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The plant growth regulator ethephon (2-chloroethylphosphonic acid) inhibits human butyrylcholinesterase (BChE) by making a covalent adduct on the acti...
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Newly Observed Spontaneous Activation of Ethephon as a Butyrylcholinesterase Inhibitor Mariya S. Liyasova,† Lawrence M. Schopfer,*,† Sean Kodani,‡ Stephen R. Lantz,‡ John E. Casida,‡ and Oksana Lockridge† †

Department of Environmental, Agricultural & Occupational Health and Eppley Institute, University of Nebraska Medical Center, Omaha, Nebraska 68198-5950, United States ‡ Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, University of California, Berkeley, California 94720-3112, United States ABSTRACT: The plant growth regulator ethephon (2chloroethylphosphonic acid) inhibits human butyrylcholinesterase (BChE) by making a covalent adduct on the active site serine 198. Our goal was to extend earlier studies on ethephon inhibition. Addition of freshly prepared ethephon to BChE in buffered medium, at pH 7.0 and 22 °C, resulted in no inhibition initially. However, inhibition developed progressively over 60 min of incubation. Preincubation of ethephon in pH 7−9 buffers increased its initial inhibitory potency. These observations indicated that ethephon itself was not the inhibitor. About 3% of the initial ethephon could be trapped as a BChE adduct. Mass spectral analysis of the active site peptide from inhibited BChE showed that the inhibitor added a mass of 108 Da to the active site serine on peptide FGES198AGAAS. This result rules out a previous hypothesis that ethephon adds HPO3 to BChE (added mass of 80 Da). To accommodate these observations, we propose that in aqueous media at neutral to slightly alkaline pH about 3% of the ethephon is converted (t1/2 = 9.9 h at pH 7.0) into a cyclic oxaphosphetane which is the actual BChE inhibitor forming the 2-hydroxyethylphosphonate adduct on BChE at Ser198 while about 97% of the ethephon breaks down to ethylene (t1/2 = 11−48 h at pH 7.0) which is responsible for plant growth regulation.

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INTRODUCTION Ethephon (2-chloroethylphosphonic acid) is a plant growth regulator used for ripening fruits, vegetables, and grains. It penetrates plant tissues and decomposes into the potent plant hormone ethylene.1 In 1997, the annual use of ethephon in the U.S. was 5.5 million pounds.2 Ethephon inhibits plasma butyrylcholinesterase (BChE) in vivo upon oral administration to rats, mice, dogs, and humans3−5 and in vitro in plasma or purified preparations.3,5,6 Administration of ethephon to human subjects at 1.8 mg/kg/ day for 28 days resulted in clinical signs of subchronic toxicity that included diarrhea, urgency of bowel movements, urinary urgency, and stomach cramps. Based on these results, a lowest observed effect level of 1.8 mg/kg/day was established for ethephon. No effects were noted with regard to hematology, urinalysis, or serum chemistry. Based on animal testing, ethephon does not pose a risk of mutagenicity, genotoxicity, developmental toxicity, reproductive toxicity, oncogenicity, or acute neurotoxicity nor does it pose any acute dietary risks.4 In vitro studies demonstrated that [33P]ethephon irreversibly inhibits BChE.6 Haux et al.7 suggested that ethephon inhibits BChE by adding a HPO3 group to the active site serine. Additional experiments demonstrated that (1) purified ethephon was inhibitory but that common contaminants in commercial ethephon preparations were not;8 (2) inhibition of BChE by ethephon reduced the level of post-labeling inhibition by [3H]-diisopropylfluorophosphate,6,7 supporting the con© 2013 American Chemical Society

clusion that inhibition occurred at the active site of BChE; and (3) inhibition did not occur below pH 6, implicating the dianion form of ethephon in the inhibition process.6,7 Though these observations provided a persuasive argument for direct phosphorylation of BChE by ethephon, a couple of observations were worrisome. Notably, (1) the time course for inhibition of BChE by ethephon appeared to accelerate, suggesting a progressive increase in the inhibitory potency of the ethephon preparation;7 and (2) though the dianionic form of ethephon appeared to be responsible for inhibition, a dianion reacting with the active site of BChE was unexpected6−9 because the BChE active site normally interacts with positively charged substrates. In the present report, we extended the studies on ethephon inhibition of BChE (1) by more thoroughly characterizing the time course of inhibition, (2) by determining the stoichiometry of inhibition, and (3) by determining the mass of the adduct formed on ethephon-treated BChE. We found that (1) the time course for inhibition of BChE by ethephon showed a distinct acceleration; (2) preincubation of ethephon at pH 7−9 resulted in more rapid inhibition reaction; (3) only about 3% of the starting ethephon concentration went to inhibit BChE; and (4) the mass of the adduct formed on BChE was 108 Da, representing addition of 2-hydroxyethylphosphonate Received: December 13, 2012 Published: February 14, 2013 422

