Enhancing enzyme stability by construction of polymer-enzyme conjugate micelles for decontamination of organophosphate agents

Article abstract of DOI:10.1021/bm401531d

Enhancing the stability of enzymes under different working environments is essential if the potential of enzyme-based applications is to be realized for nanomedicine, sensing and molecular diagnostics, and chemical and biological decontamination. In this

Full text of DOI:10.1021/bm401531d

Article
pubs.acs.org/Biomac
Enhancing Enzyme Stability by Construction of Polymer−Enzyme
Conjugate Micelles for Decontamination of Organophosphate
Agents
Nisaraporn Suthiwangcharoen and Ramanathan Nagarajan*
Molecular Sciences and Engineering Team, Natick Soldier Research, Development and Engineering Center, 15 Kansas Street, Natick,
Massachusetts 01760, United States
*
S Supporting Information
ABSTRACT: Enhancing the stability of enzymes under different working environments is essential if the potential of enzyme-
based applications is to be realized for nanomedicine, sensing and molecular diagnostics, and chemical and biological
decontamination. In this study, we focus on the enzyme, organophosphorus hydrolase (OPH), which has shown great promise
for the nontoxic and noncorrosive decontamination of organophosphate agents (OPs) as well as for therapeutics as a catalytic
bioscavanger against nerve gas poisoning. We describe a facile approach to stabilize OPH using covalent conjugation with the
amphiphilic block copolymer, Pluronic F127, leading to the formation of F127-OPH conjugate micelles, with the OPH on the
micelle corona. SDS-PAGE and MALDI-TOF confirmed the successful conjugation, and transmission electron microscopy
(
TEM) and dynamic light scattering (DLS) revealed ∼100 nm size micelles. The conjugates showed significantly enhanced
stability and higher activity compared to the unconjugated OPH when tested (i) in aqueous solutions at room temperature, (ii)
in aqueous solutions at higher temperatures, (iii) after multiple freeze/thaw treatments, (iv) after lyophilization, and (v) in the
presence of organic solvents. The F127-OPH conjugates also decontaminated paraoxon (introduced as a chemical agent
simulant) on a polystyrene film surface and on a CARC (Chemical Agent Resistant Coating) test panel more rapidly and to a
larger extent compared to free OPH. We speculate that, in the F127-OPH conjugates (both in the micellar form as well as in the
unaggregated conjugate), the polypropylene oxide block of the copolymer interacts with the surface of the OPH and this
confinement of the OPH reduces the potential for enzyme denaturation and provides robustness to OPH at different working
environments. The use of such polymer−enzyme conjugate micelles with improved enzyme stability opens up new opportunities
for numerous civilian and Warfighter applications.
INTRODUCTION
The DNA sequencing performed on the opd cistron from
Pseudomonas diminuta demonstrated the presence of an amino-
terminal signal sequence, which was thought to control the
cellular location (cytoplasmic vs membrane or outer surface
associations) of the OPH activity in the native pseudomonad
bacterium. The OPH produced from this gene was observed to
be subject to variable processing depending upon the host
strain. In the native pseudomonad and flavobacterium hosts,
the enzyme was recovered in multiple molecular forms from
cell-free lysates with molecular weights from 35000 to 39000
Da. The enzyme is a homodimer and the crystal structure of
■
Organophosphorus compounds (OPs) are highly toxic
molecules that find applications as pesticides and insecticides,
and they are also among the class of lethal chemical warfare
agents. They can readily inactivate acetylcholinesterase
(
AChE), an enzyme that plays a critical role in the central
1
,2
nervous system, leading to paralysis and possibly death. Over
the last few decades, much effort has been devoted to
developing methods to detect and inactivate the OPs, and
one of the most promising is the use of the enzyme,
organophosphorus hydrolase (OPH, also known as phospho-
triesterase), to degrade the OPs.
OPH was originally isolated from soil bacteria and adapted to
Received: October 15, 2013
Revised: February 13, 2014
Published: February 24, 2014
grow on organophosphates or the extra-chromosomal plasmids
3
−5
of the bacteria host that had been exposed to the pesticide.
©
2014 American Chemical Society
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OPH exhibited the overall fold of a distorted (β/α) or TIM-
barrel. The homodimer has approximately the size of 12 × 9
EXPERIMENTAL SECTION
8
■
6
−8
Materials. The unconjugated enzyme, designated as Cys-C -
3
0
×
7 nm based on its crystal structure.
The catalytic activity of OPH was found to be associated with
mOPH, was kindly donated by the Olsen laboratory from MIT. This
was prepared for research purposes by gene cloning into pQE9
expression vector and transferring into SG13009 cells for protein
expression. This product differed from the wild type OPH through two
important modifications. One was the replacement of five amino acids,
Lys185Arg, Asp208Gly, His254Arg, Ile274Asn, and Arg319Ser (the
numbers refer to the amino acid sequence in the wild-type OPH) and
this modification was designated as mOPH. The other was the
connection of a cysteine residue through a long spacer denoted as C ,
at the N-terminus of mOPH which is located at the opposite site of the
OP binding pocket (enzyme active site). Both modifications were
carried out in the Olsen laboratory by subcloning the Cys-C30 spacer
gene into the pQE9 expression vector together with the mOPH gene.
