choline derivatives and sodium fluoride protect acetylcholinesterase against irreversible inhibition...

J BIOCHEM TOXICOLOGY Volume 9, Number 5 , 1994

Choline Derivatives and Sodium Fluoride Protect Acetylcholinesterase against Irreversible Inhibition and Aging by DFP and Paraoxon Mohamed S . Dehlawi,' Amiua T . Eldefrawi, Mohyee E . Eldefuawi, Nabil A. Anis, and James J . Valdes Biotechnology Divlsion, U S Army Edgewood RD & E Center, Aberdeen Proving Ground, MD 21010

ABSTRACT: A light addressable potentiometric sensor was used to measure acetylcholinesterase (AChE) activity in order to evaluate the protective effects of quaternary compounds and NaF against enzyme phosphorylation and aging by two organophosphates. The use of the immobilized AChE made possible the quick removal of reagents (i.e., organophosphate, 2-pralidoxime, and protectant), thereby permitting accurate determi- nation of AChE activity before and after phosphorylation and aging. Paraoxon was 15-fold more potent in inhibiting AChE than DFP, while the percent aging following phosphorylation by diisopropylfluorophosphate (DFP) was much higher. Sodium fluoride (NaF), the most effective protectant against phosphorylation and aging, and the quaternary ammonium compounds reduced significantly AChE inhibition by DFP and paraoxon, to similar degrees. Even though the percent AChE activity that was lost to aging was reduced by these agents, aging as a percent of phosphorylated AChE was not reduced. Thus, their major effect was in reducing the percent AChE phosphorylation, which consequently resulted in reduction of total aged AChE. The finding that quaternary ammonium compounds protect against phosphorylation is consonant with the proposed presence of the active site of AChE in an aromatic gorge.

KEY WORDS: Acetylcholinesterase/Protectants; Bio- sensor/Acetylcholinesterase; Organophosphate In- hibitors; Acetylcholinesterase/Aging; Paraoxon/ Acetylcholinesterase inhibition; NaF/Acetylcho-

Received March 26, 1994. Address correspondence to Dr. Mohyee Eldefrawi, De-

partment of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 635 W. Baltimore St., Baltimore, MD 21201. Tel. (410) 706-3564; Fax (410) 706- 8012.

'Permanent address: Department of Biological Sciences, College of Science, King Abdulaziz University, Jeddah, Saudi Arabia.

0 1994 VCH Publishers, Inc.

linesterase Protection; DFP/Acetylcholinesterase Protection.

INTRODUCTION

Organophosphorus (OP) anticholinesterases (AntiChE) are irreversible inhibitors of acetylcholinesterase (AChE) (EC 3.1.1.7) and butyrylcholi- nesterase (BuChE) (EC 3.1.1.8) (1,2). They form stable phosphorylated complexes with both cholinesterases (ChEs) and have very slow sponta- neous reactivation rates (1). The phosphorylated AChE, however, can be reactivated with oxime reagents, e.g., 2-pralidoxime (2-PAM), unless the enzyme undergoes an aging process (3), which results in permanent AChE inactivation. "Aging" was first described for BuChE in 1955 by Hobbiger (4) and was found to occur much faster in acidic en- vironments and at higher temperatures and is affected by the structure of the alkyl group of the bound dialkylphosphate (5 ,6) .

The use of drugs to protect against OP poisoning has been the subject of investigation for over 40 years. Earlier studies demonstrated that carbamate AntiChEs protected AChE against inhibition by diisopropylfluorophosphate (DFP) and te- traethylpyrophosphate (7). Indeed, the injection of animals with physostigmine gave appreciable protection against poisoning by the nerve gas soman (pinacolyl methylphosphonoflouridate) (8). This was confirmed and extended to several other carbamates and against other OP nerve gases as well (9-12). Electrophysiologic evidence confirmed the protective effect of physostigmine against nerve gases (13,14). The mechanism of protection is believed to be due to their transient inhibition of AChE by carbamylating the same active site serine hydroxyl group that would other-

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262 M. S. DEHLAWI ET AL. Volume 9, Number 5, 1994

wise be irreversibly phosphorylated with the OP. Decarbamylation of AChE occurs spontaneously, thereby restoring its activity, while the OP is de- graded and its in situ dose is reduced.

