Combined effect of organophosphorus hydrolase and grime on the reactivation rate of diethylphosphoryl-acetylcholinesterase conjugates

Article abstract of DOI:10.1016/S0006-2952(97)00430-9

Reactivation of inhibited acetylcholinesterase (AChE) is essential for rapid recovery after organophosphate (OP) poisoning. However, following administration of an oxime reactivator, such as pralidoxime mesylate (P2S), in patients poisoned with certain diethylphosphorothioate pesticides, no reactivation is observed, presumably due to reinhibition by circulating anti-cholinesterase OPs. Pretreatment alone with organophosphorus hydrolases (OPH) that are capable of rapidly hydrolyzing OPs was demonstrated, in animals, to confer significant protection against OP toxicity. One strategy to augment the potentially therapeutic scope of OPHs is 3 combined post exposure treatment consisting of a drug(s) commonly used against OP toxicity and a suitable hydrolase. In this study, we examined the in vitro ability of OPH from Pseudomonas sp. (OPHps) to prevent reinhibition of P2S reactivated AChE by excess OPs. The kinetic parameters of the reactivation of a series of diethylphosphoryl AChE (DEP-AChE) conjugates, obtained by the use of various diethylphosphates, were (determined and compared with the rates of reactivation in the presence of OPHps, with and without the OP inhibitors in the reactivation medium. Extrapolation of the in vitro results to in vivo conditions suggests that an OPHps concentration as low as 1 μg/mL blond would result in a 100 fold decrease in the concentration of circulating anti AChE pesticides within less than one blood circulation time, thereby minimizing reinhibition of the reactivated enzyme. Thus, for DEP based pesticidcs, the combination of P2S-OPH treatment can significantly improve clinical recovery after OP intoxication. In addition, it is shown here for the first time that an OPH can effectively hydrolyze quaternary ammonium containing OPs. This indicates that hydrolysis of phosyhorylated oximes, toxic side products of oxime treatment, may also be accelerated by OPHs.

Full text of DOI:10.1016/S0006-2952(97)00430-9

Biochemical Pharmacology, Vol. 55, pp. 159–168, 1998.
1998 Elsevier Science Inc. All rights reserved.
ISSN 0006-2952/98/$19.00 ϩ 0.00
©
PII S0006-2952(97)00430-9
Combined Effect of Organophosphorus Hydrolase
and Oxime on the Reactivation Rate of
Diethylphosphoryl-Acetylcholinesterase Conjugates
Yacov Ashani,*† Haim Leader,* Nathan Rothschild‡ and Carlos Dosoretz‡
ISRAEL INSTITUTE FOR BIOLOGICAL RESEARCH, NESS-ZIONA, ISRAEL; AND ‡MIGAL-GALILEE TECHNOLOGICAL
CENTER, KIRYAT SHMONA, ISRAEL
*
ABSTRACT. Reactivation of inhibited acetylcholinesterase (AChE) is essential for rapid recovery after
organophosphate (OP) poisoning. However, following administration of an oxime reactivator, such as
pralidoxime mesylate (P2S), in patients poisoned with certain diethylphosphorothioate pesticides, no reactiva-
tion is observed, presumably due to reinhibition by circulating anti-cholinesterase OPs. Pretreatment alone with
organophosphorus hydrolases (OPH) that are capable of rapidly hydrolyzing OPs was demonstrated, in animals,
to confer significant protection against OP toxicity. One strategy to augment the potentially therapeutic scope
of OPHs is a combined post-exposure treatment consisting of a drug(s) commonly used against OP toxicity and
a suitable hydrolase. In this study, we examined the in vitro ability of OPH from Pseudomonas sp. (OPHps) to
prevent reinhibition of P2S-reactivated AChE by excess OPs. The kinetic parameters of the reactivation of a
series of diethylphosphoryl-AChE (DEPOAChE) conjugates, obtained by the use of various diethylphosphates,
were determined and compared with the rates of reactivation in the presence of OPHps, with and without the
OP inhibitors in the reactivation medium. Extrapolation of the in vitro results to in vivo conditions suggests that
an OPHps concentration as low as 1 ␮g/mL blood would result in a 100-fold decrease in the concentration of
circulating anti-AChE pesticides within less than one blood-circulation time, thereby minimizing reinhibition
of the reactivated enzyme. Thus, for DEP-based pesticides, the combination of P2S–OPH treatment can
significantly improve clinical recovery after OP intoxication. In addition, it is shown here for the first time that
an OPH can effectively hydrolyze quaternary ammonium-containing OPs. This indicates that hydrolysis of
phosphorylated oximes, toxic side products of oxime treatment, may also be accelerated by OPHs. BIOCHEM
PHARMACOL 55;2:159–168, 1998. © 1998 Elsevier Science Inc.