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final ethephon concentration in each inhibition mixture was 0.125 mM. Formation and degradation of the inhibitor was visualized by plotting the [initial rate for inhibition of BChE] versus the [time for incubation of ethephon]. The results are shown in Figure 3. Determination of the Stoichiometry for Ethephon Inhibition of BChE. A 200 mM ethephon solution in acetonitrile was diluted in McIlvaine buffer (pH 7.0) to concentrations ranging from 0.1 to 20 mM and used immediately to inhibit 0.16 μM BChE in McIlvaine buffer (pH 7.0). Final ethephon concentrations in the BChE/ ethephon reaction mixtures were 1, 2, 3, 4, 5, 6, or 7 μM. The mixtures were incubated at 22 °C. After various time intervals, a 5 μL aliquot was taken from the BChE/ethephon incubation mixture for measurement of residual BChE activity. Inhibition was followed until the BChE activity stabilized. The results are shown in Figure 4. Measurement of Spontaneous Reactivation of EthephonInhibited BChE. One milliliter of BChE (157 units or 2.5 nmol) in 20 mM potassium phosphate (pH 7.6) containing 1 mM EDTA was mixed with 5 μL of 50 mM fresh ethephon (250 nmol in acetonitrile). The sample was incubated at 22 °C in the dark. BChE activity, tested 24 h later, was below the limits of detection. Excess ethephon was removed by dialysis against 4 L of 10 mM ammonium bicarbonate buffer (pH 8.0) in a Pierce Slide-A-Lyzer dialysis cassette (7000 molecular weight cutoff, from Thermo Scientific, Rockland, IL) for 48 h. The dialyzed BChE was stored at 22 °C, and activity was monitored at intervals for 3 months. An uninhibited sample was run in parallel. Determination of the Mass of the Ethephon Adduct on BChE. One hundred units of BChE (1.6 nmol, 0.14 mg) in 0.1 mL of 10 mM ammonium bicarbonate (pH 8.1) was treated with a 125-fold molar excess of ethephon (200 nmol). The inhibition mixture was incubated for 24 h at room temperature. Residual BChE activity was below the limit of detection. Inhibited BChE was acidified with 2 μL of 25% TFA (v/v) and then digested with 250 μg of freshly prepared pepsin (25 μL of 10 mg/mL pepsin dissolved in 5% formic acid, v/v) for 2 h at 37 °C. Peptides were separated by HPLC and analyzed on the MALDITOF/TOF 4800 mass spectrometer as described below. Test for Aging of Ethephon-Inhibited BChE. The possibility was tested that the adduct formed by reaction of ethephon with BChE would age. Thirty units of BChE (0.49 nmol) was treated with 1600fold molar excess of ethephon (800 nmol). After 3 h at room temperature, residual BChE activity was 0.6% of control. An aliquot containing 0.24 nmol inhibited BChE was digested with pepsin, while the rest of the sample (0.24 nmol) was aged at 37 °C for 64 h before it was digested with pepsin. Both samples were analyzed by MALDITOF mass spectrometry. Expected masses for the labeled active site peptides in negative mode were 902 Da for FGESAGAAS with an added mass of 108 for the 2-hydroxyethylphosphonate adduct and 874 for FGESAGAAS with an added mass of 80 Da for a phosphate adduct. Test for More than One Type of Inhibitor. The possibility was tested that ethephon produced more than one type of inhibitor and that the inhibitor in a freshly prepared ethephon solution might be different from the inhibitor in an activated ethephon solution that had maximum inhibition potency. One hundred thirty units of BChE (2.1 nmol, 0.18 mg) in 0.5 mL of 10 mM ammonium bicarbonate buffer (pH 8.1) was treated with a 1000-fold molar excess of fresh ethephon (5 μL of 0.5 M ethephon in acetonitrile prepared from solid ethephon immediately before use) or with an ethephon solution that had been preincubated in 10 mM ammonium bicarbonate buffer (pH 8.1) for 24 h. It took 80 min to get 99.8% inhibition with fresh ethephon but less than 5 min with the preincubated ethephon solution. Each 0.5 mL sample was divided into two tubes. One set was digested with 40 μg of pepsin and the second set with 4 μg of trypsin. Samples intended for digestion with trypsin were denatured in a boiling water bath before addition of trypsin. Digests were analyzed in a MALDI-TOF/TOF 4800 mass spectrometer as described below. HPLC Purification of Peptic Peptides. BChE treated with ethephon, as well as untreated controls, was digested with pepsin. The digests were diluted to 0.5 mL with water and injected into a Waters 625 liquid chromatography system with a Phenomenex Prodigy 5 μm C18 100 × 4.60 mm column. Peptides were eluted with a 60 min gradient

OP(CH2CH2OH)(OH) or equivalent to the active site serine. These observations strongly suggest that ethephon is activated before it can inhibit BChE. The structure of the activated BChE inhibitor is tentatively assigned as 2-oxo, 2hydroxy-1,2-oxaphosphetane.

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MATERIALS AND METHODS

Materials. Ethephon (CAS number 16672-87-0) was from Chem Service Inc. (West Chester, PA, cat. number PS-325). Ethephon was 99% pure by HPLC analysis. It was dissolved in acetonitrile at concentrations up to 500 mM and stored at −20 °C. The following were from Sigma-Aldrich (St. Louis, MO): porcine pepsin (10 mg/ mL, P-6887), S-butyrylthiocholine iodide (B-3253), 5,5′-dithiobis(2nitrobenzoic acid) (D-8130), formic acid (subsidiary Fluka, 94318), and 2,5-dihydroxybenzoic acid (DHB) (subsidiaries Fluka, 85707, and Acros, 165200050). Trifluoroacetic acid (TFA) (A11650) and acetonitrile (DNA sequencing grade, BP1170) were from Fisher Scientific (Fair Lawn, NJ). Sequencing grade trypsin (V5113) was from Promega (Madison, WI). BChE was purified from outdated human plasma as described.10 Methods. BChE Activity Assay. Activity was measured at 25 °C in 0.1 M potassium phosphate buffer (pH 7.0) containing 1 mM butyrylthiocholine and 0.5 mM 5,5′-dithiobis(2-nitrobenzoic acid) in a final volume of 2 mL. The increase in absorbance at 412 nm was monitored in a Gilford spectrophotometer using quartz cuvettes with a 1 cm path length. The extinction coefficient for the product was 13 600 M−1 cm−1.11 One unit of BChE activity was defined as the amount that hydrolyzes 1 μmol of butyrylthiocholine in 1 min. BChE activity was converted to BChE concentration using a specific activity of 720 units/mg12 and a subunit molecular weight of 85 000 g/mol.13 A 0.16 μM BChE solution has an activity of 9.8 units/mL. Buffers for Inhibition Studies. McIlvaine buffers (0.2 M Na2HPO4 titrated with 0.1 M citric acid) at pH 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 were prepared as described.14 For pH 9, the buffer was 0.2 M glycine/ NaOH. For pH 11.0, the buffer was 0.2 M Na2HPO4. All buffers were autoclaved and stored at room temperature (22 °C). BChE Inhibition by Ethephon. A 200 mM ethephon solution in acetonitrile was diluted to 5 mM in McIlvaine buffer, pH 7.0. Aliquots were then mixed with BChE in McIlvaine buffer (pH 7.0) to give a final BChE concentration of 0.16 μM and final ethephon concentrations of 0.25, 0.5, or 1 mM. A 5 μL aliquot of each freshly prepared BChE/ethephon mixture was immediately assayed for BChE activity. Additional 5 μL aliquots of the BChE/ethephon mixture were assayed for BChE activity every few minutes until complete inhibition was achieved. The incubations were performed at 22 °C. The results are shown in Figure 1. Effect of pH on Ethephon Inhibition Potency. Ethephon solutions (5 mM) were prepared by mixing 5 μL of 200 mM ethephon in acetonitrile with 195 μL of buffers at pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 11.0. Each freshly prepared ethephon solution was immediately tested for inhibition potency by determining its ability to inhibit BChE. Five microliters of ethephon solution was added to 95 μL of BChE (0.16 μM BChE in McIlvaine buffer pH 7.0) to make a final ethephon concentration of 0.25 mM. Inhibition of BChE was followed by removing 5 μL aliquots of the BChE/ethephon mixture and assaying the BChE activity every few minutes until complete inhibition was achieved. A plot of [log % BChE activity] versus the [time for incubation of BChE with ethephon] gave the inhibition time course. The initial rate from that time course was used as a measure of inhibitor potency. The original ethephon solutions were reassayed for inhibition potency after 24 and 168 h of incubation at 22 °C. A representative result for pH 9.0 is shown in Figure 2. Time Course for Activation and Degradation of the Inhibitor. Freshly prepared ethephon (5 mM) was incubated in 600 μL of McIlvaine buffer (pH 7.0) at 20 °C. Periodically, the potency of the inhibitor was tested by mixing a 5 μL aliquot of the ethephon solution with 195 μL of BChE (0.4 μM BChE in McIlvaine buffer pH 7.0, 20 °C) and determining the rate of BChE inhibition, as described in the Methods section “Effect of pH on Ethephon Inhibition Potency”. The 423