Sodium fluoride (NaF) was also reported to protect against OP nerve gas poisoning when given prophylactically together with atropine (15). The administration of NaF to poisoned animals resulted in better recovery of ChE activity and res- toration of blocked neuromuscular function. How- ever, there is no direct evidence that NaF, which is a reversible inhibitor of AChE, protects the enzyme against irreversible phosphorylation by an OP. Neither is there information on whether reversible inhibitors of AChE (e.g., quaternary ammonium compounds) also protect AChE from phosphoryla tion.

Recent technologic developments have made possible the study of the molecular mechanism of action of reversible AntiChE protectants against AChE phosphorylation and agng. Knowledge of the molecular biology of AChE, its X-ray structure, molecular modeling, and site-directed mutagene- sis have revealed that AChE has a strong electrostatic dipole that is generated by the overall charge distribution throughout the enzyme, and also that the catalytic site of AChE (i.e., a triad of serine, histidine, and glutamate) lies in the bottom of a deep gorge that is lined predominantly with aromatic residues (16,17). Accordingly, the cationic ACh is driven down the gorge by serial electrostatic low-affinity binding. Therefore, a reversible inhibitor occupying space inside the gorge is expected to hinder access of an acylating agent to the esteratic site. At the same time, the use of immobilized AChE in fiber optic and potentiometric biosensors made it possible to monitor AChE activity and to detect AntiChEs (19,20), and also to measure the degree and rate of AChE phosphorylation and reactivation in the absence of inhibitor or reactivator. The present study was initiated using a light addressable potentiometric sensor (LAPS) (Molecular Devices, Menlo Park, CA) to evaluate acetylcholine (ACh), carbamylcholine, choline, and the reversible inhibitors tetramethylammonium and NaF, to evaluate them as protectants of AChE against phosphorylation and agng by DFP and paraoxon.

MATERIALS AND METHODS

Chemicals Biotinamidecaproate N-hydroxysuccinimide

ester, streptavidin (SA), 2-PAM methiodide, DFP,

and the chloride salts of acetylcholine, carbamylcholine, choline, and tetramethylammonium were purchased from Sigma Chemical Co. (St. Louis, MO). Paraoxon (>99% pure) was purchased from Chemical Service (West Chester, PA).

Immobilization of AChE Eel AChE (from Sigma Chemical Co.) was bio-

tinylated as previously described (18,19). Briefly, AChE (1 mg) was incubated for 2 hours at 23°C with biotinamide caproate N-hydroxysuccinimide in Krebs phosphate buffer. The biotinylated AChE (B-AChE) was separated from the free reagent by chromatography on Sephadex G-50, then was immobilized onto biotinylated nitrocellulose capture membranes using SA. B-AChE (15 ng) was mixed with 1 pg SA in 1 mL of buffer (10 mM Na,HPO,, pH 7.0, 150 mM NaC1, 0.1% bovine serum albu- min, 0.05% NaN3) and captured by filtration over the biotinylated capture membrane using a low filtration rate (0.13 mL/min). The capture membrane was then washed with 1 mL of buffer containing 0.05% Tween 20 at a high flow rate (0.75 mL/min), thereby removing all excess reagents and nonim- mobilized enzyme. An eight-compartment sample holder was placed on top of the capture membrane in an eight-channel filtration unit to provide eight assay sites per membrane. The LAPS instru- ment has four eight-channel units, and thus is ca- pable of high-volume sample processing. One site on each membrane does not receive enzyme and is used as a blank, and another site receives enzyme and is left untreated as a control, thereby providing six treatment sites on each membrane.

Measuring AChE Activity before and after -Phosphorylation AChE activity, based on the rate of hydrolysis

of ACh (pV/s) was measured in the Threshold sil- icon-based LAPS system as previously described (18,19), before and after exposure to inhibitors. The capture membrane carrying the immobilized enzyme was exposed to DFP or paraoxon, in the presence or absence of a protecting chemical, by filtration of 1 mL of assay buffer over the membranes at a very low flow rate (0.06 mL/min). In other words, AChE was exposed to the OP for 16.67 minutes during the assay. Preliminary experiments indicated that this method of exposure to 10 pM DFP resulted in 88 ? 2% inhibition of AChE activity. The same concentrations of AChE and DFP incubated in bulk solution prior to immobilization gave 96 k 0.5% inhibition. Phosphorylation of the

Volume 9, Number 5, 1994 ACETYLCHOLINESTERASE PROTECTANTS 263

immobilized enzyme was irreversible with a buffer wash. The exposure to DFP or paraoxon, in the presence or absence of a protective agent, was followed by a wash step with 1 mL buffer at the high flow rate (this wash step was completed in 1.3 minutes) to remove excess and unreacted reagents.

