Conjugated polyelectrolyte based real-time fluorescence assay for phospholipase C

Article abstract of DOI:10.1021/ac701672g

A fluorescence turnoff assay for phospholipase C (PLC) from Clostridium perfringens is developed based on the reversible interaction between the natural substrate, phosphatidylcholine, and a fluorescent, water-soluble conjugated polyelectrolyte (CPE). The fluorescence intensity of the CPE in water is increased substantially by the addition of the phospholipid due to the formation of a CPE-lipid complex. Incubation of the CPE-lipid complex with the enzyme PLC causes the fluorescence intensity to decrease (turnoff sensor); the response arises due to PLC-catalyzed hydrolysis of the phosphatidylcholine, which effectively disrupts the CPE-lipid complex. The PLC assay operates with phospholipid substrate concentrations in the micromolar range, and the analytical detection limit for PLC is <1 nM. The optimized assay provides a convenient, rapid, and real-time sensor for PLC activity. The real-time fluorescence intensity from the CPE can be converted to substrate concentration by using an ex situ calibration curve, allowing PLC-catalyzed reaction rates and kinetic parameters to be determined. PLC activation by Ca²⁺ and inhibition by EDTA and fluoride ion are demonstrated using the optimized sensor.

Full text of DOI:10.1021/ac701672g

Anal. Chem. 2008, 80, 150-158
Conjugated Polyelectrolyte Based Real-Time
Fluorescence Assay for Phospholipase C
Yan Liu, Katsu Ogawa, and Kirk S. Schanze*
Department of Chemistry, University of Florida, P.O. Box 117200, Gainesville, Florida 32611-7200
A fluorescence turnoff assay for phospholipase C (PLC)
from Clostridium perfringens is developed based on the
reversible interaction between the natural substrate,
phosphatidylcholine, and a fluorescent, water-soluble
conjugated polyelectrolyte (CPE). The fluorescence inten-
sity of the CPE in water is increased substantially by the
addition of the phospholipid due to the formation of a
CPE-lipid complex. Incubation of the CPE-lipid complex
with the enzyme PLC causes the fluorescence intensity
to decrease (turnoff sensor); the response arises due to
PLC-catalyzed hydrolysis of the phosphatidylcholine, which
effectively disrupts the CPE-lipid complex. The PLC
assay operates with phospholipid substrate concentra-
tions in the micromolar range, and the analytical detection
limit for PLC is <1 nM. The optimized assay provides a
convenient, rapid, and real-time sensor for PLC activity.
The real-time fluorescence intensity from the CPE can be
converted to substrate concentration by using an ex situ
calibration curve, allowing PLC-catalyzed reaction rates
and kinetic parameters to be determined. PLC activation
amplification that results from the superquenching behavior of
CPEs.1
,2,17-22
Such behavior is attributed to a combination of
several factors, including delocalization and rapid diffusion of the
singlet exciton along the CPE backbone to the quencher “trap
site”, as well as the electrostatic ion pairing of quenchers and with
oppositely charged CPE side chains.1
,17,19,23-25
It has also been demonstrated that water-soluble CPEs interact
with surfactants resulting in significant but reversible changes in
17,20,26,27
the photophysical properties
of the polymer. Studies
indicate that the CPE and surfactants form a polymer-surfactant
complex due to a combination of electrostatic interaction between
the polymer ionic side groups and the surfactant head groups, as
well as hydrophobic interactions between the surfactant tails and
the conjugated polymer backbone. Formation of the polymer-
surfactant complex induces the CPE chains to become more
extended, and more importantly, polymer-polymer interaction
17,20,26
(aggregation) is disrupted.
The changes in the microenvi-
ronment of the polymer in the polymer-surfactant complex result
(11) DiCesare, N.; Pinto, M. R.; Schanze, K. S.; Lakowicz, J. R. Langmuir 2002,
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2
+
by Ca and inhibition by EDTA and fluoride ion are
demonstrated using the optimized sensor.
(
12) Achyuthan, K. E.; Bergstedt, T. S.; Chen, L.; Jones, R. M.; Kumaraswamy,
S.; Kushon, S. A.; Ley, K. D.; Lu, L.; McBranch, D.; Mukundan, H.;
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Several recent studies have shown that water-soluble, fluores-
cent π-conjugated polyelectrolytes (CPEs) provide a useful plat-
form for the development of highly sensitive fluorescence-based
(
13) Kumaraswamy, S.; Bergstedt, T.; Shi, X. B.; Rininsland, F.; Kushon, S.; Xia,
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1
-4
1,5-7
4,8-10
sensors for biomolecules
such as proteins,
DNA,
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carbohydrates, and enzymes.12-16 The high sensitivity of CPE
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1
7510.
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assays takes advantage of the intrinsic fluorescence signal
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004, 101, 15295-15300.
*
To whom correspondence should be addressed. Tel: 352-392-9133. Fax:
52-392-2395. E-mail: kschanze@chem.ufl.edu. Web: http://chem.ufl.edu/
kschanze.
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8
150 Analytical Chemistry, Vol. 80, No. 1, January 1, 2008
10.1021/ac701672g CCC: $40.75 © 2008 American Chemical Society
Published on Web 11/29/2007

in a narrowing and blue-shift of the fluorescence, coupled with a
significant increase in the fluorescence quantum yield.17,20,26
The earlier studies of CPE-surfactant complexes provide the
basis for the study presented herein, where we explore the
interactions between an anionic CPE and phospholipids. Phos-
pholipids are naturally occurring amphiphiles that serve as the
which results in low catalytic turnover of enzymes and reduced
reaction rates.