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from 0.1% TFA (v/v) in water to 60% acetonitrile/0.09% TFA (v/v) at a flow rate of 1 mL/min. One milliliter fractions were collected from 6 to 31 min. A 1 μL aliquot from each fraction was analyzed in the MALDI-TOF/TOF mass spectrometer as described below. Mass Spectrometry on MALDI-TOF/TOF 4800. Essentially salt-free 1 μL aliquots from peptic digests of BChE or from fractions of HPLCpurified peptic digests were spotted onto a 384-well Opti-TOF sample plate (Applied Biosystems, Foster City, CA), dried in air, and overlaid with 1 μL of DHB matrix (20 mg/mL DHB in 50% acetonitrile, 0.1% TFA, 1% phosphoric acid, v/v). MALDI mass spectra were acquired on a MALDI−TOF/TOF 4800 mass spectrometer (Applied Biosystems, Framingham, MA) in negative reflector mode with the laser energy adjusted to 6000 V. Data collection was controlled by 4000 Series Explorer software (version 3.5). Each mass spectrum was the average of 500 laser shots. The mass spectrometer was calibrated using standard peptides (Cal Mix 5 from Applied Biosystems). MS/MS fragmentation spectra were taken by collision-induced dissociation in positive mode at 1 kV collision energy using ambient air as collision gas (at 2 × 10−6 Torr) and with metastable ion suppression on. Each MS/MS spectrum consisted of 500 laser pulses taken with the laser energy adjusted to yield optimal signal-to-noise. MS/MS calibration used the fragmentation spectrum of angiotensin 1. Spectra were analyzed with Data Explorer Software. Mass spectra were examined manually for the presence of adducts resulting from treatment with ethephon solution. The amino acid sequences of the candidate peptides were determined by manual inspection of the MS/MS fragmentation spectra, with the aid of the MS-Product algorithm from Protein Prospector (http://prospector. ucsf.edu/prospector/cgi-bin/msform.cgi?form=msproduct) and the Proteomics Toolkit from DB Systems Biology (http://db. systemsbiology.net:8080/proteomicsToolkit/FragIonServlet.html).

concentration. When I is much greater than E0 and thus is essentially constant with time, the plot of [log(residual BChE activity, %)] versus [inhibition time] yields a straight line.15 Under the conditions of this experiment, the reaction of ethephon with BChE would be expected to be first-order which would have made the time courses in Figure 1 linear. On the contrary, the time courses were convex. Figure 1 shows that for each ethephon concentration there was very little inhibition during the first few minutes. The inhibition curves accelerate with increasing time of incubation, indicating that the concentration of the active inhibitor increases as the reaction proceeds. It took almost 40 min for a 6250-fold molar excess of ethephon (1 mM) to completely inhibit BChE. Preincubation of Ethephon at Alkaline pH Increases the Production of the Active Inhibitor. The acceleration of the inhibition curves in Figure 1 raised two questions: (1) Does pH affect production of the active inhibitor? (2) Does production of the active inhibitor require the presence of BChE? To answer these questions, 5 mM ethephon was prepared in buffers with pH ranging from 3.0 to 11.0 and incubated at 22 °C. The inhibition potency of each ethephon preparation was measured immediately after preparation, after 24 h incubation, and again after 168 h (in most cases). Preincubation of ethephon at pH 3.0, 4.0, or 5.0 for 24 h did not affect the inhibitory properties of ethephon; that is, the inhibition time courses, assayed at pH 7.0, followed the same accelerating profile before and after incubation (similar to that shown in Figure 1), indicating that the inhibitor was not produced at acid pH. Preincubation of ethephon for 24 h at pH 6.0 resulted in a minor enhancement in inhibitory power, while incubation at pH 7.0, 8.0, or 9.0 resulted in a progressive increase in inhibitory power as the pH increased. Figure 2 demonstrates this phenomenon for ethephon preincubated at pH 9.0. With freshly prepared ethephon, the inhibition time course followed an accelerating profile, but after the 24 h preincubation, the time course was faster and linear. At the 15 min time point, BChE treated with preactivated ethephon was inhibited 99%, whereas BChE treated with fresh ethephon was inhibited only 30%. When ethephon was preincubated at pH 11.0 for 24 h, the inhibitory potency decreased slightly compared to freshly prepared ethephon, suggesting that the ethephon was activated and then degraded by 24 h at this pH. For preparations at pH 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0, the incubations were continued for 168 h. There was little effect of this increase in incubation time on the inhibition potency for the pH 3.0, 4.0, and 5.0 preparations. However, the pH 6.0, 7.0, and 8.0 preparations were much less potent after 168 h of

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RESULTS Ethephon Itself Does Not Inhibit BChE. To establish kinetic parameters for BChE inhibition by ethephon, 0.16 μM BChE in McIlvaine buffer (pH 7.0) was treated with 0.25, 0.5, and 1 mM ethephon and incubated at 22 °C. Control BChE solutions were treated with equivalent amounts of McIlvaine buffer (pH 7.0). At various time intervals, an aliquot was removed from the inhibition mixture to measure residual BChE activity. Figure 1 is a plot of the [logarithm (log) of percent residual BChE activity] against the [time of inhibition] at various concentrations of ethephon. The percent residual activity was calculated from the expression (Ait/A0t) × 100, where Ait is the activity of BChE incubated with ethephon for time t, and A0t is the control BChE activity measured at time t. Irreversible BChE inhibition by organophosphorus compounds generally follows first-order kinetics expressed by the equation E = E0e−ki·I·t, where ki is the inhibition constant, I is the concentration of inhibitor, E is the enzyme concentration at a given inhibition time (t), and E0 is the initial BChE

Figure 2. Comparison of the inhibitory power of 0.25 mM fresh ethephon, made in 0.2 M glycine/NaOH (pH 9), to ethephon preincubated in the same buffer at 22 °C for 24 h. [Logarithm of % residual BChE activity] is plotted versus [time of inhibition] as for Figure 1.