Reactivation of Phosphorylated AChE with 2-PAM The phosphorylated AChE (immobdized on the

capture membrane) was activated by exposure to 2-PAM (1 mM unless otherwise stated) during washing of each site on the capture membrane with 1 mL buffer containing 2-PAM (filtration rate: 0.06 mL/min). This step was followed by a fast wash with 1 mL buffer (filtration rate: 0.75 mL/min) to remove excess 2-PAM. A single wash with 1 mM 2-PAM proved sufficient to yield complete reactivation. Additional washes with 2-PAM did not in- crease reactivation.

RESULTS

Phosphorylation and Aging of Immobilized AChE after Exposure to DFP An initial study was undertaken in order to se-

lect the appropriate concentrations of DFP and 2- PAM to study the protective effects of choline derivatives and NaF against AChE phosphorylation and aging. DFP gave almost 100% inhibition at concentrations above 10 pM (Figure 1A) and 1 mM 2-PAM produced maximal reactivation of the AChE (Figure lB), which was inhibited by exposure to 10 pM DFP. Exposure of the immobilized AChE to 10 pM DFP over a period of 16.7 minutes (with the time required to vacuum filter 1 mL through the capture membrane at a filtration rate of 0.06 mL/min), then washing the capture membrane with 1 mL of buffer quickly (1.3 minutes), resulted in near maximal inhibition (-90%) of AChE activity. Reactivation by 1 mM 2-PAM over a period of 16.7 minutes resulted in maximal reactivation of the phosphorylated AChE. Accordingly, 10 pM DFP and 1 mM 2-PAM were selected as the working concentrations to use in subsequent experiments designed to study the protection of AChE against phosphorylation and enzyme aging by choline derivatives and NaF.

The immobilized AChE was slightly less sen- sitive to phosphorylation and aging by DFP than

AChE in solution (Table 1). The effect of incubat- ing DFP (10 pM) with AChE in solution for different periods (15-240 minutes) before AChE immobilization on .the capture membrane was compared to its effect on the immobilized AChE by vacuum filtration for 16.7 minutes, and measuring AChE activity after the indicated time intervals. It is clear that immobilization of AChE significantly reduced its phosphorylation by DFP ( p < 0.05). Reactivation of phosphorylated AChE by 1 mM 2-PAM was also significantly higher with the immobilized AChE than the enzyme exposed in solution prior to immobilization ( p < 0.05). In both cases, the larger the interval between exposure to DFP and reactivation by 2-PAM, the lower the reactivation.

Protection of AChE against DFP-Induced Phosphorylation and Aging Aging of AChE, following exposure to DFP, is

defined as the percent of phosphorylated enzyme that is insensitive to reactivation by 1 mM 2-PAM. Protecting agents at 10 mM were added to the 1 mL wash buffer containing 10 pM DFP, thereby allowing simultaneous exposure of immobilized AChE to DFP and the protecting agent during the vacuum filtration step of 16.67 minutes. Percent protection against phosphorylation was calculated by dividing the difference in percent enzyme inhibition, in absence and presence of a protective agent, over the percent inhibition in absence of a protective agent and multiplying by 100.

Exposure of the immobilized AChE to 10 pM DFP resulted in 82.9 i 2% inhibition (mean of three experiments) and reactivation with 1 mM 2-PAM resulted in reactivation of 52% of the inhibited activity and percent aging of 30.9 5 2%. Exposure of the immobilized AChE to 10 mM of the protective agent alone had no effect on AChE activity. The presence of choline derivatives or NaF during exposure to 10 pM DFP sigruficantly reduced AChE phosphorylation by DFP (Figure 2, Table 2). Ace- tylcholine and NaF were the two most potent protecting agents against phosphorylation; reducing it by 88.8 and 90.0%, respectively. However, their presence did not provide additional significant reduction of percent aging of the phosphorylated AChE (58.9 and 27.0%, respectively (Table 2)). Al- though the choline derivatives and NaF reduced the enzyme activity lost as a result of aging as a percent of control, with NaF as the most potent (Table 2), they actually gave no protection against aging of the DFP-phosphorylated enzyme, except for carbamylcholine. This choline derivative gave


