Biomedical Engineering Reference
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Fig. 9.9 Gramicidin pores can report the activity of membrane-active enzymes that alter the
surface charge of membranes. Transient formation of ion pores by gramicidin dimers resulted in
the conductance of monovalent cations through the pore. Activity of the enzyme phospholipase D
resulted in the conversion of zwitterionic lipids to negatively charged lipids. The subsequent
accumulation of cations near the negatively charged membrane was detected by an increase in the
single-channel conductance through gA pores. Adopted from Majd et al
.
with permission [
28
]
Table 9.2 Quantification of enzyme activity using nanopore recordings
Enzyme
Substrate
Pore
Parameter measured
a
HL
K
m
¼
59.2
10
6
M
k
cat
¼
4.43 s
1
Phospholipase D
a
Phosphotidylcholine Gramicidin Pseudo-
k
f
¼
1.3
10
4
s
1
Alkaline phosphatase gA-Phosphate
b
Gramicidin
K
m
¼
3.0
0.4
10
7
M
k
cat
¼
3.1
0.2
10
3
s
1
a
Due to the heterogeneous nature of catalysis on a lipid bilayer surface, the
K
m
and
k
cat
constants
could not be determined. The method provided a pseudo-first order rate constant,
k
f
, with the rate
of formation of product, d
P
/d
t ¼ k
f
[E] where [E] is the concentration of enzyme. Additional
constants specific to interfacial enzymes were estimated; see ref. [
28
] for more details
b
Trypsin
A
b
10-20
Gramicidamine derivative carrying a glycolic acid-
O
-phosphate [
3
]
conductance through the pores [
3
,
27
]. Macrae et al
.
employed this concept to
detect the activity of alkaline phosphatase and a protease, anthrax lethal factor. For
the detection of alkaline phosphatase, the authors modified the ion channel forming-
peptide to contain a phosphate group at the entrance of the pore, gA-phosphate. At
low ionic strength, the negatively charged phosphate resulted in increased conduc-
tance of monovalent cations through the gA-phosphate pore compared to a gA pore
with an electrically neutral entrance. Alkaline phosphatase removed the phosphate
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