KEY WORDS. organophosphates; inhibition; reactivation; acetylcholinesterase; phosphotriesterases; detoxifi-
cation
The toxicity of OP§ pesticides and nerve agents is attrib-
uted primarily to the inhibition of AChE (EC 3.1.1.7) at
important physiological sites. OPHs (EC 3.1.8.1) from
Pseudomonas sp. (OPHps) and Pseudomonas diminuta MG
atropine treatment [7]. The antidotal efficacy of OPHs
depends on the affinity of the OP substrates (K ), and the
m
catalytic efficiency (kcat) of the enzymic-enhanced hydro-
lysis (Fig. 1) [1–8]. Calculations based on the results of
protection experiments established a correlation between
the kinetic constants of the enzymic hydrolysis of OPs and
the blood–hydrolase concentration needed to rapidly hy-
drolyze a variety of potent anti-ChE OPs [1, 2, 4, 5]. These
observations, taken with the reported wide range of values
of the affinity and the kinetic constants, indicate that in
order to confer a meaningful protection with low doses of
an OPH against broad types of OPs, more than one kind of
enzyme may be required. Pretreatment with ChEs alone has
also been shown to protect animals against lethal doses of
highly toxic OPs [9–12]. However, the stoichiometry of
scavenging by ChEs is limited to a single turnover [11–13].
Thus, OPHs have the advantage of being able to detoxify a
large amount of OPs by a relatively small dose of enzyme in
less than one blood-circulation time.
(OPHpd) were shown to confer significant protection
against OP toxicity when given alone [1–5], in combina-
tion with atropine [6], or in conjunction with a carbamate–
†
Corresponding author: Dr. Yacov Ashani, Israel Institute for Biological
Research, P.O. Box 19, Ness-Ziona, Israel. Tel. 972-8-9381455; FAX
72-8-9401094; e-mail: yashani@netvision.net.il.
Abbreviations: AChE, acetylcholinesterase; ChE, cholinesterase;
9
§
DECP, O,O-diethyl chlorophosphate; DEFP, O,O-diethyl flurophosphate;
DEP, diethylphosphoryl; DEPQ, 7-(O,O-diethylphosphinyloxy)-1-meth-
ylquinulinium methylsulfate; HuBChE, human serum butyrylcholinester-
ase; MEPQ, 7-(methylethoxyphosphinyloxy)-1-methylquinulinium io-
dide; OP, organophosphate, organophosphorus; OPH, organophosphorus
hydrolase; OPHps, OPH from Pseudomonas sp.; P2S, 2-(hydroximinom-
ethyl)-1-methylpyridinium methansulfonate; 2-PAM, iodide salt of P2S;
POX, phosphoryl oxime; phospholine iodide, S-[2-(N,N,N-trimethyl-
ammonio)ethyl]-O,O-diethylphosphorothiolate iodide; rMoAChE, re-
combinant mouse AChE; and TEPP, O,O,OЈ,OЈ-tetraethyl pyrophos-
phate.
One approach to amplify the potentially therapeutic
scope of OPHs is a combined treatment consisting of a
Received 21 April 1997; accepted 2 July 1997.

1
60
Y. Ashani et al.
FIG. 1. Chemical pathways of the OPH-cata-
lyzed hydrolysis of diethylphosphates and the
inhibition and P2S-induced reactivation of
DEPOAChE.
commonly used drug against OP toxicity and a suitable OP
hydrolase. The accepted approach to prevent and alleviate
OP toxicity is based on pretreatment with pyridostigmine,
a carbamate capable of partial and temporary masking of
AChE, followed by administration of a cholinergic antag-
onist, such as atropine, and an oxime reactivator of the
inhibited enzyme [14]. Combined treatment with a reacti-
vator and OPH offers a promising approach with dual
therapeutic action. First, it has been shown that the
continuous and slow release of anti-ChE metabolites of
pesticides, such as parathion and related phosphorothio-
ates, into the blood renders standard therapy management
quite difficult [15]. Willems et al. [15] demonstrated the
dependence of the therapeutic effect of the oxime P2S
scavengers of OPs, produce potential complications in the
interpretation of the results from in vivo experiments. We
therefore embarked first on the in vitro characterization of
the efficacy of an oxime–OPH combination. The kinetic
parameters of P2S-induced reactivation of O,O-diethyl-
phosphorylated AChE (DEPOAChE), obtained by using a
variety of O,O-diethylphosphates, were determined and
compared with the rates of reactivation of the same
conjugates in the presence of OPHps, with and without the
OP inhibitors in the reactivation medium. Extrapolation of
the in vitro results to in vivo conditions suggests that the
proposed combination can significantly improve clinical
recovery after intoxication with certain OPs. We also
present for the first time evidence that an OPH can
effectively hydrolyze potent ChE inhibitors that contain a
quaternary ammonium moiety. The observation that
charged OPs are substrates of an OP hydrolase indicates
that the detoxification of the structurally related POX may
also be accelerated by OPHs.
(pralidoxime mesylate) on the persistence of anti-ChEs in
the blood of patients intoxicated with OP pesticides.