80

60

I c 1 10 100 8

DFP Concentration (pM)

~ _ _ ~~ ~

0.01 0.1 1 10

2-PAM Concentration (mM)

FIGURE 1. Inhibition of immobilized AChE by DFP and reactivation with 2-PAM. (A) Percent AChE inhibition by exposure to different concentrations of DFP (0) and percent aging (a), i.e., AChE activity that did not recover after reactivation with 10 mM 2-PAM. (B) Potency of 2-PAM in reactivating AChE that had been exposed to 10 mM DFP. One hundred percent AChE activity represents the control level prior to exposure to DFP. Symbols and vertical bars are means 2 SEM of triplicate experiments. A symbol without a bar reflects an SEM that is less than the size of the symbol.

TABLE 1. The Effect of AChE Immobilization on Its Inhibition by DFP (10 pM) and Aging of the Phosphorylated Enzyme

Time (minutes) Immobilized' In Solutiond Immobilized' In Solution'

0 82.9 f 2.0 94.1 2 0.6 30.9 f 2.0 40.1 t 1.0 15 88.0 ? 1.0 96.3 2 0.5 38.8 2 1.5 44.3 -t 2.0 30 90.0 * 0.5 96.1 2 0.5 39.6 2 1.0 48.2 2 2.0 60 91.0 2 0.4 97.7 ? 0.6 43.9 2 1.0 50.5 t_ 1.0

120 91.0 2 0.4 98.6 2 0.2 48.5 2 2.0 57.9 2 1.0 240 87.7 5 0.6 98.1 2 0.2 54.5 2 1.0 65.6 2 2.0

Percent Inhibition" Percent Aging

Control 0 0 0 n ~~

"The percent inhibition and aging of AChE in solution is significantly ( p < 0.05) higher than the immobilized AChE at every time period.

bPercent aging is the percent of inhibited AChE activity that is not reactivated by exposure to 1 mM 2- PAM over 16.6 minute. The percent aging, resulting from exposure of AChE to DFP in solution, was significantly higher ( p < 0.05) than for the immobilized enzyme at every time period.

'Immobilized AChE was exposed to DFP during the vacuum wash step and enzyme activity was measured after the indicated time intervals.

solution, B-AChE was incubated with DFP for the indicated interval before capturing the enzyme on the nitrocellulose membrane. The enzyme activity was measured immediately after enzyme capture and removal of excess DFP.

the least protection against DFP-induced phosphorylation (32.8 -+ 2% occurred), but was the most effective in protecting against aging of the DFP- phosphorylated enzyme (13.7 ? 0.9%).

The protective effects of NaF (the most potent protecting agent in this study and a reversible inhibitor of AChE) and choline (with little or no

known effects on AChE) against DFP-induced phosphorylation and aging were concentration dependent (Figure 2). Sodium fluoride, though toxic compared to choline at the concentrations used, was much more effective in protecting AChE against both phosphorylation and aging by DFP at all concentrations (0.1-10 mM).


0

A

P B

FIGURE 2. The concentration- dependent protective effects of (A) choline and (B) NaF against inhibition and agmg of AChE by 10 PM DFP. Choline and NaF (at the indicated concentrations) were added to the assay buffer along with 10 ,uM DFP. Aging is the percent of phosphorylated enzyme that was not reactivated by 1 mM 2-PAM. Symbols and bars are the same as in figure legend 1.

0.1 0.3 1 3 10 0.1 0.3 1 3 10

Concentration of Protectant (mM)

TABLE 2. Protection of AChE from Irreversible Phosphorylation and Aging by 10 pM DFP or 1 pM Paraoxon with Choline Derivatives and NaF

Phosphorylation (as a percent of control) Protectant Compound (10 mM)

DFP 82.9 2 2.2 30.9 i 1.8 + Tetramethylammonium 21.1b 2 1.3 41.7 2 1.7 + Carbamylcholine 32.8b t 2.1 13.7' 2 0.9 + Choline 27.2' 3- 1.4 58.7 2 2.7 + Acetylcholine 11.2' i 1.2 58.9 5 2.3 + NaF 10.0' 2 0.8 27.0' i- 1.4