Figure 1. Inhibition of BChE activity by different concentrations of ethephon in 0.2 M Na2HPO4/0.1 M citric acid buffer (pH 7.0) at 22 °C. [Logarithm of % residual BChE activity] is plotted versus [time of inhibition]. The BChE concentration was 0.16 μM. 424

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Fraction of Ethephon Converted into a BChE Inhibitor. To determine the percentage of ethephon that is converted into the BChE inhibitor, we incubated 0.16 μM BChE in McIlvaine buffer (pH 7.0) with 0, 1, 2, 3, 4, 5, 6, and 7 μM freshly prepared ethephon, at 22 °C, and measured BChE activity of the mixture after various time intervals until the reactions went to completion (Figure 4A). BChE activity was fully inhibited in the 6 and 7 μM ethephon reactions and partially inhibited for the other concentrations. The time courses for all concentrations of ethephon are essentially the same, suggesting that the rate for formation of the inhibitor is not dependent on the concentration of ethephon. The endpoint activity levels were plotted versus the ethephon concentration used in the reaction (Figure 4B). From the equation depicted in the graph, the intercept on the x-axis was equal to 5.3 μM. Thus, complete inhibition of 0.16 μM BChE required 5.3 μM of fresh ethephon. This means that only 3.0% (0.16 × 100/5.3) of fresh ethephon was converted into and reacted as the active BChE inhibitor.

incubation than after 24 h of incubation. The pH 8.0 preparation was less potent than the pH 6.0 preparation and less potent than it originally was at time zero. It was concluded that production of the active inhibitor is independent of the enzyme, but that it requires neutral to alkaline pH (pH 7.0 to 9.0 being most productive). Furthermore, prolonged incubation at pH 6.0 or above results in degradation of the inhibitor. It is important to note that these experiments required strong buffering to maintain constant pH throughout the process. Time Course for Activation and Degradation of the Inhibitor. Next, we determined the full time course for activation of ethephon and degradation of the resulting inhibitor, at pH 7.0 (as described in Methods). Figure 3 shows a plot of the [initial rate for inhibition of BChE] versus the [time for activation/degradation of the inhibitor] in the ethephon mixture. The initial rate for BChE inhibition is a measure of the amount of inhibitor in the ethephon incubation mixture. The amount of inhibitor increased progressively over the first 20 h (activation) and then declined (degradation). Both the activation and degradation can be treated as firstorder, irreversible reactions. Fitting of the time course to an irreversible, double-exponential expression yielded values of 0.070 h−1 for the rate constant of the fast phase (activation, gain in potency) and 0.023 h−1 for the rate constant of the slow phase (degradation, loss of potency). These rate constants correspond to t1/2 values of 9.9 and 30 h, respectively. A similar analysis performed at pH 9.0 (in 0.2 M glycine/NaOH buffer) and 22 °C produced values of 2 h for t1/2 of activation and 20 h for t1/2 of degradation.

Figure 4. Calculation of the stoichiometry for the reaction between BChE and ethephon. (A) BChE (0.16 μM) was mixed with 0, 1, 2, 3, 4, 5, 6, or 7 μM ethephon in 0.2 M Na2HPO4/0.1 M citric acid buffer (pH 7.0) and incubated at 22 °C. Residual BChE activity was determined daily for 7 days. The lines are simply connecting the points. (B) [Limiting BChE activity (as % of control)] remaining after 1 week is plotted versus the [initial concentration of ethephon]. The line is a linear fit to the points that corresponds to the equation inset into panel B. The intercept on the x-axis of 5.3 μM indicates that 3% of the initial ethephon concentration produced an inhibitor that irreversibly inhibited 0.16 μM BChE.

Figure 3. Time course for activation and degradation of the inhibitor created from ethephon at pH 7.0. The initial rate for inhibition of BChE in the presence of the inhibitor is a measure of inhibitor concentration. The initial rate is the slope from a plot of [BChE activity] versus [time of incubation of activated ethephon with BChE]; see Methods. The [initial rate of BChE inhibition] is plotted versus the [time for BChE inhibitor activation/degradation]. The data points are the measured time course. The line is a fit of the data to Iobs = [Ao] e−k1t(IA − IC) + ([Ao]k1/(k2 − k1))(e−k1t − e−k2t)(IB − [IC]) + [Ao]IC that was derived from the irreversible, double-exponential expression, [C] = [Ao][1 + (1/(k1 − k2))(k2e−k1t − k1e−k2t)].16 IA, IB, and IC are the inhibiting potencies of species A (unactivated inhibitor), B (activated inhibitor), and C (degraded inhibitor); k1 is the rate constant for the fast phase, and k2 is the rate constant for the slow phase; t is time; and Ao is the total amount of activated inhibitor. Fitting was made with Sigma Plot 2001, version 7.101 (Systat Software Inc., San Jose, CA) using optimized settings and parameters (step size = 0.01, tolerance = 0.01, iterations = 100, initial AoIB = 0.06, initial k1 = 0.06 h−1, initial k2 = 0.02 h−1, constants AoIA = 0 and AoIC = 0, constraints AoIB > 0, k1 > 0 and k2 > 0). The fitted values (and standard error) were AoIB = 0.146 (0.019), k1 = 0.070 h−1 (0.013), and k2 = 0.023 h−1 (0.004) with an overall coefficient of determination (R2) of 0.960.