Reactivation was not observed when parathion and methyl
parathion blood levels exceeded 30 ng/mL. Second, one of
the drawbacks of oxime therapy is the possible accumula-
tion of POXs, both inside and outside the active site gorge.
POXs are substantially more toxic than the parent OPs
MATERIALS AND METHODS
Materials
(Fig. 1) [16–19]. The origin of the POX is 2-fold: (i) direct
reaction between the oxime and OPs (k , Fig. 1); and (ii)
p
a possible intermediate formed during the reactivation of
phosphorylated ChEs. Therapy with OPH may benefit from
substantial reduction in the concentration of these toxic
by-products.
Several reactions can occur simultaneously among OPs,
ChEs, oxime reactivators, and an OP hydrolase (Fig. 1).
These reactions, together with the activity of endogenous
MEPQ [20], DEPQ [21], and TEPP [22] were prepared as
described elsewhere [20–22]. DEFP was prepared by fluori-
nation of DECP as described by Saunders and Stacey [23].
DECP was obtained from the Aldrich Chemical Co. and
distilled before use. O,O-Diethyl p-nitrophenyl phosphate
(paraoxon) was purchased from the Sigma Chemical Co.
and used as obtained. The structure and the purity (Ͼ96%)

Reactivation by Oxime and OPH
161
1
31
of all OPs were confirmed by H NMR and P NMR
spectroscopy and gas chromatography. TEPP contained less
than 5% O,O,O-triethyl phosphate. Phospholine iodide
was obtained from Ayerst Laboratories. P2S and its iodide
salt, 2-PAM, were purchased from Sigma.
enzyme was dialyzed six times against 2 L of 10 mM Tris,
pH 7.8, over 4 days at 5°.
Reactivation of DEPOAChE and DEP-HuBChE
OPH from Pseudomonas sp. was purified as previously
described [24]. Briefly, the enzyme was obtained by precip-
itation of cell-free extract with ammonium sulfate (50%
saturation) and fractionated on DEAE-cellulose and Seph-
adex G-100 columns. These purification steps resulted in a
single protein band with a molecular mass of 35 kDa, as
analyzed by SDS–PAGE [24]. OPHps was stored at Ϫ20° in
The pH of the inhibited enzymes was readjusted to 7.8 prior
to the initiation of the reactivation. Stock solutions of 1–50
mM P2S or 2-PAM in deionized water adjusted to pH 7.8
were diluted into the inhibited enzyme that was preincu-
bated at 29°, to a final concentration of 0.01 to 0.5 mM.
The rate of reactivation was followed at 29° by diluting the
reactivation medium 150 times in the assay cuvette. Non-
specific hydrolysis of the substrate was subtracted. Reacti-
vation was practically completed within 6 hr for DEP-
AChE and 16 hr for DEP-HuBChE. The reactivation in the
presence of OPH, with or without OPs (at 1–3 ␮M), was
carried out as described above. The OP was first added to
the DEPOAChE in the reactivation medium, followed by
simultaneous dilution of stock solutions of oxime and OPH,
both in 10 mM Tris, pH 7.8.
5
0 mM Tris-50% glycerol buffer containing 1% Triton
X-100, pH 8.0. Enzyme was diluted 100- to 1000-fold into
the reactivation medium. Purified wild-type rMoAChE
(ϳ14 nmol/mg) was donated by Prof. P. Taylor and Dr. Z.
Radic from the Department of Pharmacology at the Uni-
versity of California, La Jolla. HuBChE (ϳ12 nmol/mg)
was purified by procainamide-Sepharose 4B gel affinity
chromatography [25]. Active-site concentrations of
rMoAChE and HuBChE were determined by titrations
with both MEPQ [12, 20] and DEPQ [21].
Hydrolysis of DEPQ and MEPQ by OPH
The equilibrium and kinetic constants of OPHps-induced
hydrolysis of DEPQ were determined by measuring the
initial velocity of the release of 7-hydroxyquinulinium
cation (7-HQ) at 406 nm, in 50 mM phosphate buffer, pH
8.0, at 25° [21]. The same approach was used to monitor the
hydrolysis of OPHps-catalyzed hydrolysis of MEPQ [20].
Enzyme Assays
The release of p-nitrophenol (400 nm) from 1 mM para-
oxon was used to measure OPH activity in 50 mM phos-
phate buffer, pH 8.0, at 25°. The specific activity was
approximately 1000 units/mg. OPH-catalyzed hydrolysis of
phospholine iodide was monitored by using Ellman’s re-
agent, 5,5Ј-dithio-bis-2 nitrobenzoic acid (DTNB), as pre-
viously described [26]. The activity of AChE and HuBChE
was determined by the method of Ellman et al. [27], using
Determination of the Inhibition Rate Constants
of rMoAChE
0
.5 mM acetylthiocholine iodide and butyrylthiocholine
Standard stock solutions of OPs (0.1 to 50 ␮M) were freshly
prepared in deionized water and diluted at time zero into
AChE solution (0.2 to 2 nM) in 10 mM Tris, pH 7.8, 29°.
