Biomedical Engineering Reference
In-Depth Information
9.5 Quantifying Enzyme Activity with Nanopores
Monitoring the activity of enzymes with nanopores is a very recent development in
the field of nanopore sensing. Using
-hemolysin, Cockroft et al
.
detected the
addition of single nucleotides to a DNA template due to the activity of DNA
polymerase [
9
], and Clark et al
.
detected single nucleotides as a result of exonucle-
ase activity [
8
]. In this section we will focus on three recent publications that
explored strategies of using nanopores to extract
quantitative
information about
the activity of enzymes.
In this context, Guan's group described a method that detected protease
activity by measuring the translocation of the resulting peptide products though
a nanopore [
55
] (see Chapter 13 also). These authors used a mutant version
of
a
-hemolysin, called (M113F)
7
, to detect the translocation of a fragment of
amyloid-
a
) peptides (residues 10-20) and to report the activity of trypsin,
an enzyme that cleaves peptide bonds after arginine or lysine. Upon addition of
trypsin, the authors observed translocation of the enzymatic products and the
original A
b
(A
b
fragment. These three molecules could be distinguished based on
their different molecular volumes, which resulted in different magnitudes of
b
DI
(Fig.
9.8
). From the fraction of events due to each molecule, the authors could
monitor the formation of enzymatic products as a function of time. The authors
then calculated the Michaelis constant,
K
m
, and the catalytic rate constant,
k
cat
,for
the reaction between trypsin and A
(10-20) (Table
9.2
).
5
This method was hence
b
used to study protease kinetics.
Majd et al. described a second assay for enzyme activity in which the conduc-
tance through gramicidin, gA, pores that are embedded in a lipid bilayer reported
the activity of membrane-active enzymes [
28
].
6
In this method, the surface charge
of the lipid membrane affected the conductance through gA pores [
4
], and the
activity of the enzymes phospholipase D (PLD) and phospholipase C (PLC) altered
the net surface charge of the membrane. For example, the substrate for PLD is the
electrically neutral lipid phosphotidylcholine (PC), while the enzymatic products
are choline and a negatively charged lipid, phosphatidic acid (PA). Figure
9.9
illustrates this reaction. Conversely, the substrate for phospholipase C is the nega-
tively charged lipid phosphotidylinositol, and the enzymatic products are inositol-
phosphate and a neutral lipid, diacyl glycerol. Thus, the enzymatic activity of these
phospholipases changed the surface charge of the membrane, and in an electrolyte
5
The catalytic rate constant
k
cat
describes the rate at which the enzyme-substrate complex is
converted to the free enzyme and free product. The Michaelis constant,
K
m
, is the concentration of
substrate that results in the half-maximal velocity of the enzymatic reaction.
6
Gramicidin is a peptide consisting of 15 amino acids that spans one leaflet of a bilayer. If gA
peptides are present in both leaflets of a bilayer, they can transiently form a dimer, which conducts
monovalent cations through a central pore with diameter of ~4
˚
. These transient ion channels result
in discrete current values that reflect the number of ions passing through individual gA pores in a
planar lipid bilayer at a given instant. Antonenko and coworkers first characterized the
K
d
for the
interaction of two monomers of gA that bind to form dimeric gA ion channels in a lipid bilayer [
37
].
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