Formation and disposition of diethylphosphoryl-obidoxime, a potent anticholinesterase that is hydrolyzed by human paraoxonase (PON1)

Article abstract of DOI:10.1016/j.bcp.2005.04.003

The potential of pyridinium-4-aldoximes, such as obidoxime, to reactivate diethylphosphorylated acetylcholinesterases is not fully exploited due to the inevitable formation of phosphoryloximes (POX) with high anticholinesterase activity. Mono(diethylphosp

Full text of DOI:10.1016/j.bcp.2005.04.003

Biochemical Pharmacology 69 (2005) 1853–1867
www.elsevier.com/locate/biochempharm
Formation and disposition of diethylphosphoryl-obidoxime,
a potent anticholinesterase that is hydrolyzed by
human paraoxonase (PON1)
D. Kiderlen a, P. Eyer a, , F. Worek
*
b
a Walther-Straub-Institute of Pharmacology and Toxicology, Ludwig-Maximilians-University,
Goethestrasse 33, 80336 Munich, Germany
b Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937 Munich, Germany
Received 2 March 2005; accepted 7 April 2005
Abstract
The potential of pyridinium-4-aldoximes, such as obidoxime, to reactivate diethylphosphorylated acetylcholinesterases is not fully
exploited due to the inevitable formation of phosphoryloximes (POX) with high anticholinesterase activity. Mono(diethylphosphoryl)
obidoxime (DEP-obidoxime) was isolated for the first time showing remarkable stability under physiological conditions (half-life
13.5 min; pH 7.1; 37 8C). The half-life was considerably extended to 20 h at 0 8C, which facilitated the preparation and allowed isolation
by HPLC. The structure was confirmed by mass spectrometry and the degradation pattern. DEP-obidoxime decomposed by an elimination
reaction forming the intermediate nitrile that hydrolyzed mainly into the pyridone and cyanide. The intermediates were prepared and
confirmed by mass spectroscopy. DEP-Obidoxime was an extremely potent inhibitor of human acetylcholinesterase approaching a
second-order rate constant of 109 Mꢀ1 minꢀ1 (pH 7.4; 37 8C). The nitrile and the pyridone were still good reactivators. In the presence of
human plasma DEP-obidoxime was hydrolyzed into parent obidoxime. Calcium-dependence and sensitivity towards chelators,
substitution pattern by other divalent cations and protein-modifying agents all pointed to human paraoxonase (hPON1) as the responsible
protein with POX-hydrolase activity. Subjects, probably belonging to the homozygous 192arginine subtype, were virtually devoid of POX-
hydrolase activity while a highly purified hPON1 of the homozygous 192glutamine subtype exhibited particularly high POX-hydrolase
activity. Two parathion-poisoned patients with high and low POX-hydrolase activity responded well and poorly, respectively, to
obidoxime treatment although the former patient had higher plasma paraoxon levels than the poor responder. Hence, the POX-hydrolase
associated PON1 subtype may be another contributor that modulates pyridinium-4-aldoxime effectiveness.
# 2005 Elsevier Inc. All rights reserved.
Keywords: Phosphoryl oximes; Acetylcholinesterase; Paraoxonase; Obidoxime; Paraoxon; Species
1. Introduction
climate of developing countries, pest control has been
considered indispensable to meet food requirements.
OPs have two major advantages over DDT and related
organochlorine compounds: they are readily degraded and
do not accumulate either in humans or in food chains.
However, this advantage is paid for dearly, taking into
account the much higher acute toxicity of OPs to mam-
mals. The rate of accidental, suicidal and homicidal intox-
ications is high and the number of victims worldwide is
estimated to exceed 300,000 per year [1].
With the curtailed use of organochlorine insecticides,
organophosphorus compounds (OPs) became extensively
utilized throughout the world. Particularly in the hot
Abbreviations: AChE, acetylcholinesterase (E.C.3.1.1.7); DEP-obi-
doxime, (O-(O,O-diethylphosphoryl)-obidoxime; DTNB, 5,50-dithiobis(2-
¨
nitrobenzoic acid); Ery-AChE, erythrocyte AChE; HLo7, 1-[[[4-(amino-
carbonyl)-pyridinio]methoxy]methyl]-2,4-bis[(hydroxyimino)methyl]pyri-
dinium dimethanesulfonate; MOPS, 3-(morpholino)propane sulfonate; p-
OHMB, p-(hydroxymercuri)benzoate; PB, phosphate buffer; PMA, phe-
nylmercury acetate; POX, phosphoryloxime; TMB-4, (1,10-trimethylene
bis[4-(hydroxyimino)methyl]pyridinium dibromide
OPs act primarily by inhibiting acetylcholinesterase
(AChE), thereby allowing acetylcholine to accumulate
at cholinergic synapses and disturbing transmission
at parasympathetic nerve endings, sympathetic ganglia,
neuromuscular endplates and certain CNS regions.
* Corresponding author. Tel.: +49 89 218075 702;
fax: +49 89 218075 701.
E-mail address: peter.eyer@lrz.uni-muenchen.de (P. Eyer).
0006-2952/$ – see front matter # 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2005.04.003

