Chemistry Reference
In-Depth Information
binding curve to display the Scatchard representation of the mass action law
(Figure 7.1E, inset) according to:
P
y
i
;
X
K
i
n
i
y
i
L
0
R
0
P
y
i
R
0
¼
ð
7
:
4
Þ
ð
1
Þ
where R
0
is the total receptor concentration at the cell surface, Ki
i
is the association
constant of the ligand
-
receptor complex, and ni
i
is the number of binding sites per
receptor. Assuming ni¼
i
1, R
0
is equivalent to B
max
.
The speci
city and kinetics of ligand
-
receptor binding can be analyzed by
competitive displacement of the bound ligand by an excess of non-labeled ligand.
A simple monoexponential dissociation model is given by:
X
y
i
¼
¼
y
unspecific
þ
y
0
exp
ð
k
diss
t
Þ;
ð
7
:
5
Þ
where y
unspeci
c
is the fraction of non-speci
c binding, y
0
is the fraction of speci
c
binding, and k
diss
is the dissociation rate constant. The association rate constant of the
complex, k
ass
, is calculated by multiplying Ki
i
by k
diss
. More complex binding
reactions, such as those involving several binding sites of different af
nities as
observed in ligand-binding isotherms and Scatchard plots, or multi-exponential
displacement curves, can also be measured by FCS (Table 7.1).
In the particular case of large ligand molecules (i.e. with a similar mass as the
receptor), binding to membrane receptors may not produce a suf
cient effect on
the diffusion time monitored by FCS. In this case
fluorescence cross-correlation
spectroscopy (FCCS) is themethod of choice. In dual-color FCCS, the two interacting
partners are labeled with spectrally separated
fluorophores. Cross-correlation analy-
sis of the two signals allows us to assess binding properties independently of the
relative diffusion times [63].
7.4.2
FCS at High Fluorophore Concentrations
In a classical FCS experiment, the diffraction-limited observation volume restricts
fluorophore concentrations to the pico and nanomolar ranges. Two different strate-
gies have recently been presented to reduce the effective observation volume, thus
enabling single-molecule detection at micromolar concentrations:
(i) The excitation light was con
ned at the bottom of subwavelength apertures that
act as zero-mode waveguides made of a metal
film on a fused silica
coverslide [67]. The technique has been successfully applied to probe the
dynamics of lipids and proteins on arti
cial and cell membranes [68
-
70].
(ii) The effective detection volume was reduced by allowing the
uorescent
molecules to
flow in submicrometer-sized
fluidic channels [71].
With these methods, ligand
-
receptor interactions with micromolar equilibrium
binding constants can now be analyzed with high spatial and temporal resolutions.