The final concentration of the inhibitors was sufficiently
high to establish pseudo-first-order reaction conditions. At
selected time intervals, aliquots were diluted into the
Ellman’s mixture for measurements of residual activity.
Data for the time–course of the inhibition were analyzed by
nonlinear regression analysis using a monoexponential
decay equation.
iodide, respectively. Assays were carried out in 50 mM
phosphate buffer, pH 8.0, at 25°.
Preparation of DEPOAChE and
DEP-HuBChE Conjugates
Two to ten microliters of a concentrated solution of
paraoxon, DEFP, and TEPP (2 ␮M each, in isooctane) was
added by stirring into 3 mL AChE (1–3 nM) in 10 mM
Tris, containing 0.05% BSA, pH 7.8, at 25°. DEPQ and
phospholine were diluted from a stock solution in deionized
water to a final concentration of 3 and 10 nM, respectively.
DECP-inhibited enzyme was obtained by dropwise addition
of 10 mM DECP in isooctane over a period of 20 min (total
of 10 ␮L). The decrease in enzymic activity was monitored
until inhibition was almost total. To remove traces of
unreacted OPs, the inhibited enzyme was dialyzed two
times against 2 L of 10 mM Tris, pH 7.8, for 48 hr at 5°.
A stock solution of approximately 50 ␮M HuBChE in
3
1
NMR Spectrometry of TEPP
Spectra were recorded with a GN 300WB NMR instrument
3
1
(General Electric) at 121.65 MHz ( P) for 14 mM TEPP in
0.1 M Tris, pH 7.8, at room temperature. A D O ampule
2
immersed in the tested solution served for field frequency
lock. Spectral data were accumulated at 30° pulse width,
8 kHz spectral width, and 2-sec pulse delay between
consecutive scans. One hundred transients were accumu-
3
1
1
0 mM Tris, pH 7.8, containing 0.05% BSA was incu-
lated for each run. P NMR chemical shifts were assigned
to external 1% trimethyl phosphate in C D that was set to
bated for 15 min with 0.4 mM paraoxon. To ascertain
removal of excess paraoxon, 1 mL of the inhibited
6
6
0 ppm.

1
62
Y. Ashani et al.
TABLE 1. Biochemical parameters of the reactivation of (C H O) P(O)X-inhibited AChE by 2-(hydroximinomethyl)-1-
2
5
2
methylpyridinium cation*
KOX
†
k؅
†
؊1kr†
؊1
max
(min
؊
1
OP
X
(mM)
)
(M min
)
DECP
Cl
0.142 (Ϯ0.017)
0.206 (Ϯ0.056)
0.157 (Ϯ0.038)
0.136 (Ϯ0.038)
0.254 (Ϯ0.051)
0.189 (Ϯ0.030)
0.222 (Ϯ0.006)
0.250 (Ϯ0.036)
0.211 (Ϯ0.020)
0.216 (Ϯ0.024)
0.278 (Ϯ0.027)
0.263 (Ϯ0.034)
1563
1213
1344
1588
1094
1391
DEFP
F
Paraoxon
TEPP
Phospholine§
DEPQ
p-Nitrophenoxy
OP(O)(C H )
SCH CH N (CH )
7-Oxyquinulinium-1-methyl
2
5 2
ϩ
2 2 3 3
࿣
*
Obtained by use of either P2S or 2-PAM (10 mM Tris, pH 7.8, 29°).
†
‡
Obtained from nonlinear regression analysis in accordance with Equation 1. Figures in parentheses are SEM (N ϭ 5–13).
kr ϭ kЈmax/KOX
.
§
Iodide salt.
Methylsulfate salt.
࿣
RESULTS
crepancies are likely to arise from differences in either the
experimental protocols, the purity of the enzyme used, or
the source of the AChEs.
Reactivation of Diethylphosphoryl-AChE Conjugates
The main goal of this study was to define the capacity of
OPHps to prevent reinhibition of P2S-reactivated enzyme
by a variety of (C H O) P(O)X (Fig. 1; Table 1). In several
Reactivation depends on the affinity of the oxime to the
inhibited enzyme (K ) and the subsequent nucleophilic
ox
2
5
2
displacement (kЈmax), as described below in Scheme 1:
cases, the inhibited enzyme was spiked with a different
diethylphosphate than the OP that was used to obtain the
DEPOAChE conjugate. Thus, it was important first to
ascertain that all DEPOAChEs display the same reactiva-
tion characteristics. The data of the time–course of the
reactivation were best fitted by a single exponential kinetic
equation (upper curve, Fig. 2A). The homogenous class of
reactivatable conjugates is attributed to the extensive
dialysis that removes the excess of unreacted inhibitors, and
to the low molar concentration of the inhibited enzyme in
the reactivation medium (1–3 nM) that minimizes reinhi-
bition by POX. Results from a recent study [28] showed a
slight deviation from monoexponential kinetics of the
reactivation of DEPOAChE that was obtained by the use
of paraoxon. In addition, earlier investigations with bovine
AChE demonstrated significant deviations from first-order
reactivation profiles of DEPOAChE [16, 29]. These dis-
SCHEME 1. Oxime-induced reactivation.