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D. Kiderlen et al. / Biochemical Pharmacology 69 (2005) 1853–1867
While atropine represents the mainstay in the treatment of
dysfunctions at muscarine sensitive receptors, it fails at
nicotine sensitive synapses. Here oximes (OX), acting as
specific antidotes, may reactivate the inhibited AChE and
improve the clinical picture.
to be markedly longer at lower temperatures, e.g. 10 8C,
where the biphasic reactivation kinetics indicated a half-
life of hours. Since we were interested in the POX-destroy-
ing activity of human plasma [13], we tried to isolate DEP-
obidoxime in order to facilitate the quantitative determina-
tion of the enzyme activity. To this end, we took advantage
of the supposed stability of DEP-obidoxime at low tem-
peratures and succeeded in isolation, characterization and
long-term storage of the desired DEP-obidoxime.
The value of oximes, 4-pyridinium aldoximes in parti-
cular, is still a matter of controversy. Enthusiastic reports
on outstanding antidotal effectiveness, substantiated by
laboratory findings of reactivated AChE and improved
neuromuscular transmission, contrast with many reports
on disappointing results (for review see [2]). One reason
for the partial ineffectiveness of 4-pyridinium aldoximes
may be the formation of highly potent and sufficiently stable
inhibitors during the process of reactivation. In fact, the
reactivation reaction of the phosphylated AChE (EP) by an
oxime (OX; Scheme 1) is reversible and the free phosphyl-
oxime (POX) formed is in itself a powerful phosphylating
agent of the liberated enzyme (E) (Scheme 1).
This paper deals with the AChE-inhibiting activity of
DEP-obidoxime, its stability and non-enzymatic degrada-
tion as well as with the enzymatic disposition by a plasma
enzyme that was characterized as a POX-hydrolase, which
is most probably identical with the polymorphic 192gluta-
mine subtype of human paraoxonase 1 (hPON1, AA).
2. Materials and methods
The identification of some phosphyloximes was achieved
by synthetic means [3–7]. There is, however, a large body of
data showing that POXs are also generated in reactivation
media, and a recent study confirmed formation of ethoxy
methyl-POX, arising during reactivation of the respective
AChE conjugate with obidoxime and TMB-4 [8].
2.1. Chemicals
Ion pairing reagents (PIC B71 and PIC A1 low UV) for
HPLC-analysis were obtained from Waters (Milford,
USA). All other chemicals were procured from Sigma
(Deisenhofen, Germany), E. Merck KGaA, (Darmstadt,
Germany) or from suppliers as mentioned previously [13].
A high inhibitory potency of the POXs towards AChE has
been demonstrated, which often exceeds the inhibitory
potency of the parent OP by one or two orders of magnitude
[4,9–12]. This holds particularly true for the product formed
in the reaction of obidoximewith paraoxon-inhibited AChE.
The resulting diethylphosphoryl-obidoxime (DEP-obidox-
ime) caused a significant deviation from the expected first-
order reactivation kinetics and was particular prominent at
high AChE concentrations as found, e.g. on erythrocyte
membranes (Ery-AChE) of human blood. Curiously, such a
behaviorwas not found when concentrated, paraoxon-inhib-
ited Ery-AChE was incubated with obidoxime in the pre-
sence of one of the author’s plasma (DK) [13]. Besides
BChE as a scavenger, we identified some POX-sequestering
principle in human plasma, which was not inhibited by
OPs, was not albumin and required Ca2+ ions for enzymatic
activity. Such a behavior was reminiscent of paraoxonase/
arylesterase. To rule out any interference of BChE, plasma
samples were pretreated with soman that inactivates BChE
irreversibly [14]. Still, this plasma retained its activity to
inactivate DEP-obidoxime.
2.2. Enzyme preparations
2.2.1. Acetylcholinesterase (AChE) from electric eel
The AChE stock solution (type III; 950 U/mg protein;
Sigma, Deisenhofen, Germany) was diluted with 10
volumes of 0.1 M sodium 3-(morpholino)propane sulfo-
nate (MOPS) buffer, pH 7.4, containing 0.02% gelatine,
before inhibition took place with paraoxon ethyl (0.2 mM;
Dr. Ehrenstorfer, Augsburg, Germany) at 37 8C for 40 min.
The surplus of paraoxon was adsorbed on MeOH-condi-
tioned Baker Bond C18, (Baker, Deventer, The Nether-
lands) followed by centrifugation.
2.2.2. Paraoxon-inhibited human red cell AChE
Red blood cells in freshly drawn heparinized human
blood (blood group 0) were washed 4-times with 0.1 M
sodium phosphate buffer, pH 7.4, (PB) to give a final
concentration of 150 g Hb/L and incubated with 0.2 mM
paraoxon at 37 8C for 35 min, followed by five washings
with PB at 6 8C and adjustment to 150 g Hb/L. Hemolysis
was achieved by freezing at ꢀ20 8C and thawing in ice–
water.
Isolation of DEP-obidoxime failed until now, since its
stability was considered too low [8]. Earlier experiments in
this laboratory, however, [13] caused some doubt on this
assumption and suggested a half-life in the order of 10 min
under physiological conditions. This half-life was expected
2.2.3. Soman-inhibited human plasma
Heparinized human plasma from various donors was
incubated with 10 mM soman at 37 8C for 30 min where-
upon BChE was completely inhibited. Subsequent dialysis
against the desired buffer was performed at 6 8C overnight
to remove excess soman and to adjust the pH.
Scheme 1. Simplified reaction scheme of reactivation of a phosphorylated
enzyme (EP) by an oxime (OX) with formation of free enzyme (E) and the
powerful inhibiting phosphoryloxime (POX), leading to a topochemical
equilibrium.

D. Kiderlen et al. / Biochemical Pharmacology 69 (2005) 1853–1867
1855
2.2.4. Isolation of paraoxonase and POX-hydrolase by
affinity chromatography of soman-pretreated plasma
on Cibacron Blue 3GA
the reaction media with protein-modifying agents and
selected cations, buffers had to be adapted. Hence,
buffer solutions, pH values and reaction temperatures
were not always consistent, but all comparisons were
made under identical conditions each [17].
According to the method of Gan et al. [15], affinity
chromatography on Cibacron Blue 3GA was employed for
binding the paraoxonase–HDL-complex of human plasma
exhibiting high POX-hydrolase activity (DK), followed by
elution with 50 mM Tris–HCl/1 mM CaCl2/5 mM EDTA/
0.1 M Na-desoxycholate, pH 8.0, at 6 8C within 7 h.
Arylesterase activity was determined in the fractions.
For determination of POX-hydrolase activity, the fractions
with arylesterase activity were pooled, dialyzed against
1 mM PB/1 mM CaCl2 at 6 8C, followed by lyophilization,
resolubilization in distilled water and repeated dialysis
against PBS, pH 7.4, and CaCl2 (1 mM). The resulting
specific activities of paraoxonase and POX-hydrolase were
increased about 30fold (yield 14%). Human PON1, type
AA was a generous gift from Prof. P. Masson (La Tronche,
France). Animal plasma samples were obtained from
Charles River (Sulzfeld, Germany) and the German Pri-
ꢁ Method B: quantification of generated obidoxime: DEP-
obidoxime (6–8 mM) was hydrolyzed enzymatically at
37 8C and pH 7.4 by incubation with soman-pretreated
plasma (dialyzed against 25 mM Tris–HCl/1 mM
CaCl2/0.15 M NaCl/0.1% Triton X-100, pH 8.0). Obi-
doxime formed was quantified by HPLC analysis
(method C) and the rate of its generation was used to
quantify POX-hydrolase activity (nmol/min).
2.3.1.3. Determination of paraoxonase activity. The rel-
ease of p-nitrophenol from paraoxon (2 mM) was mea-
sured at 405 nm (e = 16 ꢂ 103 Mꢀ1 cmꢀ1). The assay was
carried out at 37 8C both in 0.1 M Tris–HCl/ 1 mM CaCl2
(pH 8.5) and in 0.1 M Tris–HCl/ 1 mM CaCl2/1 M NaCl
(pH 8.5). The quotient of both paraoxonase-activities was
used for determination of the NaCl-factor as a coarse
means of phenotyping of paraoxonase [18].
¨
mate Center (Gottingen, Germany).
2.3. Methods
2.3.1.4. Determination of arylesterase activity. The
release of phenol from phenyl acetate (4 mM) was deter-
mined at 270 nm (e = 1.45 ꢂ 103 Mꢀ1 cmꢀ1) in 50 mM
Tris–acetate/0.3 mM CaCl2 at pH 7.4 and 37 8C [19].
2.3.1. Enzyme activity determinations
2.3.1.1. Determination of AChE activity in a modified
Ellman assay [16]. AChE activity was determined in the
presence of 0.45 mM acetylthiocholine and 0.3 mM DTNB
in 0.1 M sodium phosphate buffer, pH 7.4, at 37 8C. To
reduce hemoglobin interference, determination of the indi-
cator chromophor was performed at 436 nm. To avoid
interference with matrix SH groups, the hemolysate was
preincubated with DTNB for 10 min, which did not influ-
ence AChE activity. This step could be omitted when
electric eel AChE was used. If not otherwise indicated,
the paraoxon-inhibited, temperature-equilibrated enzyme
preparations were incubated with 10 mM obidoxime and
plasma samples at pH 7.4 for various time intervals before
small aliquots were transferred in the cuvettes to start the
modified Ellman reaction at 37 8C.
2.3.1.5. Determination of enzyme activities in the pre-
sence of Tb3+. To avoid reduction of the availability of
Tb3+, phosphate and tryptophan-containing material such
as hemoglobin and albumin had to be avoided. Therefore,
soman-pretreated, Cibacron-purified plasma was dia-
lyzed against 0.1 M MOPS buffer, pH 7.4, containing
1 mM CaCl2 and reacted with 100 mM TbCl3. POX-
hydrolase activity was determined in the presence of
100 mM Tb3+ by incubation of paraoxon-inhibited elec-
tric eel AChE, stabilized by 0.02% gelatine, with 1 mM
obidoxime in 0.1 M MOPS/1 mM Ca2+ at 10 8C for
15 min.
2.3.1.2. Determination of POX-hydrolase activity in
soman-pretreated plasma.
2.3.1.6. Determination of POX-hydrolase activity in the
presence of carbodiimides. Soman-pretreated, Cibacron-
purified plasma was reacted with 1-ethyl-3-(3-dimethyla-
minopropyl)carbodiimide (1 mM) or N,N0-dicyclohexyl-
carbodiimide (2 mM) at 37 8C, followed by dialysis
against 1 mM PB/1 mM Ca2+, pH 7.4 and lyophilization.
Finally, this preparation was incubated with paraoxon-
inhibited electric eel AChE in the presence of 0.02%
gelatine and 1 mM obidoxime at 10 8C for 20 min. Con-
trols without carbodiimide pretreatment were run by the
same procedure.
ꢁ Method A: reactivation of AChE: Paraoxon-inhibited
human hemolysates (150 g Hb/L) were incubated with
the same volume of soman-pretreated plasma and/or
buffer medium along with 10 mM obidoxime before
AChE activity was determined. For calculation of
POX-hydrolase activity the following equation was
employed (for details see [13]): %POX-hydrolase
activity = 100 ꢂ (A+ ꢀ A )/(A0 ꢀ A ), with A+ and
ꢀ
ꢀ
A
standing for AChE activity in the presence and
ꢀ
absence of plasma, and A0 for the activity of a diluted
AChE sample (1:100), when POX did not interfere with
reactivation. This test gives arbitrary activity and can be
used for comparisons only. To minimize interference of
2.3.1.7. Determination of enzyme activities in the presence
of phenylmercury acetate (PMA) and p-(hydroxymercuri)
benzoate ( p-OHMB). Soman-pretreated, Cibacron-puri-