46,47
We now introduce a sensitive and specific fluorescent turnoff
assay for PLC. Like other fluorescence assays, the method is
convenient, but it has the additional advantage of being based on
natural lipid substrates. The assay is based on the reversible
change in fluorescence properties of an anionic CPE induced by
the formation of a polymer-phospholipid complex. In particular,
the fluorescence of the CPE is enhanced and blue-shifted upon
complexation with phosphatidylcholine. Incubation of the polymer-
phospholipid complex with PLC results in a decrease of the
fluorescence, which is due to the enzyme-catalyzed hydrolysis of
the phospholipid. The PLC enzyme assay conditions are optimized,
28
major component of biological membranes. In the present study,
we demonstrate that phospholipids interact strongly with CPEs,
eliciting significant changes in the fluorescence properties of the
polymer. The effects are reversible, and consequently, the CPE-
lipid complex provides a platform for the development of a
fluorescence turnoff assay for the lipase enzyme phospholipase
2
9
C (PLC).
PLC catalyzes the hydrolysis of the phosphate ester in a
2+
and the effects of the addition of an activator (Ca ) and several
2
9
phospholipid selectively at the glycerol side, yielding a diacylg-
lycerol (DAG) and a phosphate-containing head group. The ability
to quantitatively monitor PLC catalytic activity and inhibition is
important as DAGs play a critical role in cell function and the
inhibitors of PLC are studied. The assay is calibrated for substrate
concentration, allowing the determination of the catalytic kinetic
parameters, K and Vmax.
m
3
0-33
signal transduction cascade in mammalian systems.
PLC assays have been developed based on turbidimetric,
stat titration, radiometric,
Several
MATERIALS AND METHODS
Materials. BpPPESO (an anionic PPE-type CPE, structure
3
4,35
pH-
3
6
37,38
39-48
3
and continuous fluorometric
shown in Figure 1a), was synthesized according to a literature
method.19 All substrates, enzymes, and proteins were purchased
from Sigma-Aldrich and used as received, unless otherwise noted.
Calcium chloride and ethylenediaminetetraacetic acid, disodium
salt dihydrate (EDTA) were purchased from Fisher Chemical.
Sodium fluoride was obtained from Mallinckrodt Chemical Works,
and 1,2-didecanoyl-sn-glycerol (DAG) was obtained from Cayman
Chemical. All solvents were obtained from Fisher and used
without further purification. Water was distilled and then purified
by using a Millipore purification system.
methods. However, the turbidimetric and titration assays suffer
from low sensitivity,34-36,43
and the inherent disadvantage that large
quantities of enzyme and substrate are required. Although the
radiometric assay attains the lowest detection limit for PLC that
3
49 14
50 32
51
125 38
has been reported, H-,
C-,
P-. or I- labeled phospho-
lipids are required as substrates making the approach expensive,
laborious, and time-consuming. Fluorometric assays based on the
4
6
45
determination of choline or inorganic phosphate offer high
sensitivity and are continuous. Although they are advantageous
4
3
when carrying out studies on enzyme kinetics, most of the
fluorometric assays require synthetic fluorogenic substrates,3
9-44,46-48
Solution Preparation. Buffer solutions (pH 7.4) were pre-
pared with Tris base and hydrochloric acid. A concentrated
(
(
28) Waite, M. The phospholipases; Elsevier: New York, 1987.
29) Takahashi, T.; Sugahara, T.; Ohsaka, A. Methods Enzymol. 1981, 71, 710-
3
aqueous solution of BpPPESO was diluted with buffer solution
to a final concentration ranging from 0.1 to 15 µM. The stock
solutions of substrates, enzymes, and proteins were prepared
immediately before their use in the fluorescence assay. The
enzyme substrate, 1,2-didecanoyl-sn-glycero-3-phosphocholine
(10CPC) was dissolved in methanol and adjusted to 20 mM as
stock concentration. Phospholipase C from Clostridium perfringens
7
25.
(
(
30) Exton, J. H. J. Biol. Chem. 1990, 265, 1-4.
31) Schrijen, J. J.; Omachi, A.; Vangroningenluyben, W. A. H. M.; Depont, J. J.
H. H. M.; Bonting, S. L. Biochim. Biophys. Acta 1981, 649, 1-12.
32) Exton, J. H. Eur. J. Biochem. 1997, 243, 10-20.
33) Wakelam, M. J. O. Biochim. Biophys. Acta 1998, 1436, 117-126.
34) Murata, R.; Yamamoto, A.; Soda, S.; Ito, A. Jpn. J. Med. Sci. Biol. 1965, 18,
(
(
(
1
89-202.
35) Torley, L.; Silverstrim, C.; Pickett, W. Anal. Biochem. 1994, 222, 461-
64.
(Clostridium welchii) (PLC) was dissolved in 50 mM Tris-HCl
(
4
buffer solution and adjusted to 10 µM as stock concentration, and
the assays were conducted in the same buffer. Control enzymes
(
(
36) Little, C. Methods Enzymol. 1981, 71, 725-30.
37) Lister, M. D.; Deems, R. A.; Watanabe, Y.; Ulevitch, R. J.; Dennis, E. A. J.
Biol. Chem. 1988, 263, 7506-7513.
38) Caramelo, J. J.; Delfino, J. M. Anal. Biochem. 2004, 333, 289-295.