The experiment was repeated using 2.6 μM BChE in 20 mM phosphate buffer (pH 7.0) and titrating the BChE activity with up to 2500 μM fresh ethephon in order to reduce the incubation time. At these higher ethephon concentrations, inhibition of BChE was complete in less than 24 h. It was found that 3.7% of fresh ethephon was converted to the effective BChE inhibitor. When preactivated ethephon was used, 1−7 μM with 0.4 μM BChE in McIlvaine buffer (pH 7.0) at room temperature, 2.0% of the starting ethephon went to inhibit BChE. For this experiment, ethephon was preactivated for 14 h at pH 8.0. Inhibition of BChE by Ethephon Is Irreversible. To investigate the ability of ethephon-inhibited BChE to reactivate spontaneously, 2.5 μM BChE was completely inhibited with a 100-fold molar excess of ethephon (250 μM) and dialyzed to remove excess ethephon. After dialysis, the BChE activity was 425

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0.035 units/mL (99.98% inhibition). Three months later, activity was 1.73 units/mL (98.90% inhibition). It was concluded that BChE inhibition by ethephon is essentially irreversible. This finding is consistent with the earlier report by Haux et al.7 Ethephon-Generated Inhibitor Adds a Mass of 108 Da to Serine 198 of BChE. To determine the nature of the adduct formed by ethephon on the active site serine (Ser198), we treated 1.6 nmol of BChE with a 125-fold molar excess of ethephon for 24 h at 22 °C, digested the reaction mixture with pepsin, and analyzed the peptic peptides with MALDI-TOF mass spectrometry. Control BChE treated with the equivalent amount of acetonitrile was processed along with the inhibited sample. Comparison of the mass spectra from ethephon-treated BChE (Figure 5B) with that from the control BChE (Figure Figure 6. Identification of the adduct formed by activated ethephon on Ser198 of human BChE. The MALDI MS/MS spectrum is taken for the parent ion [M + H]+ at m/z 904.2 (in positive mode). It shows major y- and b-ions consistent with the peptide FGES198AGAAS. The asterisk indicates fragments which retain the 108 Da adduct. The symbol Δ represents fragments which have undergone loss of the 108 Da adduct along with a molecule of water (minus 126 Da), thus converting the originally adducted serine into dehydroalanine. The structure of the 108 mass is hypothesized to be 2-hydroxyethylphosphonate.

Δ indicates this loss in Figure 6. Loss of the phospho-adduct along with a molecule of water from the modified serine is commonly observed in the fragmentation analysis of the active site peptide from BChE inhibited by organophosphorus toxicants. This loss from the parent ion (for example ΔMH at m/z 778.4) is generally the dominant feature in MS/MS spectra. The b5*, b6*, b7*, b8*, a4*, and y6* ions all retain the adduct, thus supporting the presence of an added mass of 108 on Ser198. The mass at m/z 402.1 is consistent with the internal fragment GES198AG where the active site has lost the 108 Da added mass but not the accompanying water. It was concluded that ethephon-inhibited BChE has an added mass of 108 Da on the active site serine 198. Figure 6 indicates the likely structure of the adduct as 2-hydroxyethylphosphonate, based on chemical arguments presented in the Discussion section. Ethephon-Inhibited BChE Does Not Age. A frequently observed phenomenon associated with organophosphorusinhibited BChE is that the adduct undergoes aging. Aging is an enzyme-mediated reaction in which an alkyl moiety is lost from the organophosphorus adduct. To test whether ethephoninhibited BChE aged, the inhibited complex was incubated at pH 8 and 37 °C for 64 h. The increased temperature (relative to 20−22 °C used for most experiments) was chosen in order to promote aging. There was no change in the mass between aged and unaged preparations for the peptic peptide carrying the adduct. Therefore, the ethephon-inhibited BChE does not age. Adduct Formed on BChE by Fresh Ethephon Is Identical to That Formed by Preincubated Ethephon. BChE was treated with fresh ethephon or ethephon preincubated at pH 8.1 for 24 h. It took an hour to get complete BChE inhibition with fresh ethephon and less than 5 min with preincubated ethephon. The mass spectra as well as MS/MS spectra were identical. Mass spectral fragmentation analysis revealed the +108 Da adduct on Ser198 in both

Figure 5. MALDI-TOF mass spectra of peptic digests for control, unmodified BChE, and ethephon-treated BChE. These mass spectra were acquired in negative mode using DHB matrix at a laser voltage of 6000 V. (A) Spectrum of control human BChE shows a peak at m/z 794.2 that corresponds to the active site peptide with the sequence FGES198AGAAS. (B) BChE treated with a 125-fold molar excess of ethephon has a new peak at m/z 902.2 that is consistent with the addition of 108 Da to serine 198, denoted by an asterisk (numbering for the human BChE protein is taken from the sequence given in accession # gi 34810860).

5A) revealed a new peak in the ethephon-treated sample at m/z 902.2 (negative mode). The appearance of the peak at m/z 902.2 was accompanied by disappearance of the peak at m/z 794.2 that corresponds to the singly charged, active site serine containing peptide FGES198AGAAS. The isotope patterns of both signals confirmed that they were singly charged. These observations strongly suggested that the 794.2 Da peptide had been converted into a 902.2 Da peptide by addition of a 108 Da mass. No peak consistent with phosphorylation of the active site serine (+80 Da) at m/z 874 was detected in the spectrum of ethephon-treated BChE. To confirm the identity of the peptide at m/z 902.2 (negative mode), it was subjected to collision-induced fragmentation analysis in the mass spectrometer. The peptide was first purified by off-line HPLC where it eluted between 13 and 17 min. The HPLC fractions were reduced in volume before aliquots were spotted onto a MALDI target plate. MS/MS fragmentation analysis of the parent ion at m/z 904.2 (positive mode) confirmed that this ion corresponds to the BChE peptide FGES198AGAAS, with an added mass of 108 Da on Ser198 (Figure 6). The following fragment ions were used to make this assignment. Ions Δb5, Δb6, Δb7, Δb8, and ΔMH have masses that exactly match the FGES198AGAAS sequence following loss of the adduct and a molecule of water from Ser198. The symbol 426