The relationship between the apparent first-order reac-
tivation constant (kobs) at oxime concentration [OX], and
the reactivation parameters Kox and kЈmax are given in
Equation 1:
Ϫ1
k
obs ϭ kЈmax͑1 ϩ Kox/[OX͔)
(1)
Assuming that the dissociation of the complex [DEP-
AChE][OX] (kϪ1) is appreciably faster than kЈmax, Kox in
FIG. 2. Reactivation of paraoxon-inhibited AChE. (A) Time–course of reactivation of paraoxon-inhibited AChE by 0.2 mM P2S in
the presence of increasing concentrations of paraoxon. Key: (■) no inhibitor; (Ç) 0.068 ␮M; (ç) 0.4 ␮M; and (F) 2.0 ␮M. (B) Plot
of kobs versus [oxime] for reactivation of paraoxon-inhibited AChE by P2S (E) and its iodide salt, 2-PAM (■).

Reactivation by Oxime and OPH
163
Equation 1 is approximated by the dissociation constant of
the complex [DEPOAChE][OX] (kϪ1/kϩ1). The bimolec-
TABLE 2. Rate constants of the approach to steady-state of
DEPOAChE reactivation in the presence of paraoxon and
P2S at 0.2 mM
ular rate constant of the reactivation, k , is the ratio
r
kЈmax/K . The constants K and kЈmax were resolved by
ox
ox
Paraoxon
k؅obs,*
k؅ ؉ k؅ †
Max. reactivation
(units/mL)
r
i
)
nonlinear regression analysis of the plots of kobs versus [OX]
as illustrated for paraoxon-inhibited AChE (Fig. 2B), and
are summarized in Table 1. As expected, P2S and 2-PAM,
which consist of the same cation nucleophile, gave the
same results. Table 1 shows that similar Kox and kЈobs
control the overall rate of reactivation of DEPOAChE
conjugates, suggesting that all the diethylphosphates used
produced the same reactivatable enzyme, irrespective of the
leaving group X. This observation permitted the use of a
variety of diethylphosphates listed in Table 1, to spike any
of the DEPOAChE conjugates, because the reinhibited
enzyme is expected to undergo reactivation at the same rate
as the starting DEPOAChE.
؊1
؊1
(␮M)
(min
)
(min
0
0
0
0.131
0.197
0.210
0.244
0.670
1.10
0.86
0.65
0.51
0.17
.068
.17
0.152
0.187
0.233
0.791
0.40
2.0
*
Observed reactivation rate constants. Calculated from Equation 3.
Ϫ1
† Predicted reactivation rate constants. kЈr ϭ 0.131 min . kЈ (corrected) was
calculated according to Equation 4.
i
t, and compared with the predicted value, which is the sum
of kЈ
ϩ kЈ
. The constant kЈ
was calculated from Equation
r
i
r
1. Calculation of kЈ
was based on the following consider-
i
ation: P2S is a quaternary ligand that binds to AChE [28].
This reversible complexation is expected to slow the
Reactivation of DEPOAChE in the Presence
inhibition. The fraction of the enzyme that is unbound to
of (C H O) P(O)X
Ϫ1
2
5
2
the oxime is (1 ϩ [OX]/KЈox) . Thus, kЈ was corrected as
i
Reactivation of 1–3 nM DEPOAChE that was spiked with
inhibitory concentrations of diethylphosphates proceeded
to a level that differed appreciably from that obtained in
the absence of inhibitor. This is illustrated in Fig. 2A for
paraoxon-inhibited AChE that was spiked with increasing
concentrations of paraoxon. Similar results were obtained
for DEPOAChE conjugates obtained from DEPC and
TEPP, and spiked with paraoxon (not shown). It should be
noted that for initial concentrations of 1–3 nM
DEPOAChE, it is assumed that possible interference of
POX (Fig. 1) is negligible.
follows:
Ϫ1
kЈ ϭ k ͓I͔͑1 ϩ ͓OX͔/KЈ ͒
i
(4)
i
ox
6
Ϫ1
Ϫ1
where k and KЈ were set at 1.65 ϫ 10 M min and
i
ox
0
.05 mM, respectively [28]. [OX] was at 0.2 mM. Results are
summarized in Table 2. The close agreement between the
observed and calculated rate constants supports the kinetic
assumptions that underlie the mechanism of the approach
to steady-state in the presence of inhibitor in the reactiva-
tion medium.
The data points of the reactivation time–course in the
presence of inhibitor were best fitted to a monoexponential
equation, in accord with a first-order opposing reaction.