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D. Kiderlen et al. / Biochemical Pharmacology 69 (2005) 1853–1867
fied plasma (dialyzed against 0.1 M MOPS/1 mM Ca2+, pH
7.4) was incubated with p-OHMB (100 mM) or PMA
(50 mM) at 37 8C for 30 min. For determination of POX-
hydrolase activity, an aliquot of this preparation was incu-
bated with paraoxon-inhibited electric eel AChE and obi-
doxime (1 mM) at 10 8C for 20 min.
2.3.1.10. Quantification of DEP-obidoxime and its
decomposition products.
ꢁ DEP-obidoxime: The concentration of DEP-obidoxime
was determined indirectly by the liberation of obidox-
ime, which was found after complete enzymatic hydro-
lysis of isolated DEP-obidoxime upon incubation with
undiluted soman-pretreated plasma (DK) at 37 8C and
pH 7.4. Quantification of obidoxime was achieved by
HPLC (method C). Thereby, DEP-obidoxime was quan-
tified and its molar extinction coefficient estimated by
UV-spectroscopy [20].
2.3.1.8. Inhibition kinetics of human Ery-AChE by
paraoxon and DEP-obidoxime. In these experiments, ery-
throcyte SH groups were masked by preincubation with
DTNB, in order to minimize further inhibition before
AChE activity could be measured. To reduce POX-decom-
position the inhibition kinetics was followed at 10 8C and
not at 37 8C, while AChE activity was determined at 37 8C.
Preincubation of AChE with DTNB did not affect the
enzyme activity.
ꢁ Obidoxime: For identification and quantification of obi-
doxime in the presence of proteins, the sample (150 mL)
was precipitated with 1 M trichloroacetic acid (45 mL),
and the supernatant (100 mL) mixed with 50 mL PIC B7-
reagent (Waters-Millipore) containing 12% NH3 (25%).
HPLC-analysis with 50 mL aliquots was performed with
method C.
2.3.1.9. Generation and isolation of diethylphosphoryl-
obidoxime (DEP-obidoxime) and its decomposition
products.
ꢁ Obidoxime monopyridone and obidoxime mononitrile:
The UV absorption of these obidoxime derivatives in
alkaline medium was assumed to be half the absorption
of obidoxime, corresponding to e354 nm = 23.4 ꢂ 103
ꢁ DEP-obidoxime: Caution: DEP-obidoxime is an extre-
mely potent anticholinesterase agent! Since it was our
policy not to prepare and handle hazardous amounts of
highly toxic organophosphates, we prepared DEP-obi-
doxime in a one-pot reaction and agreed upon a route
with low yield. To this end obidoxime was allowed to
react with equimolar paraoxon that produced DEP-obi-
doxime at a yield of about 1%. Obidoxime dichloride
(50 mM) was diluted in 10 mM sodium veronal buffer,
pH 7.5, and adjusted to pH 7.5 with NaOH. Paraoxon
(15 mM) was suspended in the same buffer. The final
incubation mixture consisted of 10 mM obidoxime and
10 mM paraoxon in 10 mM veronal, pH 7.5, and was
allowed to react overnight on ice. Subsequently, 0.1 mL
aliquots were separated by HPLC (method A) to collect
DEP-obidoxime at Rt 18 min.
M
ꢀ1 cmꢀ1, caused by the remaining monooximate func-
tion of the bispyridinium compound. This procedure
enabled standardization of the HPLC areas (method D).
ꢁ Cyanide: This product was determined photometrically
after derivatization by the Zincke–Koenig reaction in a
modification [21] of the method of Nagashima [22]. The
sample was incubated at pH 7.4 and 37 8C in a Fernbach
vessel, while cyanide was freed from interfering com-
pounds by micro diffusion into the alkali trap (0.1 M
NaOH).
2.3.2. HPLC methods
DEP-obidoxime and the degradation products were
analyzed by HPLC using a Hitachi L-6200 pump (Merck).
Peaks were quantified by peak integration with an SPD
M6A UV-VIS diode-array detector (Shimadzu) coupled
with a personal computer Compaq ProLinea 4/66; software
Shimadzu Class LC-10 1992. Identification was achieved
by comparison of retention times and UV spectra with
authentic standards or compounds isolated by HPLC.
For chromatography with ion-pairing, 5% of the ion-
pairing reagent was added to the sample to prevent peak
broadening.
ꢁ Obidoxime mononitrile: For generation of obidoxime
mononitrile, isolated DEP-obidoxime (in the mobile
phase) was adjusted to pH 6.9 with NH4HCO3 and
allowed to react at 0 8C for 8.5 h, followed by incubation
at 37 8C for 7 min (at the end of the incubation the pH
was 8.3). To avoid further decomposition of the nitrile,
the pH was adjusted to 5.2 with 1 M HCl, followed by
lyophilisation. After dissolution in H2O, the mononitrile
was isolated by HPLC (method B; Rt 6.5 min).
ꢁ Obidoxime monopyridone: To reduce formation of obi-
doxime monocarboxamide (see Section 4) conditions
were chosen to produce first the mononitrile followed by
cyanide elimination and formation of the monopyridone.
To this end, the following procedure was adopted:
Isolated DEP-obidoxime (in the mobile phase) was
brought to pH 7.4 with ammonia and incubated for
16 h on ice and subsequently at 37 8C for 4.5 h, before
concentrating the sample (speed vac) and isolating the
monopyridone by HPLC (method B; Rt 13.4 min).
2.3.2.1. Method A. For the isolation of DEP-obidoxime,
chromatography on LiChroCart Superspher 60, RP-select
B (3 mm i.d. ꢂ 125 mm; 4 mm (Merck)) without ion-pair-
ing reagent was applied. The mobile phase consisted of
10 mM ammonium acetate pH 4.5 (A) and the same buffer
containing 50% MeOH (B). Samples were eluted with a
linear gradient increasing from 0% B to 40% B in 20 min,
40% B for 10 min, linear increase to 100% B in 10 min,
100% B for 10 min, and return to 0% B in 5 min. The
detection wavelengths were set at 260 and 300 nm; the