The C30 spacer consists of nearly 320 amino acids (primary structure
shown on Scheme S1) with no secondary structure and acts like a
polyelectrolyte with high water solubility. Indeed the C30 addition to
OPH was done to make OPH highly water-soluble overcoming any
4
,5
the protein encoded by the plasmid-borne gene (opd)
together with the two divalent metal ions in the active site of
3
the OPH. OPH easily loses its activity in the presence of
chelating agents, but the activity can be recovered upon
2
+
2+
2+
2+ 3,9
incubation with Mn , Co , Cd , or Ni . Despite its high
catalytic activity, the potential use of OPH is severely hindered
because OPH has a low stability under different storage/
3
0
10
working environments. Current methods to stabilize OPH
1
1−13
10,13−15
rely on immobilizations,1
6
encapsulation,
17,18
or com-
and poly-
plexation with hydrogel, fire-fighting foams,
1
1,19
electrolytes.
However, the challenge to develop a robust
protocol which can preserve the OPH activity and
conformation and stabilize the OPH under different working
environments remains unfulfilled.
In this study, we describe our facile approach to stabilize
OPH using covalent conjugation with amphiphilic block
copolymers leading to the formation of block copolymer−
OPH conjugates, which can exist as micelles even at low
concentrations. The thermoresponsive Pluronic (trade name)
triblock copolymer F127 (which is a symmetric triblock
hydrophobicity. In the text below, we have referred to Cys-C -mOPH
as simply OPH and this sample was used as the candidate for the block
copolymer conjugation studies reported here.
Pluronic F127 (MW 12450 Da, 70% polyethylene oxide block and
30
6
4 propylene oxide units) was a gift from BASF. Succinic anhydride
and methanol were purchased from Sigma Aldrich. 4-Dimethylami-
nopyridine (DMAP) and N-hydroxysuccinimide (NHS) were
purchased from Alfa Aesar. Triethylamine, 1,4-dioxane, and dichloro-
methane were purchased from Sigma Aldrich. 1-Ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC) was purchased from
ACROS Chemical Company. Paraoxon (O,O-diethyl O-(4-nitro-
phenyl) phosphate) was purchased from Sigma Aldrich (EC Number
copolymer PEO100PPO PEO100, with hydrophilic polyethylene
64
oxide, PEO in the ends and hydrophobic polypropylene oxide,
PPO in the middle), was selected due to its environmental
friendliness, biocompatibility, and chemical structure, which
allows the enzyme conjugation to be easily implemented. Since
the Pluronic family includes commercially available molecules
with a wide range of molecular weights and block compositions,
having the ability to spontaneously dissolve and self-assemble in
water, it offers many potential candidates for conjugation to
enzymes. Due to its amphiphilic nature, above its critical
micelle concentration, the Pluronic block copolymer sponta-
neously forms micelles in aqueous solution with hydrophilic
PEO shell or corona and hydrophobic PPO core. The covalent
coupling of Pluronic micelles to proteins, antibodies, or
2
06−221−0). HEPEs buffer (50 mM, pH 8) containing 0.1 mM
CoCl and 50 mM NaCl was used throughout the experiment unless
2
specify otherwise.
Synthesis of Carboxylated-Pluronic F127 (F127-COOH). The
synthesis was conducted following previous reports. Briefly, F127
6.3 g; 1 mmol OH) and DMAP (122.17 mg; 1 mmol) were dissolved
20
(
in 1,4-dioxane (15 mL) with the presence of TEA (139 μL) and
allowed to stir under N for 30 min. Succinic anhydride solution (125
2
mg in 5 mL 1,4-dioxane) was then added dropwise to the Pluronic
solution while stirring. The solution mixture was allowed to stir at
room temperature for 24 h. The excess 1,4-dioxane was removed by
rotary evaporation, and the remaining samples were precipitated three
times in cold diethyl ether while stirring. The precipitate was dried
under vacuum overnight at room temperature to give the white
powder of F127-COOH. The presence of COOH was analyzed using
2
0,21
polysaccharide is beginning to be explored
for applications
in biomedicine, and this study is the first report, to the best of
our knowledge, on the preparation and evaluation of Pluronic−
OPH conjugate micelles for the decontamination application.
We hypothesize that the F127-OPH conjugate micelles can
prevent the OPH from aggregating to each other, thus,
reducing the propensity for enzyme denaturation. The
thermostability of F127 may provide robustness to the OPH
at different working temperatures. The hydrophobic core can
sequester the OPs having limited aqueous solubility and
increase the interaction between the OPs and the OPH. The
enhancement of enzyme activity following conjugation to the
block copolymer compared to conjugation with the homopol-
ymer PEG (polyethylene glycol) suggests that the propylene
oxide (PPO) block of the Pluronic may indeed interact with the
surface of OPH influencing the activity as well as stability. As a
consequence of these beneficial physical-chemical features, we
believe that the Pluronic−enzyme conjugation offers a general
route to substantially increase the stability of the enzymes while
also enhancing the enzymatic activity in comparison to the
unconjugated enzyme as well as simple PEG conjugated
enzymes.