Percent Aging"

Paraoxon 96.0 t 1.8 + Tetramethylammonium 64.0b 2 0.9 + Carbamylcholine 23.1b 2 1.1 + Choline 48.9' i 0.8 + Acetylcholine 34.5" 2 0.7 + NaF 9.2" i 0.6

11.2 i- 0.7 5.0' 2 0.6 4.0' i 0.6

19.6 2 1.8 32.6 i 2.1 31.7 2 1.4

'Percent aging equals the percent of the phosphorylated AChE that is not reactivable by 2-PAM. 'All percent phosphorylation values, in the presence of various protecting compounds, are highly sig-

'Chemicals that significantly decreased aging ( p < 0.05) caused by'DFP or paraoxon. nificant ( p < 0.001) compared to DFP or paraoxon alone.

Relative Potencies of Choline Derivatives and NaF in Protecting AChE from Inhibition and Aging by DFP and Paraoxon Paraoxon was a much more potent inhibitor of

the immobilized AChE than DFP (Figure 3 ) . The ICs0 values for inhibition of the immobilized AChE by paraoxon and DFP, determined by log probit analysis of the data of Figure 3, were 80 nM and 1.2 pM, respectively. Thus, paraoxon was 15 times more potent than DFP in phosphorylating the immobilized AChE. On the other hand, the percent aging following AChE phosphorylation by DFP was

much higher than the percent aging resulting from paraoxon. However, the percent of AChE that aged with either OP was independent of the OP concentration (Figure 3) . All choline derivatives and NaF were effective in protecting AChE against inhibition and aging by paraoxon (Table 2), with NaF and carbamylcholine as the most potent in protecting AChE from phosphorylation. On the other hand, choline and tetramethylammonium were the most potent in protecting phosphorylated AChE from aging. Neither ACh, choline, nor NaF protected against paraoxon-induced aging. Aging, calculated as a percent of nonreactivatable phos-


10 I I

0 0.01 0.1 1 10 100

Organophosphate Concentration (FM)

FIGURE 3. Relative potencies of paraoxon (A, A) and DFP (0, 0) in causing inhibition (solid symbols) of immobilized AChE and aging (open symbols) of the phosphorylated AChE. Symbols and bars are the same as in figure legend 1.

phorylated AChE, was actually higher in the case of DFP-phosphorylated AChE in the presence of choline, ACh, and tetramethylammonium and in the case of the paraoxon-phosphorylated AChE by the presence of choline, ACh, and NaF.

DISCUSSION

There are two major findings in this study that shed light on the effect of several drugs in protecting AChE activity against phosphorylation by OP AntiChEs. The first is that all the tested drugs that have the trimethylammonium group protect AChE significantly from phosphorylation by DFP and paraoxon (Figures 1 and 2, Table 2). This in- cludes drugs that form a covalent bond with the serine hydroxyl group of the esteratic site (i.e., ACh) as well as those that do not (i.e., carbamylcholine, choline, and tetramethylammonium) . Older models of the active site of AChE portrayed it as a linear stretch of amino acid residues on the surface of the enzyme with an anionic site next to the esteratic site (1-3). However, the new model (16,17) suggests that the active site lies at the bottom of a gorge, whose lumen is lined with aromatic residues. AChE has a strong electrostatic dipole, which attracts the trialkylammonium group of the substrate, choline derivatives, and edro- phonium. Alternatively, the r electrons of the aromatic residues that line the lumen of the gorge might serve as the electronegative source (as pro-

posed for potassium ion selectivity in other proteins) for coordination of the quaternary nitrogen (20). Thus, the quaternary ammonium compounds may block access of OF‘ AntiChEs to the esteratic site, and interfere with the irreversible inhibition (i.e., phosphorylation) by OP AntiChEs and pos- sibly affect enzyme aging as well.

Protection against DFP-induced phosphorylation of AChE with ACh was established many years ago (3,7), but it was assumed that protection was a result of ACh occupation of the esteratic site. However, the protectants tetramethylammonium and choline are not expected to bind to the esteratic site, but may bind to the negatively charged electrostatic dipole lining the lumen of the catalytic gorge. Yet, they are less effective protectants than the substrate ACh (Table 2). ACh may be a better protectant, because in addition to occupying the aromatic gorge like choline, it also acylates the esteratic site, even though transiently. The bulkier carbamylcholine, which is resistant to hydrolysis by AChE, is the least potent in protecting against phosphorylation by DFP. The absence of correlation between potencies of the four quaternary ammonium compounds in protecting AChE from phosphorylation with DFP compared to paraoxon (Table 2) suggests that this protection is affected by the structure of the OP in addition to that of the protectant.