Thus, the rate of approach to equilibrium (i.e. steady-state),
derived for Scheme 2, is given by Equation 2:
Effect of OPHps on the Reactivation of DEPOAChE in
the Presence of (C H O) P(O)X
2
5
2
OPHps alone did not affect the rate of reactivation of
DEPOAChE by P2S, even when the concentration of the
enzyme was increased to 4 ␮g/mL. However, OPHps signif-
icantly augmented, in a concentration-dependent manner,
the recovery of enzyme activity of DEPOAChE conjugates
that were spiked with paraoxon, DEFP, TEPP, and DEPQ
(
Fig. 3, A–D). This was attributed to the rapid hydrolysis of
SCHEME 2. Kinetic scheme of opposing reactivation–inhibition
the OPs. The amount of OPHps required to decrease the
concentration of 2 ␮M DEPQ to a level that did not affect
AChE activity (k ϭ 7.46 ϫ 10 M min ; Table 3) was
greater than the amount of OPHps needed to hydrolyze
2
1
min ), at least 10- and 2.5-fold, respectively. Our calcu-
lations show (Table 3) that kcat/Km was sufficient to
decrease 1000-fold the initial concentration of 2 ␮M
reactions.
8
Ϫ1
Ϫ1
i
Ϫ1
kЈobs ϭ kЈ ϩ kЈ ϭ t ln ͓E /͑E Ϫ E ͔͒
(2)
(3)
r
i
eq
eq
t
␮M of the less potent inhibitors, paraoxon (k ϭ 1.65 ϫ
i
6
Ϫ1
Ϫ1
5
Ϫ1
and after rearrangement
0
M
min ) and DEFP (k ϭ 3.82 ϫ 10
M
i
Ϫ1
ϪkЈobst
E /E ϭ 1 Ϫ e
t
eq
The constants kЈ and kЈ are the apparent first-order
reactivation and inhibition rate constants, respectively. Eeq
paraoxon within 0.18 min. The apparent rate constant of
r
i
Ϫ1
inhibition of AChE by 2 nM paraoxon (0.0033 min
;
and E are the activity of the free enzyme at equilibrium and
T1/2 ϭ 209 min) was very low compared with the rate of
reactivation by 0.2 mM P2S (0.131 min ; T1/2 ϭ 5.3
min). A similar reduction in the concentration of DEPQ
t
Ϫ1
at time t, respectively. The rate constant kЈobs was obtained
by nonlinear regression analysis of the plot of E /E versus
t
eq

1
64
Y. Ashani et al.
FIG. 3. Time–course of the reactiva-
tion of DEPOAChE by 0.2 mM P2S
in the presence of 2 ␮M diethylphos-
phates and various concentrations of
OPHps. Dotted lines, controls: (■) no
inhibitor, ؉ OPH; and (F) ؉ inhibi-
tor, no OPHps. Solid lines, ؉ inhibi-
tor, ؉ OPHps (␮g/mL): (E) 0.01;
(Ç) 0.025; (ᮀ) 0.1; ({) 0.25; and
(É) 1.0. (A) paraoxon; (B) DEFP;
(C) TEPP; and (D) DEPQ.
would be obtained after 0.79 min. However, the apparent
rate constant of inhibition under this condition is 1.5
min . Therefore, in order to further decrease the rate of
tive aspects of OPH-enhanced hydrolysis of diethylphos-
phates by oxime reactivators in the presence of inhibitory
concentrations of OPs.
Ϫ1
reinhibition of the reactivated AChE, higher concentra-
tions of OPHps were required for this OP than for para-
oxon. Further, a 1000-fold decrease in DEFP concentration
is expected to occur within 4.65 min. Yet, in view of its
The data in Fig. 3C clearly suggest that TEPP was also
hydrolyzed rapidly by OPHps. Since the K and kcat of
m
3
1
TEPP are unknown, P NMR was used to demonstrate its
enhanced hydrolysis by the enzyme (Fig. 4). Our results
showed that the hydrolysis of TEPP to the corresponding
O,O-diethyl phosphoric acid is accelerated in the presence
of OPHps, relative to the spontaneous breakdown by water.
Signal 3 arose from triethyl phosphate (TEP), a non
relatively small k , a 10-fold decrease of 2 ␮M is sufficient
i
to render the apparent rate constant of the inhibition below
Ϫ1
0
.08 min
(T1/2 ϭ 8.6 min). This can be completed
within 1.5 min. These calculations highlight the quantita-
TABLE 3. Equilibrium and kinetic constants of diethylphosphates versus OPHps (hydrolysis) and AChE (inhibition)*
OPHps
4
Km
(mM)
kcat ؋ 10
k
cat/Km
Time to hydrolyze
2 ␮M OP† (min)
AChE ki
؊1 ؊1
؊
1
؊1
؊1
OP
(min
)
(M min
)
(M min
)
9
6
8
5
Paraoxon
DEPQ
DEFP‡
0.032
0.079
0.25
4.45
2.38
1.34
1.39 ϫ 10
3.01 ϫ 10
5.2 ϫ 10
0.18
0.79
4.65
1.65 ϫ 10
7.46 ϫ 10
3.82 ϫ 10
8
7
*
10 mM Tris, pH 7.8, 29°.