1
FT-IR and H NMR (Figure S1).
Synthesis of NHS-Terminated F127 (F127-NHS). In this step, the
carboxyl-terminated F127 was activated with NHS. Briefly, F127-
COOH (500 mg; 0.08 mmol) and EDC (77 mg; 0.4 mmol) were
dissolved in 5 mL of dichloromethane in a round-bottom flask. After
stirring the mixture for 30 min, NHS (46 mg, 0.4 mmol) was added,
and the solution mixture was left stirring at room temperature for 24 h.
Next day, the solution mixture was precipitated three times in cold
diethyl ether. The precipitate was further dried under vacuum
overnight at room temperature to give the white powder of F127-
NHS. The presence of NHS was confirmed using FT-IR and H NMR
(
Figure S1).
OPH attachment to F127. The strategy for attaching OPH to F127
21−25
was adapted from previous literature.
Briefly, OPH solution (1
−1
mL of 0.2 mg·mL ) was added dropwise into the F127-NHS solution
−1
(1 mL of 40 mg·mL ) while stirring. The mass ratio of F127 to OPH
was 200 and the mole ratio was approximately 1000, and such
overwhelming excess was used to improve the probability of OPH
conjugation. The reaction mixture was allowed to stir at 4 °C for 4 h.
At this temperature, the F127 block copolymer and its end
functionalized forms do not exist in micellar state (as confirmed by
dynamic light scattering, Figure S2) and therefore the conjugation
involved reaction between unaggregated F127-NHS and the OPH.
1
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a
Scheme 1. Schematic Representation of the Conjugation of OPH to Pluronic F127 to Create F127-OPH Conjugate Micelles
a
F127 was first activated to introduce the carboxyl function at the end. OPH was then covalently attached through the EDC/NHS coupling reaction.
The detailed view of the OPH dimer used shows the C30 modification (polyelectrolyte like polypeptide chain) on each monomer to increase the
aqueous solubility. Also indicated are some of the lysine residues accessible for conjugation to F127-NHS. Of the two F127 molecules conjugated to
each dimeric OPH, one or both could be inserted into the micelle core. The polypropylene oxide block from the conjugate will also have favorable
interactions with the hydrophobic surface patches on OPH.
Following the conjugation reaction, the samples were centrifuged
between 8000 and 16000g for 20 min to remove any unconjugated
OPH and the ester byproduct, followed by resuspension in buffer. This
step was repeated at least three times. The purification process was
monitored by sodium dodecyl sulfate polyacrylamide gel electro-
phoresis (SDS-PAGE) using NuPAGE Novex 12% Bis-Tris Gel
TEM Analysis. TEM images were obtained on JEOL 2010 TEM
equipped with a LaB6 (lanthanum hexaboride) filament, operated at
200 KeV with GIF 2001 spectrometer and 1 megapixel CCD camera.
For sample preparation, 10 μL of each sample was dropped on a
copper grid and allowed to evaporate overnight. Then, 10 μL of
phosphotungstic acid (PTA) solution (2% w/v) was added to the grid.
After 2 min, the solution was drawn off from the edge of the grid with
filter paper, and the grid was allowed to air-dry prior to the analysis.
Enzymatic Activity and Stability Assay. The activity of F127-
OPH conjugate in the hydrolysis of paraoxon was evaluated in HEPEs
(
Molecular Probes, Invitrogen) with MOPS running buffer (Invi-
trogen). The gel was stained with Coomassie Blue prior to
visualization. Typically, there was no indication of any measurable
presence of free OPH in the supernatant. When no more free OPH
was detected, the reaction mixture was lyophilized and kept at −20 °C.
The amount of OPH attached to F127 was determined using
standard Coomassie (Bradford) Protein Assay (Pierce, Rockford, IL).
OPH solutions with known concentrations were used as standards.
Molecular weight of the conjugate was determined using MALDI-TOF
mass spectrometer. Samples were prepared by plate spotting of the
samples, which were premixed with a sinapinic acid matrix. The
unconjugated OPH and F127 were used as reference samples.
buffer (pH 8) with the presence of CoCl and NaCl. Briefly, an
2
aqueous solution of paraoxon containing 10% v/v methanol was added
to OPH or F127-OPH with the same enzyme concentrations to give
the final concentrations of 0.075 μg/mL of OPH and 0.1 mM
paraoxon. The activity levels were measured by monitoring the release
of p-nitrophenol product spectrophotometrically at 405 nm for 10 min
using Eon BioTek Microplate Spectrophotometer. The concentration
of p-nitrophenol was determined based on the standard calibration
curve.
1
Instruments. H NMR was acquired on a Bruker ARX 500 MHz
spectrometer. Infrared spectroscopy was recorded on a Perkin-Elmer
Spectrum One Fourier Transform IR (FTIR), equipped with the ATR
accessory. The hydrodynamic diameter and size distributions were
measured with dynamic light scattering (DLS, Zetasizer Nano ZS,
Malvern Instruments). The deconvolution was accomplished using a
non-negatively constrained least-squares fitting algorithm. The
measurements were taken at 90° scattering angle. Static light scattering
was also done using the same instrument to obtain the absolute
molecular weight of aggregates. The unconjugated OPH was used as a
reference sample.