D. Kiderlen et al. / Biochemical Pharmacology 69 (2005) 1853–1867
1857
Scheme 2. Chemical structures of the compounds involved in the preparation of diethylphosphoryl obidoxime (DEP-obidoxime).
retention time of DEP-obidoxime was 18 min at a flow rate
of 0.6 mL/min.
3. Results
3.1. DEP-obidoxime
2.3.2.2. Method B. To get samples for MS analysis, obi-
doxime mononitrile and obidoxime monopyridone were
chromatographed on LiChroCart LiChrospher 60, RP-
select B (4 mm i.d. ꢂ 250 mm; 5 mm (Merck)) under
the same elution conditions as described for Method A.
3.1.1. Generation and isolation of DEP-obidoxime
Because the presumably more reactive diethyl phos-
phorofluoridate was not available to us we tried to generate
DEP-obidoxime directly by reaction of paraoxon with
obidoxime (Scheme 2). In order to prevent formation of
bis-diethylphosphoryl-obidoxime we hesitated to add para-
oxon in excess [6]. Since concentrated buffers, especially
phosphate buffer, are known to reduce the yield of POX
[6,7,23], the assay was buffered only slightly. Best yields of
DEP-obidoxime were obtained by reaction of equimolar
amounts of obidoxime and paraoxon (10 mM each) in
10 mM veronal at pH 7.5. Lower pH-values retarded the
reaction and increased the amount of by-products. Higher
pH-values accelerated the reaction by increasing the
amount of the oximate–anion, but resulted in more rapid
product decomposition (veronal pH 8.25). The stability of
DEP-obidoxime was substantially higher at lower tem-
perature, so the incubation was performed at 0 8C over-
2.3.2.3. Method C. For verification and quantification,
obidoxime was analyzed on LiChroCart LiChrospher 60,
RP-select B (4 mm i.d. ꢂ 125 mm; 5 mm (Merck) at a
flow-rate of 1.2 mL/min and detection at 285 nm; the
mobile phase for ion-pairing chromatography consisted
of PIC B7:PIC A low UV:CH3CN:H2O = 4:0.35:4:91.65
(v/v).
2.3.2.4. Method D. DEP-obidoxime, obidoxime mononi-
trile and obidoxime monopyridone were analyzed (detec-
tion and quantification) on LiChroCart LiChrospher 60,
RP-select B (4 mm i.d. ꢂ 125 mm; 5 mm (Merck)) at a
flow-rate of 1.2 mL/min with detection at 260 and 300 nm.
The mobile phase consisted of aqueous 4% PIC B7, 0.35%
PIC A low UV. Gradient elution was started with 2%
CH3CN, which was linearily increased to 12% within
20 min, held at 12% for 5 min and increased to 16% within
another 5 min before return to initial conditions within
5 min.
2.3.3. Mass spectrometry
Positive electrospray ionization mass spectrometry
(ESI-MS) was performed with a quadruple ion trap tandem
mass spectrometer (LCQ DUO, ThermoQuest, Finnigan).
The samples were introduced with a syringe pump into the
apparatus (flow = 25 mL/min) and analyzed by full-scan
MS mode. The parameters were set as follows: capillary
temperature of 170 8C and 200 8C, sheath gas flow of 20
(arbitrary units); obidoxime mononitrile was analyzed with
LC-MS by elution from LiChroCart LiChrospher 60, RP-
select B (4 mm i.d. ꢂ 250 mm; 5 mm (Merck) with 10 mM
ammonium formiate/50% MeOH, pH 6.0; MS/MS spectra
were obtained by ion trap collision-induced dissociation.
Fig. 1. HPLC-chromatogram of an incubate of obidoxime and paraoxon for
the isolation of DEP-obidoxime by method A, detection wavelength
260 nm. 4-PAO (4-pyridine aldoxime) was an impurity of the obidoxime
preparation.

1858
D. Kiderlen et al. / Biochemical Pharmacology 69 (2005) 1853–1867
night. Subsequently, portions of 0.1 mL of the reaction
mixture were separated by HPLC (method A). Fig. 1 shows
a chromatogram with the primary products, DEP-obidox-
ime (Rt 18 min) together with 4-pyridine aldoxime (4-
PAO, an impurity of the obidoxime preparation), p-nitro-
phenol and veronal.
betaine structure [24,25]. This behavior pointed to a free
oxime function as expected for mono(diethylphosphoryl)-
obidoxime. In fact, LC-MS confirmed the structure with
the molecular peak m/z 212 and its fragments of m/z 135
(mononitrile) and m/z 155 (diethylphosphoric acid). For
more details of the MS and MS/MS analysis the reader is
referred to Kiderlen [26].
The yield of DEP-obidoxime was 1%; it was stable at
ꢀ70 8C and pH 4.5 for several weeks.
Incubation of DEP-obidoxime at pH 7.4 on ice without
plasma resulted in the production of a more hydrophilic
product, without detection of cyanide, pointing to obidox-
ime mononitrile as the primary degradation product. At pH
4.75, this component revealed a maximum at 282 nm, at pH
11 the oximate-absorbance maximum at 354 nm appeared
and the complete decomposition of the mononitrile was
confirmed by HPLC-analysis. LC-MS analysis of the pre-
sumed mononitrile gave the molecular peak m/z 135.
During incubation of the obidoxime mononitrile at pH
7.4 and 37 8C the degradation to another hydrophilic
product was observed, accompanied by cyanide formation,
strongly suggesting a pyridone structure. The supposed
monopyridone at pH 4.5 revealed an absorbance maximum
at 267 nm with a shoulder at 285 nm that was attributed to
an oxime function. At pH 10.5, the oximate absorption at
354 nm appeared, which was reversible after acidification
as expected for the obidoxime monopyridone-derivative.
LC-MS analysis confirmed the structure with the mole-
3.1.2. Fate of DEP-obidoxime
DEP-obidoxime disappeared completely during incuba-
tion with human plasma at pH 7.4, while diethyl phos-
phoric acid and obidoxime were generated by hydrolytic
cleavage. This mechanism justified the name POX-hydro-
lase. After incubation of DEP-obidoxime without plasma
at pH 7.4, the inhibitor also disappeared, but the degrada-
tion pattern was completely different and virtually no
obidoxime was generated.
3.1.3. Characterization of DEP-obidoxime and its
degradation products
The UV spectrum of DEP-obidoxime at pH 4.5 showed
an absorbance maximum at 273 nm (obidoxime 285 nm).
At pH 9.2 the absorbance at 273 nm was shifted bath-
ochromically to 354 nm. Such a shift is typical of the
transition of 2- and 4-pyridiniumaldoximes into their
Fig. 2. UV-spectra of obidoxime (10 mM), mono-DEP-obidoxime (9 mM), obidoxime mononitrile (8.9 mM) and obidoxime monopyridone (6.9 mM) at acid
and alkaline pH. Spectra were recorded in 10 mM NH4 acetate containing 20% MeOH (HPLC solvent). Alkaline shift was reversible with obidoxime and
obidoxime monopyridone, only.