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at only 3% the concentration of ethephon, it is conceivable but unlikely that we have simply not yet found the correct contaminant. If the inhibitor is created from ethephon, then its formation would proceed in competition with the degradation of ethephon into ethylene, chloride, and phosphate. This could mean that the properties governing formation of the inhibitor might be similar to the properties governing formation of ethylene. In addition, the apparent rate for formation of the inhibitor would be the same as the apparent rate for formation of ethylene. These are parallel reactions, and the apparent rate for the formation of each product in a group of parallel reactions is equal to the sum of the actual rates for formation of each product.16 A defining property for the hydrolysis of ethephon to release ethylene is its pH dependence. A variety of studies have shown that the reaction is not base-catalyzed but is dependent on the presence of the dianion of ethephon.17−20 The pH dependence (pKa = 7) is consistent with the second pKa of ethephon20 (pKa2 = 7.27). Formation of the inhibitor shows similar properties. No activation occurs at pH values between 3 and 5, while a marked increase in rate occurs between pH 5 and 9. This behavior is consistent with the dianion of ethephon being required for the activation process. In addition, the rate for activation of ethephon was only about 5-fold faster at pH 9.0 than at pH 7.0. This effect of pH on the rate of activation is not large enough to support base-catalyzed activation. We propose that activation of ethephon to inhibit BChE, like hydrolysis of ethephon to release ethylene, starts with the dianionic form of ethephon. Comparison of the apparent rates for activation of the inhibitor and formation of ethylene is less straightforward. The rates seem to depend significantly on the experimental conditions. The following values have been reported for formation of ethylene from ethephon. A half-life of 58 h for hydrolysis of ethephon (in aqueous media at pH 7 and 25 °C, monitored by evolution of ethylene and phosphate) is given in technical reports from a wide variety of official agencies.21 A half-life of 24 h was reported for ethylene evolution at pH 7 in 10 mM 2-morpholinoethanesulfonic acid buffer or 50 mM citrate/phosphate buffer by Domir and Foy.19 The same value (t1/2 = 24 h) was reported by Segall et al. using 31P NMR to monitor conversion of ethephon to phosphate in water at pH 7.4 and 25 °C.8 Biddle et al. followed the formation of ethylene in phosphate buffer pH 7.0, at temperatures between 30 and 55 °C, from which they constructed an Arrhenius plot.20 Extrapolation of the Arrhenius plot to 20 °C yields a t1/2 of 32 h. We measured a half-time value of 9.9 h for activation of ethephon in 0.2 M phosphate/citrate buffer (pH 7), 20 °C, and a value of 2 h in 0.2 M glycine/NaOH (pH 9.0), 22 °C. However, we found that activation was significantly slower at pH 9 in Tris or borate buffers than in phosphate or carbonate buffers. In view of this range of values and the dependence of the results on experimental conditions, we suggest that our value of t1/2 = 9.9 h for activation of ethephon is within experimental error of the t1/2 values from the literature for conversion of ethephon into ethylene and phosphate. Though not definitive, this result supports the hypothesis that ethephon is the source of the BChE inhibitor. Decay of the Inhibitor. At pH 7.0, the inhibitor decays with a t1/2 of 30 h, which is about 1/3 the rate at which it appears to be formed. At pH 9.0, the t1/2 for decay is 20 h.

samples. Tryptic and peptic peptides showed the same added mass of 108 Da on the active site serine. We conclude that only one type of inhibitor is generated from ethephon.

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DISCUSSION Ethephon Itself Does Not Inhibit BChE. Ethephon preparations inhibit BChE activity both in vivo3,5 and in vitro.3,5−7 We propose, however, that the inhibitor is not ethephon itself. This proposal is supported by the following observations: (1) The time course for inhibition of BChE by a freshly prepared ethephon solution accelerates. Irreversible BChE inhibition by organophosphorus compounds generally follows first-order kinetics expressed by the equation E = E0e−ki·I·t. Such an exponential decay of concentration/activity yields a straight line when plotted as [log(residual BChE activity, %)] versus [inhibition time]. However, when a freshly prepared ethephon solution was used to inhibit BChE, the slope of the plot of [log(residual BChE activity, %)] versus [inhibition time] was nearly zero at the beginning of the reaction and increased with time (Figures 1 and 2). This indicates that the initial concentration of inhibitor was very low and increased with time. Therefore, the inhibitor was not ethephon itself. (2) Formation of the active inhibitor is pH-dependent. Preincubation of ethephon at pH 7.0, 8.0, or 9.0 increased its inhibitory potency, while preincubation for the same time at pH 3.0, 4.0, or 5.0 had no effect. Maximum activation was achieved after 10−20 h incubation. Treatment of BChE with activated ethephon yielded a linear plot of [log(residual BChE activity, %)] versus [inhibition time], indicating that the active inhibitor was present at the beginning of the reaction and did not significantly change in concentration during the course of the reaction (Figure 2). (3) The inhibition reaction is not stoichiometric with respect to ethephon. If ethephon were the inhibitory species, then 1 mol of BChE would be expected to be inhibited by 1 mol of ethephon. However, this was not the case. Concentrations of ethephon exceeding the BChE concentration by more than 35fold were required for complete inhibition. Thus the effective inhibitor concentration is only about 3% that of the starting ethephon. Active Inhibitor Derived from Ethephon. From the time dependence for the appearance of inhibition, it is clear that the inhibitor is not present in freshly prepared ethephon solutions. There are two logical sources from which the inhibitor could be formed. The first is that the inhibitor is created from a minor contaminant in the ethephon preparation. The credibility of this possibility rests in the low stoichiometry for the inhibitor. The other possibility is that the inhibitor is created from ethephon itself. The following discussion supports the second possibility. Segall et al. showed that technical grade ethephon contained a number of contaminants.8 However, none of them were inhibitory toward BChE. We found that a number of new mass spectral masses appeared in ethephon stock solutions during storage and handling (data not shown). Many of these were consistent with simple modifications on the ethephon structure. However, the intensity for none of these masses changed during the activation process, indicating that they were not the source of the inhibitor. Consequently, we did not pursue further investigations into their structures. On the basis of these observations, we believe that it is unlikely that the inhibitor comes from a contaminant. However, if the inhibitor is present 427

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Figure 7. Four possible 108 Da BChE adducts. The structure (b) in bold is favored based on arguments in the discussion.