The kinetic parameters for OPH and F127-OPH were obtained by
measuring initial hydrolysis rate of different paraoxon concentrations,
keeping a constant enzyme concentration. The kinetic constants were
determined by fitting the data to Lineweaver−Burk reciprocal plot. All
assays were performed in triplicate.
For the stability assay, OPH and F127-OPH solutions with the
same OPH concentrations were both incubated at 70 °C. Samples
were taken out at different time intervals and immediately stored on
ice. The enzymatic activity was measured by the hydrolysis of
paraoxon using the aforementioned procedure. All of the assays were
performed at least in triplicate, and the average was reported.
Decontamination of Chemical Agent Resistant Coating
(CARC) Surface. Paraoxon solution in methanol (25 μL; 0.8 mM)
was added to the surface of the CARC. The methanol was allowed to
completely evaporate. Then the OPH or F127-OPH solution (200 μL;
Circular Dichroism (CD). The CD measurements were made
using the circular dichroism spectrophotometer (Aviv Biomedical, Inc.,
Lakewood, NJ) with a path length of 1 mm. Measurements were
carried out on OPH or F127-OPH in diluted HEPEs buffer solution
−1
(
pH 8), keeping the enyme concentration at 100 μg·mL . Far-UV CD
−1
spectra were measured from 190 to 240 at 1 nm intervals. Three scans
were accumulated and averaged for each spectrum after the
background of diluted blank buffer was subtracted.
enzyme concentration of 2 μg·mL in both) was gently added onto
the CARC surface. The reaction was quenched after specified times
with methanol, and the extraction was allowed to proceed for another
1
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Figure 1. (a) SDS-PAGE of (1) molecular weight markers, (2) unconjugated OPH, (3) F127-OPH (crude), and (4) F127-OPH (purified). (b)
MALDI-TOF of unconjugated OPH and F127-OPH. The increase in molecular weight after a conjugation confirmed a successful conjugation of the
OPH to F127 and suggested that there was approximately one Pluronic molecule per OPH monomer. (c) UV−vis spectra of OPH and F127-OPH.
A slight blue-shift after the conjugation was observed indicating that there may be a change of the microenvironment polarity to a more apolar
environment around the tryptophan and/or tyrosine residues of OPH. (d) FTIR spectra of OPH, F127, and F127-OPH. The new peaks at 1732 and
−1
1
689 cm (red arrows) which are attributed to the carbonyl stretching vibration and the typical bands of amide carbonyl (OCNH) group, are
clearly observed, indicating that the conjugation occurred via the formation of amide bonds between OPH and F127-COOH.
hour before the measurement. The decontamination and extraction
efficiency were determined using the ε405 = 16241 M cm when a
Pluronic via a common EDC/NHS reaction. The excess EDC/
−1
−1
NHS was removed prior to the reaction with the OPH to
prevent the reaction between the enzyme itself. The F127-NHS
was then reacted with the lysine residues on the OPH, leading
to the formation of F127-OPH conjugate. The conjugation was
performed at 4 °C and since at this condition, the end
functionalized F127 does not form micelles but remains
unaggregated (as confirmed by dynamic light scattering, Figure
S2), the conjugation reaction occurs between unaggregated
F127 and the OPH. Since each OPH monomer contains eight
1:1 mixture of methanol and HEPEs buffer was used as a medium.
RESULTS AND DISCUSSION
■
F127-OPH Conjugation. The preparation of the F127-
OPH can be achieved via the chemical conjugation between
OPH and the hydrophilic ends of the F127 block copolymer.
As depicted in Scheme 1, since the Pluronic is chemically inert,
it was first activated with succinic anhydride to introduce
22
1
carboxyl groups at the termini of F127 (F127-COOH). The H
lysine residues, multiple site conjugations may occur on the
monomer and result in an abundance of polymer on the surface
of the OPH that subsequently could deactivate the enzyme. To
suppress this effect, the pH of the reaction mixture was
NMR spectrum of the F127-COOH showed a prominent peak
at ∼2.6 ppm, corresponding to the protons of the new
methylene groups obtained from succinic anhydride (Figure
S1). The NHS ester was subsequently introduced to the
maintained at ∼7.8 to keep the −NH in the unprotonated
2
1
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Figure 2. (a, b) Transmission electron microscopy images of F127 micelles and F127-OPH micelles, respectively. The samples were stained with
phosphotungstic acid prior to the analysis. (c) Dynamic light scattering of OPH, F127, and F127-OPH micelles. The average hydrodynamic size of
the conjugate micelles was shown to increase to about 100 nm in diameter. (d) Circular dichroism spectra of OPH and F127-OPH. Both spectra
exhibit two minima at 209 and 222 nm, indicating that the conjugation did not significantly affect the secondary structure of the OPH.
form and to allow the reaction to preferably take place with the
conjugate with the hydrophobic surface patches of OPH.
Further analysis of the F127-OPH conjugate was performed
using FTIR (Figure 1d). F127 showed three major peaks at
most fully solvent exposed ε-amine groups (Lys175 and
2
2
Lys294). It is possible that the other two lysines (Lys77
and Lys339), which are partially exposed to the solvent, may
still be able to conjugate with the polymer.
