could be labeled with DPH (Figure
1.18), which absorbs near 350 nm. If the protein is
bound to DPH-labeled membranes, its emission
would be quenched by resonance energy transfer to
DPH. Similarly, DNA could be labeled with DAPI
(Figure 3.23). An advantage of using RET is that
through-space quenching occurs irrespective of the
details of the binding interactions. Even if there
were no change in the intrinsic tryptophan emission
upon binding to lipids or nucleic acids, one still
expects quenching of the tryptophan when bound to
acceptor-labeled membranes or nucleic acids.
A3.2. A. The data in Figure 3.48 can be used to determine
the value of F 0/F at each [Cl – ], where F 0 = 1.0 is
the SPQ fluorescence intensity in the absence of
Cl – , and F is the intensity at each Cl – concentration.
These values are plotted in Figure 3.49.
Using Stern-Volmer eq. 1.6, one obtains K = 124
M –1 , which is in good agreement with the literature
value 182 of 118 M –1 .
B. The value of F 0 /F and τ 0 /τ for 0.103 M Cl –
can be found from eq. 1.6. Using K = 118 M –1 ,
one obtains F 0 /F = τ 0 /τ = 13.15. Hence the
intensity of SPQ is F = 0.076, relative to the
intensity in the absence of Cl – ,F 0 = 1.0. The
Figure 3.49. Stern-Volmer plot for the quenching of SPQ by chloride.
lifetime is expected to be τ = 26.3/13.15 = 2.0
ns.
C. At [Cl – ] = 0.075 M, F = 0.102 and τ = 2.67.
D. The Stern-Volmer quenching constant of SPQ
was determined in the absence of macromolecules.
It is possible that SPQ binds to proteins
or membranes in blood serum. This could
change the Stern-Volmer quenching constant
by protecting SPQ from collisional quenching.
Also, binding to macromolecules could
alter τ 0 , the unquenched lifetime. Hence it is
necessary to determine whether the quenching
constant of SPQ is the same in blood serum as
in protein-free solutions.
A3.3. A. The dissociation reaction of the probe (P B )
and analyte (A) is given by
P B = A + P B , (3.3)
where B and F refer to the free and bound forms of the
probe. The fraction of free and bound probe is related
to the dissociation constant by
K D P F
P B A
(3.4)
For the non-ratiometric probe Calcium Green, the fluorescein
intensity is given by
F = q F C F + q B C B , (3.5)
where q i are the relative quantum yields, C i the molecular
fraction in each form, and C F + C B = 1.0. The fluorescent
intensities when all the probe is free is F min =
kq FC, and then all the probe is bound in F max = kq BC,
where k is an instrumental constant.
Equation 3.5 can be used to derive expressions
for C B and C F in terms of the relative intensities:
F B F F min
F max F min
F F F max F
F max F min
(3.6)
(3.7)
The fractions C B and C F can be substituted for the
probe concentration in (3.4), yielding