Sodium fluoride was originally thought of only as a reversible inhibitor of AChE that does not provide any protection against irreversible inhibition by OP AntiChEs (7). Although later studies showed that NaF protected animals from poisoning by OF’ nerve agents (15), no investigation of the direct protective effect of NaF on AChE against OP AntiChEs has been undertaken. The afore- mentioned data show that NaF is more potent than choline and derivatives (Figure 2, Table 2), includ- ing ACh, in protecting AChE against irreversible inhibition by DFP or paraoxon. Since the mechanism of interaction of NaF with AChE is not yet known, it is difficult to explain the potency of NaF as a protectant against phosphorylation.

An advantage of the use of immobilized AChE for studying AChE phosphorylation and aging is the feasibility of measuring AChE activity prior and subsequent to exposure to the OP and removal of the unreacted OP. The same enzyme can then be reactivated with 1 mM 2-PAM and the percent reactivation measured after removal of 2-PAM. Thus, there is no interference from unreacted OP or excess 2-PAM. Older protocols using particulate enzyme preparation and centrifugation to wash the particulate enzyme at each step took hours to


complete. In addition, centrifugation is less effi- cient in removing excess unreacted agents than the vacuum filtration wash used in the LAPS instru- ment. The LAPS system made it possible to make, in less than 1 hour, eight simultaneous measure- ments of basal AChE activity, as well as activities after inhibition with the OP and after reactivation with 2-PAM. Furthermore, it is possible to study the effect of the five protective agents simulta- neously. Alternatively, one can use any number of the eight enzyme sites on the capture membranes for replication. Although the kinetic parameters (K, and V,,,) of the immobilized purified AChE are the same as those of the membrane-bound AChE (19), it is not certain whether the former retains all of its stereochemical characteristics.

The phosphorylation of AChE is followed by a time-dependent permanent loss of catalytic activity that is not recoverable by 2-PAM (i.e., aging)(4,5,6). The immobilized AChE exhibits similar time-dependent aging as does AChE in solution, even though immobilization reduces by about 10% both the percent inhibition and percent aging of AChE with DFP (Table 1). The second major finding of the study is that aging of AChE occurs as long as the enzyme is phosphorylated, since protectants reduce aging as a percent of control level, but not as a percent of phosphorylated AChE. The data suggest that the presence of choline derivatives or NaF does not interfere with dealkylation of the phosphorylated AChE that leads to aging. Thus, the drugs that are more potent in protecting against phosphorylation are the ones that are more potent against aging, calculated as a percent of control.

The finding that paraoxon is a much more potent inhibitor of AChE than DFP (Figure 3 ) con- firms previous reports (1,2,19). However, paraoxon causes less aging of the phosphorylated AChE than does DFP (Table 2, Figure 3) . This differential effect on aging is also predictable from earlier in- vestigations on the effect on aging of the OP alkyl substituent (4,5). It is also evident that the protection afforded by choline derivatives against aging of AChE, calculated as percent aging in Table 2, is different when the phosphorylating agent is DFP than when it is paraoxon. In the case of the former, tetramethylammonium provides one of the lowest recoveries of the phosphorylated AChE (41.7 +- 2% aging), while in the latter it is one of the best protectants (5 2 1% aging). However, since the most prominent effect of the protectant is against AChE phosphorylation, the difference in the percent recovery with 2-PAM is a less impor- tant factor in determining its protective potency.

This study provides evidence that millimolar NaF can significantly protect AChE from inhibition and aging by OP AntiChEs, more so than the choline derivatives, tested (Table 2). It is also possible that some of the NaF action is due to its reactivation of the phosphorylated AChE. It is clear that it is not necessary for the protectant to form a covalent bond with the serine hydroxyl group of AChE, as the carbamate protectants do.

ACKNOWLEDGMENTS

This research was partially supported by an Army Research Office grant number DAAL03-92- G-0064 (MEE), a Senior NRC Fellowship (NAA) and a Bressler award (ATE).

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