−1
960, 1092, and 1279 cm , while the unconjugated OPH
−1
showed major peaks at 1551, 1625, 2944, and 3182 cm .
When comparing the FTIR spectrum of Pluronic before and
After extensive purification to remove any unconjugated
OPH by centrifugation, the conjugation was confirmed using
SDS-PAGE, showing the molecular weight of the OPH
increasing from 66 kDa to about 80−100 kDa (Figure 1a).
The results were further confirmed by MALDI-TOF (where
the molecular weight cut off was at 100 kDa). The increase in
apparent molecular mass as determined by both SDS-PAGE
and MALDI suggested that there was approximately one
Pluronic molecule per OPH monomer (Figure 1a,b). The total
amount of enzyme in F127-OPH purified product was
−1
after conjugation, the new peaks at 1732 and 1689 cm , which
are attributed to the carbonyl stretching vibration and the
typical bands of amide carbonyl (O = CNH) group, are clearly
observed, indicating that the conjugation occurred via the
formation of amide bonds between OPH and F127-COOH.
Characterization of F127-OPH Conjugates in Solution.
The F127-OPH conjugate solution includes F127-OPH
conjugate molecules as well as free F127-NHS molecules,
since a large excess of F127-NHS was used in the conjugation.
Because of the amphiphilic character of the block copolymer,
F127-NHS forms micelles (just as F127 and F127-COOH)
with polypropylene oxide core and polyethylene oxide
estimated by the Coomassie (Bradford) Protein Assay to be
1
1
62 μg·mL− indicating that nearly 80% of the enzyme that was
used in the reaction was successfully conjugated.
28
A slight blue-shift after the conjugation was observed in the
UV−vis spectrum indicating that there may be a change of the
microenvironment polarity to a more apolar environment
around the tryptophan and/or tyrosine residues of OPH
corona. The F127-OPH molecules are expected to be
included in these self-assembled structures. Transmission
electron microscopy (TEM), dynamic light scattering (DLS),
and static light scattering (SLS) were used to characterize the
morphology and size distribution of the F127-OPH conjugate
micelles. After staining with phosphotungstic acid solution,
2
6,27
(
Figure 1c).
This could be due to the interactions of the
polypropylene oxide block of the block copolymer in the
1
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spherical micelles of the pure F127 with a diameter of about 50
nm were observed (Figure 2a) in the TEM. Upon conjugation
with OPH, the size of the micelles increases to about 100 nm in
diameter, with a narrow size distribution (Figure 2b) as seen by
the TEM and also by DLS (Figure 2c).
The secondary structure of the OPH after a conjugation was
evaluated using circular dichroism (CD). Unconjugated OPH
exhibits two strong minima of α-helix content at 209 and 222
13
nm (Figure 2d). This clearly shows that the secondary
conformation of OPH is not disturbed by the attachment of the
C30 polyelectrolyte chain which has been introduced to make
the enzyme more water soluble. The F127-OPH conjugate
shows slightly less negative peaks, yet both minima at 209 and
2
22 nm still persist, indicating that the conjugation does not
significantly affect the secondary structure of the OPH.
The particle size from DLS analysis (Figure 2c) reveals
results similar to that from TEM. The DLS results for the
unconjugated OPH suggest an hydrodynamic diameter in the
range of 10 to 20 nm. As mentioned previously, the size of the
OPH dimer estimated from its crystal structure was about 12 ×
Figure 3. Static light scattering plot for F127-OPH. The intercept is
used to calculate the absolute molecular weight of the micelles and the
slope provides the second virial coefficient representing interactions
between micelles.
9
× 7 nm. One would have to add to this the contributions
kDa (twice the value observed from the MALDI in Figure 1b),
the SLS data points to a multimolecular aggregate incorporating
one or more F127-OPH conjugate molecule and a large
number of unconjugated F127 molecules. The slope of the
Debye plot is positive indicating that the second virial
from the C30 modification made on each OPH monomer and
the resulting diameter would be comparable to what is seen
from the DLS. The DLS data for the unconjugated F127
micelles indicate a diameter of about 50 nm. This is similar to
the result from the TEM and is also comparable to the DLS
data obtained for F127-COOH (Figure S2). The DLS data for
F127-OPH shows a main population of F127-OPH micelles at
about 120 nm. If one adds the size of OPH to the unconjugated
F127 micelles, one would get a diameter of about 90 nm for
F127-OPH. Clearly, the incorporation of F127-OPH also
changes the aggregation characteristics of F127, presumably by
extending the coronal region (since the PPO core must remain
compact because of the hydrophobic nature of the domain).
This is reasonable since an extended coronal region will
correspond to increased dilution of the PEO segments in the
coefficient A is positive. A positive second virial coefficient
2
implies that the particles repel each other and prefer to be
interacting with the solvent rather than with each other. This is
consistent with the use of OPH, which has strongly hydrophilic
C30 chains attached to it, with the polyelectrolyte character of
the C30 chain dominating over any surface hydrophobicity of
the native OPH.