39) Young, P. R.; Snyder, W. R.; Mcmahon, R. F. Biochem. J. 1991, 280, 407-
and proteins include phospholipase A
2
from bovine pancreas
(
(
(
PLA ), phospholipase D from Arachis hypogaea (peanut) (PLD),
2
bovine serum albumin (BSA), avidin from egg white (AVI), and
peptidase from porcine intestinal mucosa (PEP). They were used
in place of phospholipase C in control experiments. Calcium
chloride (1.0 M), sodium fluoride (0.2 M), and EDTA (0.2 M)
were dissolved in water as stock solutions. Sodium deoxycholate
4
10.
(
(
(
(
(
40) Snyder, W. R. Anal. Biochem. 1987, 164, 199-206.
41) Thuren, T.; Kinnunen, P. K. J. Chem. Phys. Lipids 1991, 59, 69-74.
42) Wu, S. K.; Cho, W. W. Anal. Biochem. 1994, 221, 152-159.
43) Hendrickson, H. S. Anal. Biochem. 1994, 219, 1-8.
44) Durban, M.; Bornscheuer, U. Eur. J. Lipid Sci. Technol. 2003, 105, 633-
6
37.
(SDC) was dissolved in methanol as 0.2 M stock solution.
(
(
45) Hergenrother, P. J.; Martin, S. F. Anal. Biochem. 1997, 251, 45-49.
46) Hergenrother, P. J.; Spaller, M. R.; Haas, M. K.; Martin, S. F. Anal. Biochem.
Instrumentation. UV-visible absorption spectra were ob-
1995, 229, 313-316.
tained on a Perkin-Elmer Lambda 25 UV/vis spectrophotometer,
(
(
(
(
47) Kurioka, S.; Matsuda, M. Anal. Biochem. 1976, 75, 281-289.
48) Wilton, D. C. Biochem. J. 1991, 276, 129-133.
49) Miller, I. R.; M., R. J. J. Colloid Interface Sci. 1971, 35, 340-345.
50) Demel, R. A.; Geurtsvankessel, W. S. M.; Zwaal, R. F. A.; Roelofsen, B.;
Vandeenen, L. L. M. Biochim. Biophys. Acta 1975, 406, 97-107.
51) Hirasawa, K.; Irvine, R. F.; Dawson, R. M. C. Biochem. J. 1981, 193, 607-
-
1
with a scan rate of 960 nm‚min . Fluorescence spectra were
recorded on a Jobin Yvon-SPEX Industries Fluorolog-3 model
FL3-21 spectrofluorometer and corrected by using correction
factors generated in-house with a primary standard lamp. The
fluorescence cuvette was placed in a custom-built thermostated
(
6
14.
Analytical Chemistry, Vol. 80, No. 1, January 1, 2008 151

Figure 1. (a) Structures of polymer, BpPPESO3 and substrate, 10CPC, and reaction scheme for hydrolysis of 10CPC by PLC. (b) Mechanism
of PLC turnoff assay.
cell holder, which was maintained at 37 °C during the assay and
was equipped with a microsubmersible magnetic stirrer.
then the solution was placed in the spectrophotometer, and the
fluorescence intensity (Ibt) as a function of time was recorded as
a blank. (Note that the blank signal Ibt decreased slightly with
time due to photobleaching of the polymer; see Supporting
Information for more detail.) Another freshly prepared 2.0-mL
Fluorescence Turnoff Assay Procedure. The PLC enzyme
assays were carried out at 37 °C in a fluorescence cuvette with
continuous stirring. For kinetics studies, the fluorescence intensity
was measured with excitation and emission wavelengths of 400
and 460 nm, respectively. A typical assay procedure was carried
3
aliquot of BpPPESO /10CPC solution was incubated at 37 °C, and
then it was quickly pipetted into a cuvette containing a 4-µL aliquot
out as follows. First, a 2.0-mL aliquot of the BpPPESO
was allowed to thermally equilibrate at 37 °C, and then the initial
polymer fluorescence intensity (I ) at 460 nm was measured. The
substrate (10CPC) was added to a second 2.0-mL aliquot of
BpPPESO solution, this mixture was incubated for 15 min, and
3
solution
of the enzyme solution, and the fluorescence intensity of the
BpPPESO
3
/10CPC/PLC solution was monitored as a function of
was
p
time (I ). Subsequently, the sample fluorescence intensity I
t
t
corrected for photobleaching by using the blank intensity Ibt (see
Supporting Information for details and validation of the correction
3
152 Analytical Chemistry, Vol. 80, No. 1, January 1, 2008

procedure). The sample and blank fluorescence intensity (Ibt and
, respectively) were measured using the same conditions. After
correction for photobleaching, the corrected fluorescence intensity
3
BpPPESO to disrupt its aggregation. As a result, the polymer’s
fluorescence reverts to its original (aggregated) state, in which
the spectrum is red-shifted and less intense. As shown by the
I
t
Itc at each time t was derived.
photograph in Figure 1b, the changes in BpPPESO fluorescence
3
For full wavelength-spectral scans of the assays, the fluores-
that accompany the assay are easily observed by eye. After
p t
cence intensity (I , or I ) versus wavelength profiles were recorded
addition of 10CPC to the solution of BpPPESO the fluorescence
3
with excitation wavelength at 400 nm. Because fresh assay solution
was used for spectral measurements at each time t, it was not
necessary to correct for photobleaching.
is considerably brighter (compare middle vial with left vial), and
the subsequent addition of PLC causes the fluorescence intensity
to decrease again (right vial). The 10CPC/PLC assay is rendered
quantitative by applying a calibration (eq 1), which relates the
BpPPESO fluorescence intensity to 10CPC concentration. This
3
allows one to determine the enzyme reaction kinetics from
changes in polymer fluorescence intensity.