Calculated by using an approximation of the integrated form of the Michaelis–Menten equation [1]. Figures are estimates of time to decrease 1000-fold the initial
concentration of OP, by 1 ␮g/mL OPHps.
Taken from Ref. 1 (pH 7.4, 37°).
†
‡

Reactivation by Oxime and OPH
165
31
FIG. 4. P NMR spectra of 14 mM TEPP in
.1 M Tris, pH 7.8, with and without 1
g/mL OPHps. (A) no OPHps, t ؍ 0; (B) no
0
␮
OPHps, t ؍ 120 min; (C) ؉ OPHps, t ؍ 30
min; and (D) ؉ OPHps, t ؍ 85 min. Line
assignment: 1, (C H O) P(O)-O-(O)P(OC
2
2
5
2
H ) (TEPP); 2, (C H O) P(O)OH; and 3,
5
2
2
5
2
(
C H O) P(O).
2 5 3
anti-ChE stable impurity in TEPP. TEP was used as an
internal standard for monitoring the progression of the
hydrolysis of TEPP.
The reactivation time–course in the presence of DEFP
and TEPP without OPHps deviated from simple first-order
reactivation 7 inhibition kinetics (see lower dotted curves
in Fig. 3, B and C). This was attributed to a parallel
spontaneous hydrolysis of both OPs, a fact that precluded
approach to a stable equilibrium, as was observed with the
stable inhibitor paraoxon (Figs. 2A and 3A).
enzyme was available to us, it was used to examine the
possibility of reinhibition of a ChE by the putative phos-
phorylated oxime (POX, Fig. 1), and to determine whether
OPHps can counteract the observed POX-related slowed
reactivation.
The reactivation time–course was determined in 1
mM P2S. A clear dependence of the rate of reactivation
on the initial concentration of DEP-HuBChE was ob-
served (not shown). Thus, 5, 118, and 210 nM DEP-
HuBChE were reactivated at 0.058 Ϯ 0.003, 0.022 Ϯ
Based on monitoring changes in DTNB, phospholine
was found to be a very poor substrate for OPHps, as would
be expected of a phosphorothiolate [30, 31]. No reactiva-
tion of DEPOAChE could be detected in the presence of 2
Ϫ1
0
.003, and 0.010 Ϯ 0.002 min , respectively. The
observation that the reactivation depends on the initial
concentration of the inhibited enzyme strongly indicates
that POX may be accumulated in sufficient quantity to
reinhibit HuBChE and slow the overall rate of regener-
ation of enzyme activity. However, no acceleration of
the P2S-induced reactivation was detected in the pres-
ence of 1 ␮g/mL of OPHps. Although OPHps did not
accelerate the hydrolysis of the putative POX, we cannot
rule out the possibility that it may catalyze the break-
down of POX that is produced outside the catalytic
␮M phospholine, with as high as 2 ␮g/mL OPHps, and after
a 30-min incubation with 0.2 mM P2S.
Reactivation of DEP-HuBChE in the Presence
of OPHps
It has been shown that BChE of human plasma can readily
sequester OPs [12, 13]. Since a large quantity of the purified

1
66
Y. Ashani et al.
cavity of ChEs via direct reaction of OPs with oximes
Fig. 1, k ).
authors further demonstrated an OPHpd-enhanced recov-
ery of ChE activity in sarin intoxication [6]. No reactivator
was used in these experiments, and conclusions were based
on the spontaneous recovery of the inhibited enzymes. It
was reasoned that in addition to the rapid hydrolysis of OPs,
OPHpd seemed to have ChE-reactivating properties [5],
and it might hydrolyze a form of bound OP that is released
from ChE before it attacks a new AChE molecule [6]. Our
data show that OPHps, an enzyme with substrate specificity
similar to that of OPHpd, did not exhibit detectable
enhancement of spontaneous recovery of enzyme activity of
either DEPOAChE or DEP-HuBChE. It is proposed that
OPHps accelerates oxime-induced reactivation by rapid
hydrolysis of excess OPs rather than by breaking the
covalent bond that attaches the OP moiety to the active-
site serine. This conclusion is consistent with the eluci-
dated 3-D structure of AChE, which implies that the P
atom of DEPOAChE is buried inside a deep gorge that
would preclude the approach of a bulky protein nucleophile
such as OPH [33].