D. Kiderlen et al. / Biochemical Pharmacology 69 (2005) 1853–1867
1859
UV-analysis of obidoxime monopyridone at pH 11 was
used for its quantification. It was assumed that the UV-
absorption of the single pyridinium-4-aldoximate function
in obidoxime monopyridone was half the absorption of
obidoxime with its two pyridinium-4-aldoximate func-
tions. Hence, its molar extinction coefficient was calcu-
lated to be 23.4 ꢂ 103 Mꢀ1 cmꢀ1. From this the extinction
coefficients of the alkali-labile precursors could be derived
(Table 1).
3.1.5. Influences on the decomposition pattern
of DEP-obidoxime
DEP-obidoxime, incubated in 0.1 M MOPS and 1 mM
CaCl2, pH 7.4, at 37 8C decomposed completely, produ-
cing 49% cyanide and 80% of the obidoxime monopyr-
idone (percent of theory). Hydrolysis of DEP-obidoxime
into obidoxime was not observed. Incubation of DEP-
obidoxime in the same buffer at 0 8C produced nearly
exclusively obidoxime mononitrile (95%). The half-lives
for degradation of DEP-obidoxime and production of
obidoxime-mononitrile were comparable. Obidoxime-
mononitrile decomposed readily by further incubation at
37 8C into 67% obidoxime monopyridone (n = 4; S.D. = 6)
and 52% cyanide (n = 4; S.D. = 7). Besides, a second
hydrophilic product was detected with an estimated yield
of 27% (n = 2). The UV spectrum at pH 4.5 showed a
maximum of absorbance at 282 nm, compatible with a
monoisonicotinamide or monoisonicotinic acid derivative
of obidoxime. The balance was incomplete, especially with
regard to the yield of cyanide.
Scheme 3. Enzymatic and non-enzymatic degradation pathways of diethyl-
phosphoryl obidoxime and the corresponding mass units as confirmed by
mass spectrometry.
cular peak m/z 260. The UV-spectra of obidoxime and its
derivatives are shown in Fig. 2.
A completely different degradation pattern resulted after
incubation of DEP-obidoxime at 37 8C with the soman-
pretreated Cibacron-purified plasma of one of the authors
(DK). Obidoxime was generated quantitatively, while cya-
nide was only detectable in traces. Here, DEP-obidoxime
was hydrolyzed before being degraded via the nitrile
pathway.
Scheme 3 summarizes the observations: incubation of
DEP-obidoxime at pH 7.4 resulted in the generation of the
mononitrile by an eliminating mechanism, followed by
further decomposition into cyanide and the monopyridone.
In the presence of soman-pretreated plasma, however,
DEP-obidoxime was hydrolyzed enzymatically into obi-
doxime and diethylphosphoric acid.
The stability of DEP-obidoxime, approx. 5 mM, was
tested under near-physiological conditions in Ringer-phos-
phate solution, pH 7.1 and 37 8C, followed by HPLC
analysis. The compound showed a mono-exponential
decay with a half-life of 13.5 min. The stability increased
markedly when the reaction temperature was lowered. The
Arrhenius plot shown in Fig. 3 indicates an apparent energy
of activation of 85 kJ/mol (Ringer-phosphate solution, pH
7.1). Phosphyloximes are known to decompose via two
competitive pathways, a cyclic elimination reaction and a
3.1.4. Quantification of DEP-obidoxime and its
degradation products
For quantification, DEP-obidoxime was incubated with
soman-pretreated plasma at pH 7.4 and 37 8C to ensure
complete hydrolysis, followed by determination of the
obidoxime concentration by HPLC-analysis. The concen-
tration of obidoxime found was equated with the concen-
tration of DEP-obidoxime.
Table 1
Spectroscopic data of obidoxime, DEP-obidoxime, obidoxime mononitrile and obidoxime monopyridone
Obidoximea
Mono-DEP-obidoxime
Obidoxime mononitrile
Obidoxime monopyridone
lmax (nm) (e (mMꢀ1 cmꢀ1))
pH 4–5
285 (34.7)
354 (46.8)
273 (31.6)
282 (22.4)
267; 285 (S) (24.5)
354 (23.4)
pH 9–11
Degradation to pyridone
Degradation to pyridone
If not otherwise stated, spectra were recorded in 10 mM NH4 acetate, containing 20% MeOH.
a
In water, from [20]; S: shoulder.

1860
D. Kiderlen et al. / Biochemical Pharmacology 69 (2005) 1853–1867
Fig. 3. Arrhenius plot of the decomposition rate constants (minꢀ1) of DEP-
obidoxime vs. the reciprocal absolute temperature. DEP-obidoxime,
approximately 5 mM, was dissolved in Ringer-phosphate solution, pH
7.1, and incubated at 37, 25, 10 and 0 8C. The decay of DEP-obidoxime
was followed by HPLC and exhibited first-order kinetics.
Fig. 4. Inhibition of human Ery-AChE (final concentration 15 g Hb/L) by
200 nM paraoxon (~) und 2 nM DEP-obidoxime (~) in 0.1 M PB, pH 7.4,
at 10 8C. Erythrocyte suspensions were preincubated with DTNB, before
the inhibitor was added and incubated at 10 8C. After various time intervals
an aliquot was taken and AChE activity determined at 37 8C by the modified
Ellman method.
nucleophilic attack on phosphorus to regenerate the oxime.
A 4-pyridiniumaldoxime-induced elimination reaction has
been described for the conjugate of sarin, whereas the 4-
pyridiniumaldoxime-induced nucleophilic attack, leading
to re-synthesis of the POX was not observed [23]. In order
to get an impression whether therapeutic oxime concen-
trations could affect DEP-obidoxime degradation, we
tested obidoxime and TMB-4, 10 mM each. It turned out
that the decay at 25 8C was not accelerated by the presence
of obidoxime or TMB-4.
3.1.6. Inhibitory potency of DEP-obidoxime on human
Ery-AChE
Fig. 5. Reactivation potency of obidoxime mononitrile. Paraoxon-inhibited
electric eel AChE was incubated with various concentrations of obidoxime
mononitrile (closed symbols) and with 1 mM obidoxime (open symbols),
respectively, in 0.1 M MOPS/0.02% gelatine, pH 7.4, at 37 8C. After various
time intervals, AChE activity was determined in 0.1 M MOPS/0.02%
gelatine, pH 7.4, at 37 8C. Obidoxime mononitrile 2 min (&), 6 min
(~), 10 min (!), 15 min (^); obidoxime 2 min (*), 6 min (&),
10 min(~) and 15 min (*).
In order to prevent the rapid degradation of DEP-obi-
doxime, the reaction temperature was lowered to 10 8C.
Human Ery-AChE (15 g Hb/L, approximately 0.3 nM
AChE) in 0.1 M PB, pH 7.4, was pretreated with DTNB
to mask matrix SH groups and was incubated at 10 8C with
either 2 nM DEP-obidoxime or 200 nM paraoxon before
determination of AChE activity. (By diluting the mixture in
the cuvette (1:32), further inhibition of the enzyme during
measurement was sufficiently retarded to allow correct
determination.)
for comparable reactivation the incubation time with obi-
doxime mononitrile was 2.5–3 times longer than with
obidoxime. Equieffective reactivation with obidoxime
monopyridone required four times longer reaction times
than with obidoxime.
As shown in Fig. 4, the inhibition of AChE by DEP-
obidoxime (2 nM) proceeded faster than by paraoxon
(200 nM). The bimolecular inhibition rate constants were
calculated at 1.2 ꢂ 108 Mꢀ1 cmꢀ1 for DEP-obidoxime and
3.5 ꢂ 105 Mꢀ1 cmꢀ1 for paraoxon; this difference by a
factor of 350 corresponds to the report of De Jong and
Ceulen, who generated DEP-obidoxime in situ and deter-
mined the inhibition kinetics on bovine Ery-AChE [11].
3.2. POX-hydrolase
3.2.1. Isolation of POX-hydrolase
Paraoxonase/arylesterase as an HDL-associated glyco-
lipoprotein was isolated by affinity-chromatography on
Cibacron Blue 3GA, whereby the paraoxonase-HDL-com-
plex was first adsorbed on Cibacron Blue 3GA, resulting in
removal of albumin and other proteins, before desorption
of the paraoxonase-HDL-complex by Na-desoxycholate
ensued [15]. This method was exploited for isolation of
POX-hydrolase in soman-pretreated plasma, with com-
plete recovery of POX-hydrolase activity in the fractions
containing paraoxonase/arylesterase activity. The fact of
3.1.7. Reactivating potencies of obidoxime mononitrile
and obidoxime monopyridone
The reactivating properties of the degradation products,
obidoxime mononitrile and obidoxime monopyridone,
were compared with obidoxime. Paraoxon-inhibited elec-
tric eel AChE in 0.1 M MOPS/1 mM Ca2+, pH 7.4, was
reacted with the compounds at 37 8C. Fig. 5 illustrates that