Calculation of Initial Rate of Reaction (v
to substrate concentration [10CPC] as a function of time by using
eq 1, which is derived from the calibration plot of fluorescence
0
). Itc was converted
t
p
intensity ratio (I/I ) versus concentration of substrate [10CPC].
3
Effect of 10CPC on Fluorescence of BpPPESO . A series
of titrations was carried out to quantify the effects 10CPC and
polymer concentration on the polymer’s absorption and fluores-
I /I - 1
tc
p
[
10CPC] ) [10CPC]
(1)
t
0
I /I - 1
0
c
p
3
cence. Upon addition of 10CPC to an aqueous BpPPESO solution
at room temperature, the absorption of the polymer blue-shifts
by only 2 nm without a significant change in the shape of the
absorption band (see Supporting Information). However, as shown
in Figure 2a, the fluorescence spectrum changes dramatically. In
particular, addition of 10CPC (0-15 µM) to a solution of Bp-
In eq 1, [10CPC]
is the substrate concentration at time t, I
intensity of the polymer solution before addition of substrate, I0c
is the initial corrected fluorescence intensity at t ) 0, that is, the
fluorescence intensity after addition of substrate but before the
addition of enzyme and Itc is the corrected fluorescence intensity
at time t after enzyme addition. A plot of [10CPC]
then derived and the v was calculated from the slope of the plot
by using data for the region where hydrolysis of 10CPC is less
than 5 µM.
0
is the initial substrate concentration, [10CPC]
is the fluorescence
t
p
3
PPESO (c ) 1 µM) induces a blue-shift of the fluorescence
maximum from 503 to 436 nm combined with a 50-fold increase
in the fluorescence intensity at 436 nm. The significant change of
the fluorescence intensity suggests that 10CPC inhibits the
aggregation of the polymer as has been reported for the addition
of other surfactants to CPEs.2
t
vs time was
0
0,27
Figure 2b illustrates the plots of the relative fluorescence
intensity at 436 nm as a function of concentration of added 10CPC
at different BpPPESO concentrations. For each plot, the fluores-
3
cence intensity increases gradually at low 10CPC concentration.
After the concentration of 10CPC reaches a certain point, the
intensity increases sharply and linearly until reaching a plateau,
at which point further addition of 10CPC causes little additional
RESULTS AND DISCUSSION
Overview of Turnoff Assay. The structure of the conjugated
polyelectrolyte used in the assay, BpPPESO
3
, is shown in Figure
1a. The synthesis and characterization of the polymer is described
in the Supporting Information. Previous studies on structurally
similar, anionic PPE-type CPEs have shown that in water these
polymers exist as aggregates. The characteristic feature of the
aggregated state of CPEs is that the fluorescence emission appears
as a broad, structureless band that is Stokes shifted significantly
from the absorption band. Like the previously studied CPEs, in
change in the intensity. (The plot for 5 µM BpPPESO
b only shows the effect of added 10CPC below the concentration
range where the sharp increase is observed.) Note that as the
BpPPESO concentration increases, more lipid is needed to induce
3
in Figure
2
3
3
water, BpPPESO exists in an aggregated state, as is clearly
the fluorescence change. This is consistent with the involvement
of a polymer-lipid complex in the observed fluorescence intensity
enhancement. Importantly, over the concentration range in which
the fluorescence intensity increases sharply, the relative increase
signaled by the broad, structureless fluorescence band that is
flr
Stokes shifted from the absorption band (λmax ) 503 nm and
abs
λ
max
) 419 nm).
The cartoon shown in Figure 1b illustrates the mechanism of
in fluorescence intensity (I/I
0CPC concentration. This suggests that is should be possible to
create a calibration curve that relates fluorescence intensity to
p
) is nearly linearly proportional to
the PLC turnoff assay. Although 10CPC is a zwitterion, electro-
static (ion-dipole) and hydrophobic forces induce the formation
of a polymer-lipid complex between the phospholipid and
1
1
0CPC concentration.
3 3
BpPPESO . As a result, the backbone of BpPPESO is more
extended and aggregation of the polymer is reduced. The lipid-
induced changes in polymer conformation and aggregation state
are signaled by a blue-shift and enhancement in the polymer’s
fluorescence intensity. Introduction of PLC to the polymer-lipid
complex induces hydrolysis of 10CPC. The PLC catalyzes hy-
drolysis of the zwitterionic head group from the hydrophobic tail,
disrupting the amphiphilic structure of the lipid, which is a key
factor allowing 10CPC to complex with the polymer. One of the
Prior to developing a calibration allowing one to follow the
10CPC substrate concentration during PLC hydrolysis, a study
was carried out to determine the solution conditions under which
the PLC activity was optimized, and yet the polymer’s response
to 10CPC was still acceptable. This survey demonstrated that the
optimum solution conditions include the use of Tris-HCl buffer
2+
(c ) 50 mM, pH 7.4) along with added Ca as an activator for
the PLC.29 Figure 3 shows the effect of 10CPC on the fluorescence
2
+
1
0CPC hydrolysis products, DAG, is hydrophobic and charge
of 15 µM BpPPESO
°C. Note that, under these solution conditions, upon addition of
10CPC the fluorescence of BpPPESO is blue-shifted and en-
3
in 50 mM Tris-HCl with 2 mM Ca at 37
neutral while the second product, phosphorylcholine, has a net
negative charge. Apparently neither of these species interacts with
3
Analytical Chemistry, Vol. 80, No. 1, January 1, 2008 153

Figure 2. (a) Fluorescence spectroscopic changes of a solution of 1 µM BpPPESO3 in water observed upon addition of 10CPC at 25 °C,
λex ) 400 nm. (b) Fluorescence intensity increase at 436 nm upon titration of 10CPC at different concentrations of BpPPESO3 in water, λex )
400 nm.
