Biomedical Engineering Reference
In-Depth Information
Fig. 9.5 Detecting the binding of a protein to a ligand that was attached to the entrance of a
a
-hemolysin pore by a polymer chain. Translocation of the polymer resulted in rapid, transient
current decreases. Binding of the protein to the end of the polymer chain resulted in a steady
current value as long as the protein remained bound, yielding a time to dissociation of
t
off
. The
interval between binding events,
t
on
, as a function of the protein concentration was used to
calculate
k
on
with (
9.8
). Adopted from Movileanu et al
.
with permission [
33
]
Conversely, the average time interval of rapid current fluctuations that occured
between the steady-state current values,
t
on
(s), was related to
k
on
and the concen-
tration of free protein, [P], by [
33
]:
1
t
on
ΒΌ
[P]
k
on
:
(9.8)
Howorka et al
.
expanded on this method by covalently linking two ligand-
modified polymers within the pore in order to distinguish between monovalent
and bivalent binding of a carbohydrate binding protein, lectin, to the disaccharide
Gal-
-1,3-GalNAc [
21
]. Upon addition of lectin, the stochastic response of this
assay permitted the determination of two distinct values for
k
off
. The two values for
k
off
resulted from monovalent or bivalent binding of lectin to the disaccharide and
permitted the authors to calculate two values of
K
d
b
(Table
9.1
). Although only
demonstrated in genetically engineered
-hemolysin pores, this concept could be
applied to solid-state nanopores if a
single
polymer could be immobilized in the
pore. Unlike methods that measure protein-ligand complexes under equilibrium
conditions, this method has the unique ability to determine values of
K
d
a
for
monovalent and bivalent binding.
Robertson's group demonstrated another approach to calculate kinetic
parameters. In this method ligand-modified polymers were dissolved freely in the
bulk solution [
24
]. Again, in the absence of protein, the translocation of polymers
through the pore resulted in rapid current blockages. The frequency of these
transient current blockages was related to the concentration of unbound, ligand-
modified polymer. Depending on the length of the polymer, binding of a protein to
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