D. Kiderlen et al. / Biochemical Pharmacology 69 (2005) 1853–1867
1861
free MOPS buffer (4 8C) immediately before incubation of
samples with various ions for 1 h at 37 8C. Aliquots (1/30
and 1/100) were assayed for POX-hydrolase and arylester-
ase activity.
Table 2 shows that the activities of arylesterase and POX-
hydrolase are influenced comparably: Sr2+ did stabilize both
activities, Ba2+ and Mn2+ did stabilize arylesterase but not
POX-hydrolase. Mg2+ and Ni2+ did slightly inhibit both
enzyme activities which remained stable over 1 h. Incuba-
tion with Zn2+-ions resulted in strong inhibition, which was
reversed by addition of EDTA (under conditions:
[Zn2+] < [EDTA] < [Ca2+]). This effect pointed to rever-
sible inhibition. (It should be noted that the final concentra-
tions of the salts examined was 1.5 mM in the arylesterase
and 400 mM in the POX-hydrolase assay. While AChE was
not affected by these salts, their influence on POX-hydrolase
in the assay cannot be excluded.)
Fig. 6. Influence of calcium deprivation on the activities of POX-hydrolase
and arylesterase. Purified plasma was lyophilised, dialyzed against 10 mM
PB/1 mM Ca2+/ 0.9% NaCl, pH 7.4, and incubated with EDTA (2 mM) at 37
8C for the time intervals indicated. Thereafter, aliquots were incubated with
1 mM excess Ca2+ (over EDTA) at 10 8C for 20 min before POX-hydrolase
(method A; &) and arylesterase (~) determination.
The lanthanide and Ca2+-analogue Tb3+ was suitable to
detect the involvement of tryptophan, which may play
some role in the active site of paraoxonase/arylesterase
[28]. Paraoxonase/arylesterase was reversibly inhibited by
Tb3+ (100 mM) even in the presence of Ca2+ (1 mM). This
inhibition could be reversed by EDTA as long as Ca2+ was
in excess. Also the enhanced reactivation of paraoxon-
inhibited electric eel AChE by Cibacron-purified plasma
was nearly completely abolished by the addition of
100 mM Tb3+.
the simultaneous elution of paraoxonase/arylesterase and
POX-hydrolase raised the question of a common protein.
For this purpose the effect of various modifiers on the
Cibacron-purified preparation was examined based upon
known characteristics of paraoxonase/arylesterase [27–
29].
3.2.2. EDTA and the influence of various cations
Ca2+-ions are necessary for both catalytic activity to
hydrolyze arylesters and for enzyme stability [30]. As
shown in Fig. 6, incubation with EDTA (2 mM) resulted
in a progressive inhibition of arylesterase and POX-hydro-
lase and also in the inhibition of paraoxonase (not shown).
It appeared that the inactivation proceeded in a biphasic
way.
3.2.3. Influence of protein modifying reagents
Preincubation of Cibacron-purified enzyme with
0.2 mM N-bromosuccinimide, another reagent for trypto-
phan-modification [29,31], in 0.1 M MES/0.1 mM Ca2+,
pH 6.5 at 37 8C for 15 min, followed by addition of
0.2 mM tryptophan to scavenge free NBS, resulted in a
decrease of arylesterase-activity to 7% and of POX-hydro-
lase to 11%.
In order to characterize the cation sensitivity, soman-
pretreated plasma was dialyzed against 0.1 M MOPS
buffer/1 mM Ca2+ pH 7.4 at 4 8C. Calcium ions were
removed by flash chromatography of the dialyzed material
(2 mL) on chelex 100-resin (1 mL) equilibrated with Ca2+-
Carbodiimideslikedicyclohexylcarbodiimide(DCC)and
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC)are
reagents for modification of peptide-bonded aspartate and
Table 2
Influence of various salts (1 mM) on arylesterase and POX-hydrolase activities
CaCl2
MnCl2
SrCl2
BaCl2
MgCl2
NiSO4
ZnSO4
Arylesterase
Stable
Stable
Stable
Stable
Stable
Stable
Slightly inhibited
Slightly inhibited
Slightly inhibited
Slightly inhibited
Inhibited
Inhibited
POX-hydrolase
Inactive
Inactive
Table 3
Influence of various modifiers on paraoxonase/arylesterase and POX-hydrolase activity
Inhibitor
% Paraoxonase/arylesterase
% POX-hydrolase
Reaction with
EDTA
Tb3+
No activity
No activity
7%
No activity
No activity
11%
e.g. Ca2+
Tryptophan
N-bromosuccinimide (NBS)
Dicyclohexylcarbodiimide (DCC) or
1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC)
Phenylmercury acetate (PMA) or
p-(hydroxymercuri)benzoate ( p-OHMB)
Tryptophan
30%
30%
Aspartate glutamate
No activity
No activity
SH groups

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D. Kiderlen et al. / Biochemical Pharmacology 69 (2005) 1853–1867
Table 4
Arylesterase and paraoxonase activities in different subjects
Subject
Aryl-esterase
(U/mL)
Paraoxonase
(mU/mL)
Paraoxonase
(mU/mL)
Paraoxonase
paraoxonase
NaCl-factor
(with NaCl)/
(without NaCl)/
arylesterase ꢂ 1000
(without NaCl)
(with NaCl)
arylesterase ꢂ 1000
PE
213.5
118.5
198
502.5
241
1256
615.6
570.3
290.5
5.88
5.19
2.88
1.38
2.35
2.03
1.28
0.71
2.5
GH
2.55
2.25
1.9
DK
253
hPON1 AA
210.4
149.7
The NaCl-factor (activity with1 M NaCl vs. without) of paraoxonase may be used for phenotyping the various plasma samples [18].
Table 5
Influence of POX-hydrolase of various subjects on the degradation of DEP-obidoxime
Subject
Arylesterase (U/mL)
Obidoxime (mM)
Obidoxime-nitrile (mM)
DEP-obidoxime (mM)
S (mM)
PE 1:10
9
90
48
8
–
–
7.1
6.1
6.8
6.3
–
7.1
7.4
7.2
7.6
7.6
7.5
7.1
9
PE conc.
1.3
0.4
1.3
7.6
2.3
5.6
–
–
GH conc.
–
DK 1:10
–
DK conc.
80
9
–
hPON AA 1:10
hPON AA conc.
Dilution buffer
–
5.2
1.5
8.1
90
–
0.9
DEP-obidoxime (8 mM) was incubated in the presence of various plasma sources in 25 mM Tris–HCl, 1 mM Ca2+, 0.15 M NaCl, 0.1 % Triton X-100 at pH 7.4
and 37 8C for 60 s.
glutamate [32]. Addition of 0.6 mM DCC or 1 mM EDC
resulted in the inactivation of arylesterase, its extent being
dependentonthe incubationmedium. In order to avoid some
inhibiting effect on AChE activity during the determination
of POX-hydrolase activity, Cibacron-purified enzyme was
first incubated with the carbodiimide at 37 8C, followed by
dialysis against 1 mM PB/ 1 mM Ca2+, pH 7.4, before
arylesterase and POX-hydrolase activities were determined.
Both activities were reduced to 30% of the expected value.
Phenylmercury acetate (PMA) and p-(hydroxymercuri)-
benzoate ( p-OHMB) completely blocked arylesterase and
POX-hydrolase, indicating free SH groups being important
for catalytic activity of plasma for both reactions. Table 3
gives an overview on the influence of the protein-modifying
reagents on paraoxonase/arylesterase and POX-hydrolase
activity.
DK and to the purified hPON AA, when related to their
arylesterase activities.
3.2.5. Paraoxonase, arylesterase and POX-hydrolase
distribution in different species
Paraoxonase, arylesterase and POX-hydrolase activities
were determined in soman-pretreated plasma samples of
different species and compared with human values. As
shown in Fig. 7 marked species differences in the activities
of paraoxonase and arylesterase were observed. Interest-
ingly, stimulation of paraoxonase by NaCl was only
observed in the two human plasma samples. POX-hydro-
lase activities in plasma samples of identical arylesterase
activity (Fig. 8) were calculated by following the decrease
of DEP-obidoxime after correction for non-enzymatic
hydrolysis. Highest POX-hydrolase activities were found
in primates and humans.
3.2.4. Experiments with soman-pretreated plasma of
various donors and purified human PON1
The potency of soman-pretreated plasma of various
donors and of purified human PON1, type AA, to degrade
DEP-obidoxime was investigated. Table 4 characterizes
the plasma activities towards phenyl acetate and paraoxon
as well as the stimulation of paraoxonase by NaCl [18]. For
determination of POX-hydrolase activity, DEP-obidoxime
was incubated with enzyme preparations of various ary-
lesterase activities at pH 7.4 and 37 8C for 60 s, followed
by analysis of the degradation products by HPLC. Table 5
presents the quantities of the degradation products after
DEP-obidoxime incubation at 37 8C in the presence of
various enzyme sources. It is apparent that subjects PE and
GH have low POX-hydrolase activity compared to subject
4. Discussion
4.1. DEP-obidoxime
To the best of our knowledge, mono(diethylphosphoryl)-
obidoxime (pyridinium, 1-[[[[4-(diethoxy-phosphory-
loxy)imino]methyl]pyridinio]methoxy]methyl-4-[(hydro-
xyimino)methyl)]-diacetate) was isolated and charac-
terized for the first time. Notwithstanding, the existence
of this intermediate has already been postulated several
times [7,11,13,33], because the reactivation of diethylpho-
sphorylated AChE by obidoxime followed biphasic
kinetics in the case of highly concentrated enyzme. On