Figure 3. (a) Fluorescence spectroscopic changes for a solution of BpPPESO3 (15 µM) observed upon addition of 10CPC in 50 mM Tris-HCl
2+
(
pH 7.4) with 2 mM Ca at 37 °C, λex ) 400 nm. (b) Fluorescence intensity increase at 460 nm upon titration of 10CPC, λex ) 400 nm. Inset:
calibration plot of relative fluorescence intensity increase as a function of 10CPC concentration.
hanced to a lesser extent in comparison to the change in
fluorescence for the polymer seen in pure water (see Figure 2a).
This difference arises because Ca induces aggregation of
used to derive eq 1, which is used to determine [10CPC]
the PLC assay.
t
during
2
+
PLC Turnoff Assay. In order to demonstrate the feasibility
2,52
BpPPESO
to influence the extent of BpPPESO
that under these solution conditions more 10CPC is needed to
elicit a strong fluorescence response, at [BpPPESO ] ) 15 µM, a
0-fold fluorescence enhancement is observed upon addition of
00 µM 10CPC. In particular, Figure 3b illustrates the increase
3
,
and this effect partially offsets the ability of 10CPC
of using BpPPESO as the basis for a PLC turnoff assay, we initially
3
3
aggregation. Despite the fact
examined the effect of added 10CPC and PLC on the polymer’s
fluorescence intensity in water without added buffer or Ca . The
results of this initial experiment are shown in Figure 4. In
particular, the initial fluorescence from a solution BpPPESO
2+
3
1
1
3
(c
)
1 µM) is blue-shifted and significantly enhanced in intensity
in fluorescence intensity at 460 nm (λex ) 400 nm) as a function
of added 10CPC. This plot features a trend similar to that shown
by addition of 10 µM 10CPC (change indicated by arrow 1). After
introduction of PLC (c ) 2.3 nM) to the solution, hydrolysis of
in Figure 2b, with the BpPPESO
nearly liner intensity increase until reaching a plateau for [10CPC]
250 µM. The inset in Figure 3b shows a calibration plot of the
relative fluorescence intensity increase (I/I ) as a function of
10CPC]. The plot features a good linear relationship over the
concentration range 0-100 µM with a y-intercept of 1, and
consequently, the [10CPC] is directly proportional to (I/I - 1)
at any time during a PLC hydrolysis reaction. This calibration is
3
exhibiting a significant and
10CPC causes a red shift of the fluorescence band and a decrease
in intensity with increasing incubation time (indicated by arrow
>
2
). The BpPPESO
3
fluorescence decreases ∼50% within 1 min
p
2
even in the unbuffererd solution and without added Ca activator,
which demonstrates the potential for the PLC turnoff assay.
In order to optimize the conditions for the PLC assay, the effect
of the Ca2 activator was investigated when assays were conducted
in 50 mM Tris-HCl buffer (pH 7.4). Calcium ion serves as an
activator for PLC because Ca2 interacts with the substrate
[
p
+
+
(
52) Jiang, H.; Zhao, X. Y.; Schanze, K. S. Langmuir 2006, 22, 5541-5543.
54 Analytical Chemistry, Vol. 80, No. 1, January 1, 2008
1

3
at 37 °C. Figure 6a illustrates plots of the BpPPESO fluorescence
intensity monitored at 460 nm as a function of time for solutions
containing PLC at concentrations ranging from 0 to 50 nM. Note
that the rate of decrease in the BpPPESO
increases as the concentration of PLC increases. Figure 6b
illustrates a plot of v as a function of PLC concentration for the
3
fluorescence intensity
0
range 0-75 nM, and it is evident from this presentation that the
initial catalyzed reaction rate varies linearly with enzyme concen-
tration. The analytical detection limit for PLC is 0.5 nM (22
-
1
-4
-1
ng‚mL , 6.6 × 10 unit‚mL , one unit is defined as the amount
of enzyme hydrolyzing 1.0 µmol of 10CPC/min at pH 7.4 at
3
7 °C) and was obtained from the calibration curve in Figure 6b.
This sensitivity is comparable to values obtained by using different
assays (see Discussion section).5
5,56
3
An interesting observation is that the BpPPESO /10CPC/PLC
solution becomes turbid as the reaction proceeds when solutions
containing g35 µM 10CPC undergo PLC-catalyzed hydrolysis.
Under these conditions, at longer reaction times, a greenish-yellow
precipitate forms and the fluorescence of the polymer decreases
sharply. This observation suggests that the product DAG produced
by PLC-catalyzed hydrolysis of 10CPC coprecipitates with Bp-
Figure 4. Fluorescence spectroscopic changes observed in the PLC
turnoff assay. (s) Initial fluorescence of 1.0 µM BpPPESO3 in water
at 25 °C; (• •) fluorescence after step 1: addition of 10 µM 10CPC;
fluorescence intensity as a function of time after step 2: addition of
2
.3 nM PLC and incubate for 1 (- -), 5 (- - ), 20 (s s), and 45
(- ‚ -) min, λex ) 400 nm.