(
p
DISCUSSION
In this work we aimed to demonstrate the in vitro efficacy of
an oxime-OPH combination against the toxicity of the
diethylphosphates because a large number of widely used
pesticides (e.g. parathion, chlorpyrifos, diazinon, couma-
phos) are metabolized to potent AChE inhibitors that
eventually produce DEPOAChEs. The results clearly sug-
gest that a combination of a standard oxime reactivator,
such as P2S, and a low dose of an OPH can significantly
improve clinical recovery after poisoning with diethylphos-
phate-based pesticides that are characterized by a long
persistence of anti-ChE activity in the blood. A concen-
tration as low as 1 ␮g/mL of OPHps was sufficient to
detoxify a 2 ␮M concentration of potent diethylphosphate
inhibitors, and to enable rapid reactivation of the inhibited
enzyme within a short period of time.
These observations are of particular importance to the
reactivation of inhibited AChE in vivo because reactivation
of DEPOAChE in peripheral tissues is essential for rapid
recovery after OP poisoning. Willems et al. [15] reported
that a clear-cut reactivation of blood ChE by P2S was
accompanied by marked improvement in the clinical con-
dition of patients treated for intoxication with commercial
pesticides. Assuming similar reactivatability of DEP-inhib-
ited MoAChE and human AChE [32] under physiological
conditions, the in vitro data offer the following speculation:
OPHps rapidly hydrolyzed paraoxon, DEFP, TEPP, and
DEPQ so as to render 2 ␮M inhibitor practically inactive
within the time frame of reactivation by 0.2 mM P2S.
While 2 ␮M is a realistic blood concentration of an
anti-ChE following diethylphosphorothioate intoxication,
the average therapeutic plasma level of P2S in humans is
ϳ0.02 mM [15]. This suggests that in the absence of
inhibitory activity the expected T1/2 of reactivation of
DEPOAChE is approximately 16 min at 37°*, compared
with a half-life of 5 min in the presence of 0.2 mM P2S at
Since the observed increase of the rate of recovery and
the enzymic activity of AChE at equilibrium depends on
the acceleration of the OP-catalyzed hydrolysis by OPHps,
it is possible to generalize the results from the in vitro
experiments as follows: in most cases the concentration of
the circulating OPs in human blood is significantly smaller
than the corresponding K , and their enzymic hydrolysis by
m
OPH (khyd) is approximated by a first-order reaction, with
khyd ϭ (kcat/K )[OPH] . We can predict that a blood
m
0
concentration of 1 ␮g/mL (Ϸ29 nM) of an even moderately
7
Ϫ1
Ϫ1
active OPH (kcat/Km Ϸ 1 ϫ 10 M min ) would be
sufficient to rapidly detoxify anti-ChE OPs (k ϭ 0.29
hyd
Ϫ1
min ; T1/2 ϭ 2.4 min). Thus, together with an oxime
reactivator, OPH can preserve AChE levels that are nec-
essary for maintaining normal physiological functions of
important sites (e.g. diaphragm), even when anti-ChE
metabolites are released continuously into the blood. Ob-
viously, to achieve this therapeutic goal a reasonable
plasma concentration of the oxime and OPH will have to
be maintained by one of the techniques of controlling drug
administration.
2
9°. It has been reported that in patients intoxicated with
parathion, reactivation following administration of P2S
could not be observed if the pesticide blood levels exceeded
A surprising observation was made with DEPQ. OPHps
was capable of hydrolyzing this quaternary ammonium-
8
3
0 ng/mL (ϳ0.1 ␮M) [15]. A 100-fold decrease in the
containing OP with a remarkably high kcat/Km (3 ϫ 10
Ϫ1
Ϫ1
concentration of parathion or paraoxon can be effected by
a suitable OPH within less than one blood-circulation time.
At 0.001 ␮M paraoxon, reinhibition of AChE should be
negligible* compared with the rate of P2S-induced reacti-
vation.
Touvinen et al. [5] showed that OPHpd hastened the
reactivation of paraoxon-inhibited serum ChE in mice, and
of paraoxon-inhibited human serum ChE in vitro. The same
M
min ). MEPQ [20], a charged methylphosphonate
homologue of DEPQ, also was hydrolyzed rapidly by OPH
(data not shown). The rapid hydrolysis of these OP sub-
strates would suggest that detoxification of the positively
charged POX may be enhanced by OPH. However, results
of reactivation of a high concentration of DEP-HuBChE in
the presence of OPHps did not provide evidence to support
this hypothesis, at least not with respect to the POX that is
expected to be produced within the active-site gorge during
reactivation. Since phosphorylation of P2S can also pro-
ceed via a direct reaction of the OPs with the oxime outside
the active site gorge, this issue has to be investigated
further, under experimental conditions that may allow
3
Ϫ1
*
Assuming a 2-fold increase per ⌬10°, k is estimated at 2.2 ϫ 10 M
r
Ϫ1
min (37°).
*
6
Ϫ1
Ϫ1
Assuming k ϭ 2.6 ϫ 10
M
min (37°), T1/2 is estimated at 265
i
min.

Reactivation by Oxime and OPH
167
meaningful characterization of the OPH-induced hydrolysis
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