The increased size of the F127-OPH micelle compared to
F127 micelles observed from DLS, the positive second virial
coefficient representing stronger micelle-water affinity over
micelle−micelle affinity observed from SLS, the expectation to
finding OPH in the aqueous domain because of the presence of
the polyelectrolyte like C attachment are all supportive of the
corona in order to allow the penetration of the C hydrophilic
3
0
attachment on the OPH. The DLS data for F127-OPH also
shows a small population at ∼10 nm. This may belong to some
residual free OPH (the free OPH was mostly removed by the
centrifugation method as indicated on the SDS-PAGE) since
the intensity plots of DLS can capture even very small
concentrations of scattering particles.
The size of F127-OPH micelles was also determined from
static light scattering (Figure 3). The intensity versus
concentration data can be used to estimate the absolute
molecular weight of the micelles in solution as well as the
second virial coefficient representing the nature of interactions
between the micelles. The data are plotted in the form of the
classical Debye plot given by the relation
30
concept that the OPH is located on the corona surface of the
micelle, in spite of the hydrophobic patches on the surface of
the native OPH. Indeed, native OPH shows the tendency to
rapidly aggregate (data not shown here), indicating the
attractions arising from the contacts between the surface
hydrophobic patches. We speculate that the polypropylene
oxide blocks of the F127 conjugated to OPH may protect these
hydrophobic regions, which are forced to exist in the aqueous
domain because of the water solubility provided by the C30
modification.
Enzymatic Activity of the F127-OPH Micelles. It has
been reported that the enzyme conjugation or immobilization
22
KC
P(θ) = M1 + 2A C
can result in a decrease in the enzymatic activity. In that
study, OPH was conjugated with linear and branched methyl-
2
R
θ
PEO -NHS esters (of molecular mass from 333 to 2420 Da). In
n
where K is the optical constant of the instrument, C is the
all cases, the PEGylated OPH displayed a decreased maximal
catalytic rate, though substantial activity was still retained.
Therefore, PEGylation as a method to improve stability was
found to reduce the activity. In our work, to investigate whether
the OPH is still active after conjugating to F127 micelles, the
activity of OPH and F127-OPH micelles was tested against
paraoxon, a V-type neurotoxin stimulant. Both OPH and F127-
OPH micelles have shown to catalyze the hydrolysis of
paraoxon and produce p-nitrophenol, a fluorogenic product
that can be easily detected using a spectrophotometer. Using
concentration of the solution, R is the Rayleigh ratio directly
θ
connected to the measured scattering light intensity, P is the
shape or form factor (taken to be that for a spherical particle),
M is the molecular weight, and A2 is the second virial
coefficient.
From Figure 3, we estimate the absolute molecular weight to
be approximately 1500 kDa. Recognizing that F127 has a
molecular weight of about 13 kDa and the molecular weight of
F127-OPH conjugate in the dimeric protein form is about 170
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OPH solution as a blank spectrum, the peak at 280 nm was
attributed to the paraoxon molecules. The addition of OPH
leads to a decrease of the peak at 280 nm and a simultaneous
increase of the peak at 405 nm, which is attributed to the p-
nitrophenol molecules, with an isosbestic point at 310 nm
micelles to inactivate dried paraoxon on a flat surface, a solution
of paraoxon in methanol was added onto a polystyrene
substrate, and the solvent was allowed to completely evaporate
at room temperature. Once the thin film of paraoxon was
created, the OPH or F127-OPH solution at the same enzyme
concentration was added directly to the dried paraoxon film,
and the hydrolytic activity was measured. As shown in Figure
4a, it was found that F127-OPH exhibits a ∼20% increase in
activity when the dried paraoxon coating on a solid surface was
used at 25 °C. When the temperature was raised to 37 °C, a
(
Figure S3). Similar results were observed with a higher
hydrolytic rate when F127-OPH micelles were used, implying
that the enzymatic activity after conjugating was preserved. The
unmodified OPH and F127-OPH micelles displayed Michae-
lis−Menten kinetics (Table 1), with kcat values of the conjugate
1
0% increase in activity was observed in comparison to that at
Table 1. Kinetic Parameters of Unmodified OPH and F127-
OPH Micelles
25 °C. While the activity of the unmodified OPH was shown to
be similar under both liquid and dried paraoxon, the F127-
OPH exhibits about ∼1.5-fold enhancement in activity. This
observation clearly suggests the great potential of using F127-
OPH micelles as an effective candidate for decontamination
agents.
a
k /K (s− M−1
1
)
sample
OPH
F127-OPH
Km (mM)
cat
m
0.0274 ± 0.007
0.0270 ± 0.003
1.02E6 ± 2.2E5
1.49E6 ± 1.3E5
a
Values represent means of triplicate experimental data, as indicated
Stability of the F127-OPH Micelles. To investigate the
thermal stability of the F127-OPH conjugates at elevated
temperature, the samples along with the unconjugated OPH
were incubated at 70 °C (a typical condition used in the
literature to allow OPH or other proteins to be denatured and
by the standard deviation (mean ± st dev).
micelles being about 1.5 times higher than that of the
unmodified OPH (Figure S4). Since paraoxon (0.1 mM) was
relatively soluble in an aqueous solution, no change in the Km
value was observed. However, the increase in kcat clearly
suggests that F127-OPH micelles can be used as effective
decontamination agents.