3
PPESO and this effect interferes with the sensor response. In
order to prove that DAG is the origin of the precipitation, its effect
on the assay was investigated. Thus, various concentrations of
DAG were added to a solution containing BpPPESO
3
and 10CPC
(c ) 15 and 50 µM, respectively). It was found that DAG dissolves
with a negligible effect on the fluorescence intensity when added
at low concentration (c < 10 µM). However, with increasing DAG
concentration, the fluorescence intensity gradually decreases
followed by a coprecipitation of the DAG/polymer complex at
higher concentration of DAG (c > 30 µM). The precipitation
results in a significant decrease in the fluorescence intensity.
Therefore, quantitative analyses, including the calculations for
initial rate of reaction and determination of kinetic parameters,
were conducted at a DAG concentration less than 5 µM.
Determination of PLC-Catalyzed 10CPC Hydrolysis Ki-
Figure 5. Effect of concentration of activator, [Ca2+], on the initial
rate of hydrolysis (v0) of 10CPC by PLC. Experiment conditions: 15
µM BpPPESO3, 80 µM initial 10CPC, and 20 nM PLC in 50 mM Tris-
HCl (pH 7.4) at 37 °C, λex ) 400 nm.
netic Parameters. The BpPPESO
used to determine the kinetic parameters (K
PLC-catalyzed hydrolysis of 10CPC. The kinetics experiments
3
-based fluorescence assay was
m
and Vmax) for the
were carried out using solutions containing BpPPESO
3
(c ) 15
µM), PLC (c ) 20 nM), Ca2 (c ) 2 mM), and Tris-HCl buffer
+
(
50 mM, pH 7.4) at 37 °C. The initial concentration of 10CPC was
providing charge density, which enhances binding of the enzyme
with the substrate.53 The dependence of the initial enzyme-
varied from 0 to 90 µM, which is the range corresponding to the
linear range in the calibration curve shown in Figure 3b (inset),
2+
catalyzed rate (v
was conducted with a solution containing BpPPESO
0CPC (cinitial ) 80 µM), and PLC (c ) 20 nM). Note that v
increases almost linearly until it reaches a maximum rate when
0
) on [Ca ] is shown in Figure 5. The experiment
and the v
10CPC] (see Supporting Information for the plot). Using a
nonlinear regression routine, the plot of v versus [10CPC] was
fitted with the Michaelis-Menten equation (eq 2, where v is initial
rate of reaction and [S] is the initial 10CPC concentration)
yielding values for K and Vmax of 28 ( 3 µM and 19 ( 0.7
µmol‚min ‚mg , respectively. Unfortunately, there are no other
reports of the values for K and Vmax with 10CPC as substrate.
However, using natural egg lecithin (a mixture of several different
0
values were obtained and plotted as a function of
3
(c ) 15 µM),
[
1
0
0
2
+
0
[
Ca ] ) 2 mM, and at higher concentrations, v
0
decreases. The
2
+
0
observed dependence of v
in previous kinetic studies of PLC activity.
0
on [Ca ] is similar to that observed
4
1,54
m
-1
-1
In a series of investigations, we examined the kinetics of the
PLC-catalyzed hydrolysis of 10CPC at varying PLC concentration.
These real-time kinetic assays were carried out with a solution
m
phospholipids including phosphatidylcholine), K
to be 1657 or 28
m
was reported
containing BpPPESO
3
(c ) 15 µM) and 10CPC (cinitial ) 30 µM)
41,57
µM and Vmax was 68 µmol‚min-1‚mg by
57
-1
in Tris-HCl buffer (50 mM, pH 7.4) in the presence of 2 mM Ca2
+
(55) Holdsworth, R. J.; Parratt, D. J. Immunoassay 1994, 15, 293-304.
(
53) Bangham, A. D.; Dawson, R. M. C. Biochim. Biophys. Acta 1962, 59, 103-
15.
54) Ohsaka, A.; Sugahara, T. J. Biochem. 1968, 64, 335-345.
(56) Yamakawa, Y.; Ohsaka, A. J. Biochem. 1977, 81, 115-126.
(57) Casu, A.; Nanni, G.; Pala, V.; Monacell, R. Ital. J. Biochem. 1971, 20, 166-
1
(
178.
Analytical Chemistry, Vol. 80, No. 1, January 1, 2008 155

Figure 6. (a) Changes in fluorescence emission intensity (Itc) at 460 nm during the PLC turnoff assay as a function of reaction time for various
concentrations of PLC: 0 (black), 5 (red), 10 (green), 25 (yellow), and 50 (blue) nM. Itc was corrected for photobleaching by blank fluorescence
intensity Ibt. Experiment conditions: 15 µM BpPPESO3 and 30 µM initial 10CPC in 50 mM Tris-HCl (pH 7.4) with 2 mM Ca2 at 37 °C, λex ) 400
nm. (b) Dependence of initial rate of reaction (v0) on PLC concentration.
+
monitoring the hydrolysis kinetics by phosphorus determination57
or by a fluorometric method with dye-labeled lecithin as sub-
41,57
strate.
the BpPPESO
agreement with values obtained using other assays; however, the
max value is smaller. It is likely that the lower Vmax arises due to
This comparison shows that the K
m
value obtained from
3
-based fluorescence turnoff assay is in good
V
complex formation between 10CPC and the polymer, which
effectively decreases the binding of the lipid to PLC.
Vmax[S]0
ν0 )
(2)
K + [S]0
m
Inhibition of the PLC Catalysis. In order to further demon-
strate that the observed fluorescence intensity decrease that is
induced by addition of PLC to the BpPPESO /10CPC solution
3
Figure 7. Inhibition of PLC turnoff assay. Plot illustrates the initial
rate of reaction (v0) versus inhibitor concentration: F (black b), EDTA
-
arises due to PLC-catalyzed hydrolysis of the 10CPC, we tested
the effect of known PLC inhibitors on the fluorescence assay. Two
(
red 1). Experiment conditions: 15 µM BpPPESO3, 50 µM initial
10CPC, and 20 nM PLC in 50 mM Tris-HCl (pH 7.4) with 2 mM Ca2+
at 37 °C, λex ) 400 nm.
