Biomedical Engineering Reference
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from the entrance of gA, which resulted in a gA molecule with an electrically
neutral entrance and a decreased single-channel conductance. Consequently, this
approach made it possible to distinguish the formation of pores due to the substrate,
gA-phosphate, from those due to the uncharged gA (product). This ability enabled
the authors to monitor the fraction of events due to the enzymatic substrate and
product over time, and consequently, calculate
K
m
and
k
cat
(Table
9.2
).
For the detection of anthrax lethal factor, Macrae et al. linked nine amino acid
residues covalently to gA peptides. Anthrax lethal factor is a protease produced
by the bioterrorism agent Anthrax, and it cleaves a specific amino acid sequence
(Fig.
9.10
). The activity of LF removed eight of these residues and resulted in a
significant increase in the formation of gA pores and the subsequent transport of
charge through the membrane (Fig.
9.10
). Thus, this signal could be used to detect
and quantify the activity of anthrax lethal factor.
This technique exploited the amplification characteristics of ion channels in that
the cleavage of
one
phosphate group on a
single
gA molecule reduced the flux of
ions through the pore by
thousands
of ions per second. This method also used only
picomolar concentrations of enzyme in volumes that resulted in the use of femto-
moles of protein.
All three of these nanopore-based, enzymatic assays open the door for generating
miniature platforms to monitor rapidly the activity of enzymes
in situ
and with native
substrates.
Fig. 9.10 Detecting enzyme activity with gA peptides that are modified covalently with substrate.
Anthrax lethal factor (LF) cleaves nine amino acids from the entrance of a modified gA peptide.
LF activity resulted in the formation of gA pores and a significant increase in the transport of
charge across the membrane over time. Adopted from Macrae et al
.
with permission [
27
]
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