13
aggregated ), and the activity was measured at different time
intervals. As depicted in Figure 4b, OPH completely lost its
activity after 10 min of the incubation time, whereas the activity
of the F127-OPH was retained to about 37%. A significant
decrease in activity for F127-OPH was observed during the first
We rationalized that the increase in the catalytic activity of
the OPH after conjugation maybe attributed to multiple
1
0 min of the incubation time, then the activity only slowly
29
dropped. After 120 min of the incubation time, the activity of
the F127-OPH still remained at about 15%.
reasons: (i) the local concentration of enzyme is increased;
ii) the substrate, organophosphate agent, can be solubilized in
the micelle core increasing its proximity to the enzyme; (iii) the
(
The enhanced freeze−thaw stability was demonstrated in
Figure 4c. After five freeze−thaw cycles, the F127-OPH retains
more than 80% activity while the unconjugated OPH showed
more than 50% decrease in activity after the second cycle and
completely lost its activity after the fourth cycle. The half-life
data for OPH and F127-OPH under different storage
conditions are summarized in Table 2.
3
0
crowding effect due to higher macromolecular concentration
in the proximity of the enzyme; and (iv) the protection of the
active region of the enzyme by the polypropylene oxide block
of the F127 conjugated to the enzyme. Reason (i) may not be
important because the overall concentration of the enzyme
used in the activity studies was kept very small. Reason (ii) may
not be important in this case, because of the relatively
appreciable aqueous solubility of the substrate paraoxon. The
crowding effect could play a role, but such crowding effect,
which would have been relevant even for the case of PEGylated
We believe that the conjugation between OPH and F127 and
the presence of micelles might prevent conformational
transitions of OPH at high temperature or during the freeze−
31
thaw cycles as the micelles offers benign microenvironment to
assist the conformational preservation of OPH; hence, the
F127-OPH micelles can maintain the thermal stability of the
OPH against the unfolding transition at the transition
temperature, comparing to that of the unconjugated enzyme
in the solution. In addition, it was previously shown that the
change in temperature induces the aggregation of the enzyme,
22
OPH discussed above, resulted in a decrease in the enzyme
activity rather than an increase. Therefore, we speculate that
reason (iv) based on possible interactions of the hydrophobic
polypropylene oxide block of the F127 molecule conjugated to
OPH with the hydrophobic surface domains on the OPH
surface that are close to ligand pockets may be responsible for
providing some enhancement in activity. Compared to free
F127 molecules, the conjugated F127 molecules may allow for
this interaction between PPO and OPH to occur more
effectively. Support for such interactions was provided by the
observed spectral shift shown in Figure 1c.
32,33
primarily due to the loss of tertiary structure of enzyme.
By
introducing the use of conjugate micelles, the thermal
inactivation of OPH resulting from the aggregation effect
could be minimized.
We further evaluated the stability of OPH at low
concentrations. As is well-known, many enzymes are prone to
degradation at a low concentration. The activity drop of
unconjugated OPH (0.5 ug/mL) was observed within the first
two hours when storing at 4 and 25 °C, and a complete loss of
original activity of OPH was observed after 72 h of storage time
(Figure 4d,e). Upon the conjugation with F127, the activity of
the conjugated OPH remained unchanged throughout 3
months under the same conditions, indicating that the micelles
can prolong the shelf life of the OPH (Figure 4f). To increase
the long-term stability and ease of handling and transporting,
the freeze-drying technique was applied to the F127-OPH
Since OPH can potentially be used as prophylactic against
acute OP poisoning, we evaluated the activity of the F127-
OPH-micelles at 25 and 37 °C, using paraoxon in a liquid
formulation (shown in Figure 4a with marking “l”) and also
using dried paraoxon on a surface (shown in Figure 4a with the
marking “f”). Against the liquid formulation, in comparison to
the unconjugated OPH, the enzymatic activity against paraoxon
was shown to be ∼30% higher when F127-OPH was used.
When the temperature was raised to 37 °C, the activity of
F127-OPH increased while no significant change in the activity
of OPH was observed. To measure the ability of F127-OPH
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Figure 4. Relative activities of OPH and F127-OPH (a) in the presence of liquid and dried paraoxon at 25 and 37 °C (*p-value < 0.05 vs F127-OPH
control), (b) after incubation at 70 °C, (c) after five freeze−thaw cycles, (d) after storing at 4 °C in a solution form, (e) after storing at 25 °C in a
solution form, and (f) after lyophilization and a 3-month storage. For all the data, 0.075 ug/mL enzyme concentration and 0.1 mM paraoxon were
used.
micelles, and the OPH activity of the powder was determined
periodically. As illustrated in Figure 4f, the unconjugated OPH
suffers severe activity drops after freeze-drying with less than
cost effectiveness, sample handling, and other practical
applications.
While many OP compounds are well solubilized in various
organic solvents, the OPH molecule is inactivated in the
presence of organic solvents. Here, the enhancement in the
OPH stability with the presence of organic solvents was
demonstrated by exposing both unconjugated OPH and F127-
OPH conjugates in different organic/aqueous volume ratios.