being cyclization yielding 2-oxo, 2-hydroxy-1,2-oxaphosphetane (h). A parallel reaction scheme affords a simple rationalization for our stoichiometry studies wherein only about 3% of the starting ethephon was activated to the BChE inhibitor. Such a result can be accommodated by the mechanism in Scheme 1 if the actual rate for activation of the inhibitor is about 3% the actual rate for hydrolysis of ethephon to ethylene. For parallel reactions, the yield for a given species is equal to the actual rate for formation of that species divided by the sum of the actual rates for formation of all species (the apparent rate).16 The loss of chloride upon formation of the inhibitor makes the activation process essentially irreversible. The irreversible nature of the formation of the inhibitor separates the degradation of the inhibitor from hydrolysis of ethephon to ethylene. Therefore, the downward trend in Figure 3 is strictly related to the degradation of the inhibitor. The time course might even be expected to continue after ethephon itself has been completely hydrolyzed. Note that h is negatively charged at neutral pH. As has been mentioned, the active site of BChE normally reacts with positively charged substrates and inhibitors, not negatively charged ones. However, the active site can accommodate compounds with a single negative charge. This is demonstrated by the observation that negatively charged aspirin can be hydrolyzed by BChE.24 Thus, reaction with a compound carrying a single negative charge is not as difficult to accept as reaction with a compound carrying two negative charges (e.g., the ethephon dianion). Mechanisms of BChE Inhibitor Formation and Ethylene Generation. The proposed oxaphosphetane structure (h) is not unprecedented. Oxaphosphetanes are generally accepted as transition states in the Wittig reaction between an aldehyde or ketone and phosphonium ylide to produce a phosphine oxide and an alkene. A number of functionalized oxaphosphetanes have been prepared, structurally characterized, and analyzed for degradation.25,26 The stability of the structures depends largely on the substituents of the oxaphosphetane ring and the assay conditions.25 It has not escaped our attention that the structural similarity between the proposed cyclic BChE inhibitor and the Wittig transition state implies that the oxaphosphetane could be an intermediate in phosphate and ethylene generation from ethephon, as well (h→g, Scheme 1). Hydrolytic degradation of the cyclic oxaphosphetane would produce 2-hydroxyethylphosphonate (h→i, Scheme 1), a reported minor degradation product of ethephon in aqueous solution27 and a non-enzymatic plant metabolite.28,29 Studies of 2-chloro-1-(substituted-phenyl)ethylphosphonic acids9 as well as 2-bromo- and 2-iodoethylphosphonic acids7 indicate chloride dissociation as the rate-limiting step in hydrolysis of ethephon and inhibition of BChE. A noncyclic intermediate with chloride partially dissociated from the βcarbon has been proposed,9 indicating a path from the

Though the decay process is faster at pH 9 than at pH 7, it is not sufficiently fast to constitute base catalysis. Proposed Structure of the Activated Ethephon Inhibitor Deduced from the BChE Adduct. Mass spectral analysis of BChE treated with activated ethephon established that the adduct on Ser198 has a mass of 108 Da. The phosphorus atom from ethephon is part of the adduct.7 Based on the 108 Da mass alone, four adduct structures (a−d) can be proposed (Figure 7): 2-hydroxyethylphosphonate linked through the alkyl (a) or phosphate (b) group, monoethylphosphate (c), and dimethylphosphate (d). The dimethylphosphate adduct (d) requires considerable rearrangement and is therefore unlikely. This conclusion is supported by the fact that the adduct generated by ethephon inhibition of BChE did not undergo aging under conditions where the dimethylphosphate adduct of BChE is known to age.22 Species a, b, and c all could be obtained through a cyclic intermediate formed by intramolecular nucleophilic attack on the β-carbon by a phosphate oxygen of the ethephon dianion yielding 2-oxo, 2-hydroxy-1,2-oxaphosphetane (f→h, Scheme 1). The phosphorus is the most electrophilic moiety of h and Scheme 1. Proposed Reactions of Ethephon at Physiological pH as an Ethylene Generator and BChE Inhibitor

thus most susceptible to nucleophilic attack by the BChE Ser198 hydroxyl, favoring phosphorylation (b or c) over alkylation (a) to generate the adduct. Furthermore, 2hydroxyethylphosphonate (b) is the most likely adduct after ring opening because cleavage of a P−O bond is more facile than cleavage of a P−C bond.23 Despite several attempts, we have no direct physical−chemical evidence to support structure h. We attribute that to the low yield of active inhibitor in our preparations. Direct verification for structure h is a matter of ongoing investigation. The mechanism in Scheme 1 depicts two parallel reactions for the ethephon dianion (f), one reaction being hydrolysis and yielding chloride, phosphate, and ethylene (g) and the other 428

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hydrolases,37 most of them of unknown function and all of them of unknown sensitivity to the activated ethephon inhibitor. The low yield and instability of the esterase inhibitor means it is relevant in the cells of the exposed organism but not as a residual toxicant. The focus of the current studies was on the mechanism of inhibition of BChE by ethephon not the physiological effects of ethephon. Concentrations of ethephon between 1 μM and 1 mM were used during the course of our studies. The proposed mechanism for inhibitor formation as an intramolecular cyclization is unlikely to be greatly influenced by the initial ethephon concentration. Temperatures of 20−22 °C were chosen to make the results comparable to literature studies on the hydrolysis of ethephon to ethylene. Our conditions were not designed to be physiologically relevant. The link between the physiology/toxicology of ethephon and our experiments is further blurred by the fact that for in vivo studies administration of the ethephon was either by gavage, by capsule, or in the food.4,5 Circulating levels of ethephon in the subjects were not measured. These methods of administration make it difficult to determine the circulating levels of ethephon for comparison to our in vitro studies. Nevertheless, some correlations between our studies and the toxicological studies can be drawn. In general. ethephon is lethal to rats when administered by gavage at 1000−2000 mg/kg/day for 2 weeks. 4 LD50 values of 1600−2800 mg/kg/day have been reported.4,5 At levels below 300 mg/kg/day, no adverse symptoms were observed, though plasma BChE activity was decreased.4 If we assume that all of the administered ethephon entered the circulation and was evenly distributed throughout the rat, then the circulating concentration of ethephon associated with 300 mg/kg gavage would be 2 mM. Though this number is undoubtedly high, our experiments demonstrated formation of the BChE inhibitor from ethephon levels as low as micromolar, well within the range of the toxicology studies. Maximum formation of the inhibitor, in phosphate/citrate buffer, pH 7.0 and 22 °C, required 24 h. Even though activation of the inhibitor is dependent on the reaction conditions, in vivo conditions are sufficiently similar to those in our study that formation of the inhibitor would be expected. Furthermore, in the animal studies, ethephon was re-administered daily, making it reasonable to propose that the circulating levels of BChE inhibitor would accumulate. Thus, even if the actual circulating levels of ethephon in the animal were 10-fold lower than we have assumed, it would be reasonable to expect inhibition of BChE over the course of several days.