D. Kiderlen et al. / Biochemical Pharmacology 69 (2005) 1853–1867
1863
relatively stable in H2O at pH 4.5, but decomposed quickly
to the nitrile in 10 mM Tris–buffer, pH 7.8. This POX-
derivative of 4-PAM decomposed in alkaline medium into
the pyridone and the isonicotinamide derivative [7].
In addition, ethoxymethylphosphonyl-obidoxime was
generated in substance, purified by HPLC and characterized
with 31P-NMR [8]. Upon phosphylation the absorbance
maximum of the compound was shifted hypsochromically
from 286 nm to 276 nm, in accordance with the observation
of Waser et al. [34] who noticed a hypsochromic shift of the
obidoxime absorbance maximum (284–278 nm) after reac-
tion of obidoximewith sarin. Luo et al. [8] did not succeed in
generating the DEP-obidoxime and supposed that the com-
pound was not stable enough to be isolated.
That we finally succeeded in isolating DEP-obidoxime
and its mononitrile-derivative was due to the unexpected
high stability of these compounds in the cold and the high
activation energy necessary for degradation (85 kJ/mol).
The results of mass spectrometry and the characteristic
oximate absorbance at alkaline pH pointed to mono(-
diethylphosphoryl) obidoxime and not the bisphosphory-
lated obidoxime derivative.
The spontaneous decomposition of DEP-obidoxime at
neutral pH resulted predominantly in formation of the
mononitrile as the primary degradation product. The cor-
responding half-lives for the decrease of DEP-obidoxime
and the simultaneous increase of the mononitrile support
this assumption. This reaction proceeds probably by a
synchronous cyclic elimination mechanism as suggested
by Hagedorn et al. [25]. The nitrile hydrolyzes to the
pyridone with cyanide liberation or produces the carbox-
amido-derivative [23,35]. The degradation of phosphylox-
imes was reportedly enhanced by the presence of oximes
[36]. We did not observe such an effect. Neither obidoxime
nor TMB-4 accelerated DEP-obidoxime decomposition.
Because attack of obidoxime on the phosphorus atom
would regenerate DEP-obidoxime, TMB-4 was tested
which should give DEP-TMB-4. Formation of such a
compound was not observed.
Fig. 7. Distribution of paraoxonase and arylesterase activities in soman-
pretreated plasma of various species. The plasma samples were dialyzed
against 25 mM Tris–HCl/1 mM Ca2+/0.15 M NaCl/0.1% Triton X-100, pH
8.0. Paraoxonaseactivitiesweremeasuredintheabsence(A)and presence(B)
of 1 M NaCl. Black column: paraoxonase, hatched column: arylesterase.
Activities refer to the protein-content and are presented in International Units.
the other hand the analogous compound, diethylpho-
sphoryl-4-PAM, has been isolated and characterized by
1H, 13C and 31P-NMR and additionally by SIMS (liquid
single ion mass spectroscopy). This compound was
Theenzyme-catalyzeddecompositionofDEP-obidoxime
by POX-hydrolase in human plasma [13], however, suppo-
sedly occurs by a nucleophilic attack at the phosphorus atom
followed by hydrophilic decomposition resulting in the
parent obidoxime. This behavior is comparable with the
hydrolysis of O-(O,O-diethylphosphoryl)-1-methyl-4-pyri-
diniumaldoxime by the phosphotriesterase of Pseudomonas
sp. [7]. In the presence of sufficient enzyme activity obidox-
ime is generated quantitatively. Hence, the existence of
POX-hydrolase in human plasma may contribute to optimal
reactivation of diethylphosphoryl-AChE by obidoxime;
otherwise DEP-obidoxime results in rapid reinhibition of
the reactivated enzyme thus retarding net reactivation.
Fig. 8. Distribution of POX-hydrolase activity in soman-pretreated plasma
of various species. The plasma samples were dialyzed against 25 mM Tris–
HCl/1 mM Ca2+/0.15 M NaCl/0.1% Triton X-100, pH 8.0. For quantifica-
tion of POX-hydrolase activity, the decrease of DEP-obidoxime (6.4 mM)
was followed upon incubation with equal activities of arylesterase (13.9 U/
mL) at pH 7.4 and 37 8C for 3 min. (Data are corrected for non-enzymatic
degradation of DEP-obidoxime).
4.1.1. Inhibitory activity of DEP-obidoxime
The bimolecular inhibition rate constant of human
Ery-AChE by DEP-obidoxime was determined with