Methanol and dimethylsulfoxide (DMSO) were employed as
4
5% of its original activity, and the activity slowly drops
through the 3 months while storing at 4 °C in a dry
formulation. In contrast, the activity of freeze-dried F127-OPH
was virtually unchanged throughout 3 months. A great long-
term stability of this formulation offers promising benefits in
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Table 2. Half-Life (h) of OPH and F127-OPH under
Different Storage Conditions
to disrupt the intramolecular interactions, such as hydrogen
bonds of the proteins.
a
34
Decontamination of Chemical Agent Resistant Coat-
ing (CARC) Surface. The use of F127-OPH as an effective
decontamination agent was demonstrated on the chemical
agent resistant coating (CARC) surface (Figure 6a). Briefly, the
CARC surface was first contaminated with a paraxon solution
in methanol. After the complete evaporation of methanol, the
unconjugated OPH or F127-OPH solution was added to the
contaminated surface, and the decontamination efficiency was
determined periodically with a methanol quenching and
extraction. Within 15 min of decontamination, the OPH and
F127-OPH exhibited ∼30 and 60% decontamination efficiency,
respectively, and reached up to 60 and 90% after 1 h (Figure
storage condition
OPH
F127-OPH
incubation at 70 °C
°C in a solution
5 °C in a solution
lyophilization
0.05
10.6
2.2
0.1
nd
nd
nd
nd
nd
4
2
626.9
4.7
1
1
0% (v/v) methanol
0% (v/v) DMSO
5.5
a
“
nd” indicates that the half-life was not determined because the
enzyme activity remained unaffected over the long duration of the
experiments and the experiments were not extended to allow the
determination of a half-life.
6
b,c). Although the contaminated CARC surfaces can be
polar protic and aprotic solvents, respectively. As depicted in
Figure 5, although the activities of both OPH and F127-OPH
decrease as the volumetric fractions of the organic solvents
increase, the F127-OPH retained higher enzymatic activity in
every solvent ratio in the presence of either solvent. In addition,
the F127-OPH was shown to be stable in the presence of 10%
treated with the unconjugated OPH solution, the unconjugated
enzyme itself is not stable enough for a wide-area or long-term
decontamination, since the stability of the unconjugated OPH
was shown to decrease at room temperature. The control
studies using 1:1 HEPEs/methanol showed ∼96% extraction of
paraoxon while no decontamination was observed (see
Supporting Information, Table S1), indicating that the solvents
are capable of removing the paraoxon from the CARC surface
but the contaminant would remain as a toxic waste in the
absence of enzymatic reactive decontamination. The relatively
(v/v) organic solvent even after 25-day storage (Figure 5c,d).
We believe that the presence of the micelles and also the
possible protection of OPH afforded by the PPO block of the
F127 block copolymer could prevent some parts of the OPH
from being denatured by the organic solvents, which are known
Figure 5. Relative activities of OPH and F127-OPH in the presence of different volumetric fraction of methanol (a) and DMSO (b). The long-term
stability of the OPH and F127-OPH in the presence of 10% (v/v) methanol (c) and DMSO (d). The data were normalized to F127-OPH in HEPEs.
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Figure 6. (a) Schematic representation of the decontamination procedures on the CARC surface. The yellow color is that of p-nitrophenol product
resulting from the hydrolysis of paraoxon. (b) CARC surfaces after treating with OPH, F127-OPH, and HEPEs buffer for 30 min. (c) The
decontamination and extraction efficiency of p-nitrophenol after a surface treatment with OPH and F127-OPH.
high decontamination efficiency observed with F127-OPH
clearly suggests a great potential of using the solution of
conjugate micelles as an effective decontaminant formulation.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
CONCLUSION
■
The authors gratefully acknowledge the funding from the
National Research Council, CBD Research Associateship
Award, and Defense Threat Reduction Agency (DTRA). We
are grateful to Dr. M. Kim (MIT), Dr. B. Olsen (MIT), and Dr.
Y. Xia (Stanford) for kindly providing us the Cys 30-OPH and
their helpful discussions. We thank Jason Soares and Steven
Arcidiacono (NSRDEC) for help with SDS-PAGE, John
Walker (NSRDEC) for help with Circular Dichroism, and
David Ziegler (NSRDEC) for help with the TEM.
In summary, we have developed a facile approach to prepare
robust and highly active F127-OPH conjugate micelles. The
OPH in conjugate micelles exhibited reasonable improvement
in activity and significantly enhanced stability, with elevated
heat, multiple freeze−thaw cycles, and different substrate
conditions. We believe that the F127 conjugation and the
formation of micelles may provide spatial confinement to the
OPH and promote a favorable OPH conformation, thereby
enhancing the OPH stability. Our approach may open up a way
to incorporate more functional materials onto the micelles as
the enzyme activity is still preserved. As the F127-OPH
conjugate micelle is fairly easy to prepare, inexpensive,
nontoxic, and biodegradable, our system holds promise in the
development of enzyme-based protection and decontaminant
systems for military and civilian applications.
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