Chemistry Reference
In-Depth Information
Figure 5.2 Kinetic analysis of Rh-EGF binding.
(A) A typical change in the fluorescence
intensity of Rh-EGF at the binding site is
shown (left). At time 0, 0.5 nM Rh-EGF was
added to the medium. A sudden step-like
increase in the fluorescence intensity indicates
binding of a single Rh-EGF molecule. A single
exponential function (a red line; right) was
fitted to the histogram showing the duration
of binding (
102. (B) A typical change in the fluorescence
intensity at the site of the formation of an
EGFR dimer is shown (left). Each step
represents the first and second binding event
of Rh-EGF to a dimeric binding site. Histograms
of the duration (
t
2
) are shown (right). The
total number of events was 147
163. A
function of tandem reaction (red, green and
blue lines) was fitted to the histograms. AU,
arbitrary unit.
-
t
1
). The total number of events was
quanti
cation of the total number of EGFmolecules bound on the cell surface as well
as dividing the total number of molecules into monomeric or dimeric groups.
Unitary binding processes can be observed directly in single-molecule experi-
ments (Figure 5.2). The sudden appearance of a
fluorescent spot on the cell surface
indicates that Rh-EGF fromthe solution has bound to a vacant EGFRmolecule.When
the same binding site contains two receptors, a second ligand from the solution will
bind at a certain time after the
first to produce a signaling dimer. Kinetic information
concerning EGF/EGFR association can be derived from the lengths of time required
for the
first binding to take place (
t
1) and between the binding of the
rst and second
ligand (
2) to the EGFRmolecule. The time required for the
first binding to take place
can be described by a single exponential function which provides an association rate
constant of 4.0
t
10
8
M
1
s
1
. On the other hand, the waiting time before the second
binding can take place showed a peak suggesting that there is a kinetic intermediate.
Conducting similar observations using different EGF concentrations, it was con-
cluded that the
rst process was achieved by some type of structural changewith a rate
