Chemistry Reference
In-Depth Information
compounds, usually tritium or iodine-125 isotopes. After incubating receptor-
comprising membrane preparations or whole cells with radioligands, the unbound
fraction of the labeled ligand is separated from the receptor-bound fraction. Major
drawbacks of these binding assays are their low sensitivity, which excludes the
investigation of cell surface receptors at low expression, and requires the require-
ment of long-living receptor
-
ligand complexes that should be stable during the
separation and the washing steps.
FCS is not only a powerful technique for measuring the lateral mobility of
membrane proteins, it is also a sensitive and non-invasive tool to determine the
thermodynamic and kinetic parameters of ligand
receptor interactions in living
cells with no need for the physical separation of the receptor-bound from the free
ligand fraction (Table 7.1). Moreover, the small detection volume allows measure-
ments in sub-cellular compartments such as the nucleus [64], the endoplasmic
reticulum or the Golgi [65].
Quantitative analysis of the diffusion time and fraction of the different species is
given by the following simpli
ed autocorrelation function G(
-
), which describes the
three-dimensional diffusion of free ligands in solution and the two-dimensional
diffusion of bound ligands in different populations of cell membrane receptors
having different mobilities:
t
2
4
0
@
1
A
3
5
;
!
1
1
1
=
2
!
X
y
i
X
y
i
1
N
1
1
G
ðtÞ¼
1
þ
1
þ
w
z
1
þ
t
f
2
1
þ
t
bi
w
xy
t
t
þ
f
ð
7
:
3
Þ
where w
z
is the vertical half axis of the detection volume,
t
bi
are the
diffusion time constants of the free ligands and ligands bound to the population i
of membrane receptors, respectively. It is assumed in the multicomponent
diffusion model of Equation 7.3 that the quantum yield of the
fluorescently-labeled
ligand is the same in the free and bound states. Since the diffusion time scales with
the cubic root of the molecular weight (Stokes
-
Einstein relation), the diffusion time
of free ligands is substantially lower than the diffusion time of ligands bound to
comparatively large receptors. This has been observed in binding experiments
performed on detergent-solubilized membrane receptors [66]. The two components
are more easily discernable for receptors diffusing in cell membranes due to the
higher viscosity of the lipid bilayer (Figure 7.1C). Typically, the receptor-bound ligand
diffuses more slowly, by about two orders of magnitude, than the free ligand in
solution. The fraction of different species can be further examined by evaluating the
distribution of diffusion times (Figure 7.1D and [137]).
The ligand-binding isotherm is obtained by determining at equilibrium the
fraction of bound ligand from the autocorrelation amplitude for different ligand
concentrations (Figure 7.1E). At saturation, the maximum concentration of bound
ligand B
max
equals the total number of binding sites. Assuming one binding site for
one receptor molecule, the receptor density in the cell membrane is calculated from
the average observed membrane area. It is useful to linearize the equation for the
t
f
and