5
8,59
reported inhibitors for PLC from C. perfringens are fluoride ion
and EDTA.
4
7,60
Both of these species were tested as inhibitors in
assays conducted under the same conditions used for the
kinetics studies described above using 10CPC at a concentration
of 50 µM. As shown in Figure 7, both fluoride ion and EDTA
effectively inhibit the PLC activity. While the overall inhibition
increases with increasing inhibitor concentration, the inhibition
efficiency is largest at low inhibitor concentration. Nonetheless,
the inhibition experiments provide very strong evidence that the
assay is effectively reporting on the PLC-catalyzed hydrolysis
reaction.
2
lipases. As shown in Figure 8a, PLA catalyzes the hydrolysis of
an acyl ester bond exclusively at the 2-acyl position in the
glycerophospholipid, affording a free fatty acid and a lysophos-
61
pholipid. The lysophospholipid has a surfactant-like structure,
containing a single hydrophobic carbon chain and a polar head
group. Thus, like 10CPC, the lysophospholipid produced by PLA
2
hydrolysis of 10CPC is expected to complex strongly with the
polymer preventing aggregation and quenching of the fluores-
cence. On the other hand, PLD-catalyzes hydrolysis of the
phosphate ester bond on the choline side to form choline and
Specificity of the PLC Turnoff Assay. Given that an effective
biosensor should exhibit specificity for the target enzyme, it is of
62
phosphatidic acid (Figure 8a). Although phosphatidic acid also
interest to examine the response of the BpPPESO
PLC assay to other proteins. In order to accomplish this objective,
five proteins were selected, including PLA , PLD, peptidases PEP
a commercial mixture of protease), BSA, and AVI. Three of these
3
/10CPC-based
has a surfactant-like structure, its overall negative charge might
prevent it from interacting with BpPPESO
3
. In addition, it is
2
2+
reported that 20-100 mM Ca is required for maximum activity
(
63
of PLD, along with a pH of 5.6. Therefore PLD might not achieve
2
enzymes, PLC, PLA , and PLD, belong to the group of phospho-
(61) Cottrell, R. C. Methods Enzymol. 1981, 71, 698-702.
(
(
(
58) MacFarlane, M. G.; Knight, B. C. J. G. Biochem. J. 1941, 35, 884-902.
59) Matsumoto, M. J. Biochem. 1961, 49, 23-31.
60) Krug, E. L.; Kent, C. Arch. Biochem. Biophys. 1984, 231, 400-410.
(62) Tzur, R.; Shapiro, B. Biochim. Biophys. Acta 1972, 280, 290-296.
(63) Heller, M.; Mozes, N.; Abramovi, Ip.; Maes, E. Biochim. Biophys. Acta 1974,
369, 397-410.
156 Analytical Chemistry, Vol. 80, No. 1, January 1, 2008

method can also be readily adapted to a fluorescence-based high-
throughput screening assay format.64
A comprehensive literature survey reveals that many assays
for monitoring the enzymatic activity of PLC from C. perfringens
have been previously reported.1
2,35,36,38,39,41,43,44
The previously
reported assays are based on a variety of detection methods
including turbidimetric, pH-stat titration, radiometric, and continu-
ous fluorometric methods. Concerning the sensitivity, the best
-
1
detection limit of 0.005 units‚mL for PLC from C. perfringens
was achieved by using an ELISA assay.55 A second assay that is
based on the acid-soluble phosphorus method affords a detection
-
1
56
limit of 70 ng‚mL for PLC. (Assuming the molecular mass for
PLC is 43 kDa, this corresponds to a detection limit of ∼2 nM.)
3
The BpPPESO -based PLC assay that is described herein clearly
affords sensitivity that is comparable to (or better than) that of
the earlier reports. With respect to the amount of lipid substrate
needed for the assay, substrate concentrations in the milli-
molar range are required in the previously reported PLC
assays.3
9,41,47,55-57,59,60,65
By contrast, our method not only allows
an assay to be carried out at significantly lower initial substrate
concentration but also affords the ability to carry out real-time
detection of the enzyme activity. In addition, surfactants such as
SDC have been frequently used in the presence of Ca ions to
disperse water-insoluble phospholipids to enhance the rate of
Figure 8. Specificity of PLC turnoff assay. Changes in fluorescence
emission intensity at 460 nm following 2-min incubation with PLC and
other five control proteins. Experiment conditions: 15 µM BpPPESO3,
2
+
5
0
3
0 µM initial 10CPC, and 20 nM PLC or control proteins (except for
.86 µg/mL PEP) in 50 mM Tris-HCl (pH 7.4) with 2 mM Ca at
7 °C, λex ) 400 nm.
reaction and leading to an effective increase the PLC activ-
-
1
2+
29,47,56,60
ity.
However, the addition of a surfactant does not afford
any improvement in PLC activity if a water-soluble phospholipid
is the substrate.47 The effect of SDC was also examined for the
BpPPESO /10CPC/PLC assay, and it was found that the polymer’s
3
its full activity in the optimal conditions for the PLC turnoff assay
2
+
(2 mM Ca and pH 7.4).
fluorescence response was suppressed in the presence of the
surfactant. The likely explanation for this effect is that the
surfactant influences the fluorescence intensity change by com-
plexing with the polymer (just like 10CPC), effectively counteract-
ing the effect induced by hydrolysis of 10CPC. Nonetheless, since
the formation of the polymer/lipid complex facilitates the solu-
bilization of the lipid in water, addition of a surfactant such as
SDC is not required for our assay.