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D. Kiderlen et al. / Biochemical Pharmacology 69 (2005) 1853–1867
1.2 ꢂ 108 Mꢀ1 minꢀ1 at pH 7.4 and 10 8C and was found
to be by 350 times a stronger inhibitor than paraoxon. At
37 8C, the inhibition constant is calculated as
7.5 ꢂ 108 Mꢀ1 minꢀ1, assuming the influence of tempera-
ture to be comparable for DEP-obidoxime and paraoxon.
This value approaches the constant for the inhibition of
recombinant mouse-AChE by 7-(O,O-diethylphosphinyl-
oxy)-1-methylquinolinium-methylsulfate (29 8C, 10 mM
Tris, pH 7.8 [37]) and the inhibition of human Ery-AChE
by cyclosarin (37 8C, pH 7.4 [38]) and exceeds the inhi-
bitory activity of soman [39]. As DEP-obidoxime and
reactivated AChE are produced at equimolar concentra-
tions, the half-life of reinhibition can be calculated, which
is 1.3 min for 1 ꢂ 10ꢀ9 M AChE. With the therapeutic
reactivation half time of 4 min at therapeutic obidoxime
concentration (10 mM), only one-third of AChE could be
reactivated after reaching equilibrium conditions, which
was also confirmed experimentally [13]. However, in the
presence of enough POX-hydrolase the reactivation
potency of obidoxime was fully exploited.
onase/arylesterase towards these reagents strongly
suggested that tryptophan, aspartate/glutamate and
cysteine residues are in or close to the active site and to
Ca2+-binding sites of hPON1. Meanwhile the crystal struc-
ture of a variant of PON1 obtained by directed evolution
˚
has been resolved at 2.2 A [43]. The six-bladed propeller
structure contains two calcium ions in the central tunnel,
one responsible for catalytic action and one for structural
integrity. Further efforts with site-directed mutagenesis
will shed more light on this multifunctional protein [44].
4.2.1. Substrate specificity of POX-hydrolase
As already described [13], DEP-TMB-4, dimethylpho-
sphoryl-obidoxime and diisopropylphosphoryl-obidoxime
are also substrates of POX-hydrolase though with lower
affinity, while the sarin conjugate of obidoxime is probably
not hydrolyzed by the POX-hydrolase [45].
¨
Preliminary experiments with HLo7, a bis-pyridinium-
2,4-dialdoxime, allowed the conclusion that DEP-HLo7
¨
may also be generated in situ, and was cleaved by POX-
hydrolase [26].
4.1.2. Reactivating potency of the degradation products
Interestingly, neither the nitrile nor the pyridone struc-
ture fundamentally influenced the reactivation potential of
the remaining pyridinium-4-aldoxime-function, as the
potency was only little less than expected for the remaining
oxime function. Oldiges and Schoene reported on a similar
behavior of HI 2, the 4-carboxamido analogue of obidox-
ime: Its potency to reactivate AChE in paraoxon-poisoned
mice was 2.5–2.8 times lower than that of obidoxime [40].
4.3. Identity of POX-hydrolase and human PON1?
The above results strongly pointed to the identity of
POX-hydrolase and hPON1. Serious doubts arose, how-
ever, when considering the different distribution of these
two enzyme activities in human individuals and animal
species. The known polymorphisms of human PON1 with
arginine or glutamine at position 192 and, to a lesser degree
the leucine/methionine polymorphism at position 55, are
probably responsible for the different activities of these
two forms when hydrolyzing paraoxon. Conceivably, argi-
nine in hPON1 type BB is protonated at physiological pH
thereby impeding the access of positively charged sub-
strates such as DEP-obidoxime. In case of hPON1 type
AA, however, the glutamine residue is uncharged under
physiological conditions and may render better accessi-
bility for charged molecules. This consideration could
explain why the individuals PE and GH with PON1posses-
sing higher paraoxonase activity that is also more stimu-
lated by NaCl did not exhibit significant POX-hydrolase
activity and may therefore represent the homozygous
192arginine hPON1-type BB (still awaiting sequence ana-
lysis or genotyping). The other individuals, however,
should represent the AB-mixed forms of hPON1. This
assumption is plausible since there is a difference at human
polymorphic PON1-types concerning the hydrolysis of the
various organophosphates (for review, see [46]).
4.2. Characterization of POX-hydrolase
The plasma compound which was able to inactivate
DEP-obidoxime, was considered to be an enzyme with a
molecular weight of >100 kD and was reminiscent of
human paraoxonase (hPON1) [13]. The enzyme activity,
which was tentatively termed POX-hydrolase, was not
inactivated by soman or paraoxon, thus belonging to the
family of A-esterases [41,42].
Upon purification of POX-hydrolase by chromatography
on Cibacron Blue 3 GA, co-chromatography with paraox-
onase/arylesterase was observed. The most interesting
question was the structural similarity of the active center
of POX-hydrolase and PON1.
In the active center of PON1 at least one tryptophan
should be involved, because Tb3+ (0.1 mM) completely
inhibited both the Ca2+-dependent PON1 and POX-hydro-
lase. Modification of tryptophan by N–bromosuccinimide
did also result in comparable sensitivity of paraoxonase/
arylesterase and POX-hydrolase. Experiments with carbo-
diimides as reagents for specific modification of carboxyl
groups of peptidic aspartate and glutamate and also with
PMA and p-OHMB as SH group blockers revealed that
both enzyme activities were influenced in the same man-
ner. The similar behavior of POX-hydrolase and paraox-
Comparison of the expression of paraoxonase, aryles-
terase and POX-hydrolase in the plasma of various species
elucidated fundamental differences. Referring to arylester-
ase-activity, rodents, pig and rabbit did only exhibit very
poor POX-hydrolase activity, whereas in primates this
activity seems to be comparable with the putative human
PON1-type AB (DK).

D. Kiderlen et al. / Biochemical Pharmacology 69 (2005) 1853–1867
1865
Fig. 9. Influence of the PON 1 status on the efficacy of obidoxime to reactivate inhibited AChE in two patients poisoned with parathion. Upper panel: Ery-
AChE activity in patient’s blood (&) and after reactivation of diluted blood in vitro (~), to assess reactivatability (100 mM obidoxime, 30 min). Activities refer
to the hemoglobin content of the samples [47]. Mid panel: Plasma obidoxime concentration as determined by HPLC [20]; arrows indicate beginning of the
obidoxime scheme, i.e. 250 mg bolus i.v., followed by continuous infusion at 750 mg/24 h. Lower panel: Plasma paraoxon concentration (*) as determined by
HPLC [51]. The depicted data refer to free paraoxon (Px) in plasma water. The measured amounts in organic plasma extracts were corrected on the assumption
of 55% protein binding [51]. The calculated Px values (*) were derived from the kinetic constants of inhibition and reactivation and the ratio of active vs.
inhibited AChE. (For calculation see [2].) Time scale refers to the period elapsed after poison ingestion. (A) Patient SG 25 (left column) exhibited low POX-
hydrolase activity (37%, method with AChE reactivation), correlating with poor reactivating efficacy of obidoxime in vivo and comparably higher calculated Px
concentrations. (B) Patient LR 31 (right column) exhibited high POX-hydrolase activity (89%, method with AChE reactivation), correlating with high
reactivation efficacy of obidoxime in vivo. Measured and calculated Px concentrations were virtually identical. (The residual obidoxime found in the plasma
before beginning with our scheme stem from a bolus dose of 250 mg i.v. given by the emergency physician.)
4.4. Influence of POX-hydrolase activity on the
effectiveness of obidoxime in organophosphate
poisoning
determined. With the kinetic parameters KD, kr and ki
[50] and the measured concentrations of active AChE,
inhibited AChE and obidoxime at hand, one could also
calculate the expected poison-concentration [2]. Fig. 9
represents the course of these values in two parathion-
poisoned patients. Case A, SG25, exhibited poor POX-
hydrolase activity (37%, method with AChE-reactivation),
and the calculated paraoxon concentration in plasma water
was higher than the measured one. This may be some hint
for the existence of a more potent poison such as some
accumulation of DEP-obidoxime. Somewhat different was
the progress in another parathion-poisoned patient, case B,
LR31, (Fig. 9). Here, calculated paraoxon concentration in
plasma water corresponded to the measured one with POX-
hydrolase activity being particularly high (89%, method
If one transfers these aspects to the therapeutic effec-
tiveness of obidoxime in patients, poisoned with diethyl or
dimethylphosphorylating agents, one would expect that
patients with high POX-hydrolase activity will benefit
more from the therapy with obidoxime. Some hints for
this assumption came from a study with parathion-poi-
soned patients. The therapy of these patients consisted of
atropine and obidoxime as continuous infusion. The impor-
tant parameters of Ery-AChE-activity in vivo, maximal
reactivatability in vitro [47], obidoxime concentration
[20,48] and plasma paraoxon concentration [49] were

1866
D. Kiderlen et al. / Biochemical Pharmacology 69 (2005) 1853–1867
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taken as a first hint, pointing to the relevance of POX-
hydrolase activity for optimal effectiveness of obidoxime
therapy. Thus, the PON1-polymorphism seems to be a
further factor, which may influence the therapeutic success
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The authors are indebted to Prof. Patrick Masson (La
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PON1, type AA, and to Prof. Gabriele Sabbioni (Walther-
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to R. Heilmair for her skilful and engaged technical
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