The assays were carried out with the same conditions used
for the kinetics studies described above, with 50 µM 10CPC. In
each case PLC was not added, but an aliquot containing the other
protein was added in to achieve a final concentration of 20 nM
(except for PEP, which was added to achieve a concentration of
-
1
0
.86 µg‚mL ). Figure 8b compares the fluorescence intensity
changes (460 nm) observed 2 min after addition of the protein
aliquot to the BpPPESO /10CPC/buffer solution. As expected,
the assay with PLC exhibits ∼70% decrease in fluorescence
intensity, while the solutions containing the “control” proteins
exhibit less than an 8% decrease in intensity. This result demon-
3
3
While the BpPPESO /10CPC/PLC assay is relatively easy to
implement and has many advantages, nonetheless the method still
has some disadvantages. For example, the sensitivity of the assay
is affected by various experimental conditions, such as buffer
concentration, temperature, or polymer concentration. In addition,
nonspecific interactions with various proteins and other solutes
strates that the BpPPESO
note that a small, but significant decrease in fluorescence intensity
was observed upon addition of PLA , PLD, and PEP to the
BpPPESO /10CPC/buffer solution, suggesting the existence of
a nonspecific interaction between the proteins and the BpPPESO
Although such interaction exists, a negligible decrease of fluo-
rescence intensity (<0.5%) is observed after 2-min incubation of
a blank PLC assay (20 nM PLC) in which no substrate, 10CPC,
is added. Therefore, the nonspecific effects are very small in
comparison to the effect of specific enzymatic hydrolysis of 10CPC
by PLC. Thus, it is safe to conclude that the overall interaction
between PLC and polymer/phosphatidylcholine complex consists
mainly of specific enzymatic activity as well as very small
contribution from nonspecific interaction.
3
assay is specific for PLC. However,
2
(e.g., metal ions, lipids, or surfactants) and the polymer (and lipid
3
substrate) could interfere with the sensor response, especially if
quantitative (kinetic) data are needed. Finally, the PLC turnoff
assay is limited by one of the hydrolysis products, DAG, which
leads to a precipitation of polymer at higher concentration (c >
3
.
3
0 µM). Due to these limitations, when applying this assay with
biological samples (e.g., a serum sample), the specific assay
conditions will need to be optimized in order to attain the optimal
sensor response in the presence of possible interfering species.
CONCLUSION AND OUTLOOK
This paper describes the development and application of a
Discussion. The assay described herein affords the ability to
monitor PLC activity rapidly and in real time. In addition, the
method is quite sensitive, with respect to both the amount of
enzyme and substrate required (<1 and 5 µM, respectively). The
novel fluorescence turnoff assay that affords real-time detection
(
64) Hertzberg, R. P.; Pope, A. J. Curr. Opin. Chem. Biol. 2000, 4, 445-451.
65) Moreau, H.; Pieroni, G.; Jolivetreynaud, C.; Alouf, J. E.; Verger, R.
Biochemistry 1988, 27, 2319-2323.
(
Analytical Chemistry, Vol. 80, No. 1, January 1, 2008 157

of PLC activity with good specificity and that functions at very
low substrate and enzyme concentrations. The sensor takes
advantage of the interaction between the lipid substrate and a
fluorescent, water-soluble conjugated polyelectrolyte. The high
sensitivity of the sensor is due to the well-known ability of the
fluorescent conjugated polyelectrolyte to exhibit amplified re-
sponse to aggregation or the presence of charged fluorescence
also be possible to develop a similar assay to monitor DNA
transfection, since some natural and synthetic cationic lipids have
been utilized in gene transfer.6
6,67
ACKNOWLEDGMENT
We thank Xiaoyong Zhao for synthesis and characterization
3
of BpPPESO . We also gratefully acknowledge financial support
by the U.S. Department of Energy, Basic Energy Science (Grant
DE-FG02-03ER15484).
2
,17,19,20,22,26
quenchers.
Although the work demonstrates the turnoff assay with a
specific conjugated polyelectrolyte, substrate, and enzyme, this
method is quite general and can very likely be extended to other
lipases and to different lipid substrates. For example, a similar
conjugated polyelectrolyte-based turnoff assay can be designed
to detect the activity of sphingomyelinase using sphingomyelin
as a substrate. If an anionic phospholipid, phosphatidylinositol
biphosphate, is used as a substrate to monitor phosphatidylinositol
phospholipase C activity, a cationic CPE can be utilized. It may
SUPPORTING INFORMATION AVAILABLE
Details concerning the fluorescence intensity correction for
photobleaching; synthesis and structural characterization of
BpPPESO
before and after addition of 10CPC; nonlinear regression of v
10CPC] data to derive K
3
; normalized absorption and emission of BpPPESO
3
0
vs
[
m
and Vmax. This material is available
free of charge via the Internet at http://pubs.acs.org.
(
66) Zhang, S.; Xu, Y.; Wang, B.; Qiao, W.; Liu, D.; Li, Z. J. Controlled Release
004, 100, 165-180.
67) Zhdanov, R. I.; Podobed, O. V.; Vlassov, V. V. Bioelectrochemistry 2002,
8, 53-64.
Received for review August 6, 2007. Accepted October 1,
2
2007.
(
5
AC701672G
158 Analytical Chemistry, Vol. 80, No. 1, January 1, 2008