ethephon dianion to phosphate and ethylene without forming the cyclic intermediate (f→g, Scheme 1). Precedent for the Activation of an Organophosphate. Ethephon is not the only example of an organophosphorus compound that must be activated in order to become a cholinesterase inhibitor. Trichlorphon is an insecticide that rearranges to dichlorvos under slightly alkaline conditions.30,31 Trichlorphon itself does not inhibit cholinesterases. Its inhibitory properties are due to non-enzymatic conversion to dichlorvos.32 As with ethephon, reaction of BChE with nonactivated trichlorphon results in an accelerating time course of inhibition. Preincubation of trichlorphon at pH 7 converts it into the dichlorvos inhibitor. Increasing the pH of the preincubation buffer above 7 increases the rate of conversion.32 The principal differences between the two inhibitors are that 62% of the trichlorphon is converted to its activated form,33 while only 3% of the ethephon appears to be converted to its effective activated form, and dichlorvos is stable in aqueous media. Slow Rate for the Reaction of the Activated Ethephon Inhibitor with BChE. It is worth noting that the reaction of the ethephon-derived inhibitor with BChE is slow relative to the rates for other organophosphorus compounds. The apparent rate for reaction of ethephon with BChE at the time of maximum activation (Figure 3) was about 0.09 min−1. The total amount of ethephon in those reactions was 125 μM. Assuming that 3% of the ethephon was activated to the inhibitor that corresponds to about 4 μM active inhibitor. The resultant apparent second-order rate constant would then be 2.3 × 104 M−1 min−1. This compares to second-order rate constants for most organophosphorus agents of 106 to 109 M−1 min−1.34 Addition of Phosphate to BChE by an Organophosphate Is a Unique Event. An important result from the current studies on ethephon is the demonstration that the adduct formed was not a phosphate derivative as had been originally suggested.7 A phosphate adduct would have resulted in an added mass of 80 Da not the 108 that was observed. The importance of this observation centers around our assay for exposure to tri-ortho-cresyl phosphate, an antiwear component of jet engine lubricants. That assay is based on the observation that the metabolically activated form of tri-ortho-cresyl phosphate, cresyl saligenin phosphate, covalently reacts with the active site of BChE, ultimately yielding a phosphorylated adduct with an added mass of 80 Da.35,36 In the course of developing that assay, we argued that no other organophosphorus toxicant adds phosphate to BChE, making the plus 80 Da adduct diagnostic for exposure to tri-ortho-cresyl phosphate. The studies of Haux et al. drew that argument into question.7 The results of the current study re-enforce our original contention that cresyl saligenin phosphate is the only organophosphorus compound that adds a mass of 80 Da to the active site of human BChE. This finding validates our mass spectrometry assay for exposure to tri-ortho-cresyl phosphate. Biological Activity of Ethephon. The biological activity of ethephon is primarily attributable to two different activation products and mechanisms. The most important reaction in plants is liberation of ethylene as a plant growth regulator. In mammals, it is formation of the proposed oxaphosphetane as a BChE inhibitor. Both pathways take place spontaneously at physiological pH and are therefore relevant in all organisms to varying degrees. For example, the model plant Arabidopsis thaliana contains over 50 organophosphate-sensitive serine

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CONCLUSION In conclusion, we have established that (1) ethephon does not inhibit BChE but is spontaneously converted at physiological pH to the active inhibitor, (2) only about 3% of the starting ethephon is trapped as the inhibitor, (3) the mass added to BChE upon inhibition is 108 Da, consistent with a 2hydroxyethylphosphonate adduct but not a phosphate adduct, and (4) spontaneously activated ethephon is tentatively assigned as 2-oxo, 2-hydroxy-1,2-oxaphosphetane.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. 429

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Funding

(13) Lockridge, O., and Masson, P. (2000) Pesticides and susceptible populations: people with butyrylcholinesterase genetic variants may be at risk. Neurotoxicology 21, 113−126. (14) McIlvaine, T. C. (1921) A buffer solution for colorimetric comparison. J. Biol. Chem. 49, 183−186. (15) Estevez, J., and Vilanova, E. (2009) Model equations for the kinetics of covalent irreversible enzyme inhibition and spontaneous reactivation: esterases and organophosphorus compounds. Crit. Rev. Toxicol. 39, 427−448. (16) Frost, A. A., and Pearson, R. G. Kinetics and Mechanisms: A Study of Homogeneous Chemical Reactions; 2nd ed.; John Wiley & Sons: New York, 1965. (17) Segall, Y., Toia, R. F., and Casida, J. E. (1993) Mechanism of the phosphorylation reaction of 2-haloalkylphosphonic acids. Phosphorus, Sulfur and Silicon 75, 191−194. (18) Maynard, J. A., and Swan, J. M. (1963) Organophosphorus compounds I. 2-Chloroalkylphosphonic acids as phosphorylating agents. Aust. J. Chem. 16, 596−608. (19) Domir, S. C., and Foy, C. L. (1978) A study of ethylene and CO2 evolution from ethephon in tobacco. Pestic. Biochem. Physiol. 9, 1−8. (20) Biddle, E., Kerfoot, D. G., Kho, Y. H., and Russell, K. E. (1976) Kinetic studies of the thermal decomposition of 2-chloroethylphosphonic acid in aqueous solution. Plant Physiol. 58, 700−702. (21) Bureau-REACH-RIVM. (2011) Proposal for harmonized classification and labeling; substance ethephon, Annex VI Report CLP, The Netherlands. (22) Worek, F., Diepold, C., and Eyer, P. (1999) Dimethylphosphoryl-inhibited human cholinesterases: inhibition, reactivation, and aging kinetics. Arch. Toxicol. 73, 7−14. (23) Benkovic, S. J.; Schray, K. J. Transition States of Biochemical Processes; Plenum Press, New York, 1978. (24) Masson, P., Froment, M. T., Fortier, P. L., Visicchio, J. E., Bartels, C. F., and Lockridge, O. (1998) Butyrylcholinesterasecatalysed hydrolysis of aspirin, a negatively charged ester, and aspirin-related neutral esters. Biochim. Biophys. Acta 1387, 41−52. (25) Kawashima, T. (2003) Syntheses, structures, and thermolyses of three- and four-membered heterocyclic compounds containing highly coordinate main group elements. Bull. Chem. Soc. Jpn. 76, 471−483. (26) Pascariu, A., Mracec, M., and Berger, S. (2008) Dynamic NMR study of the oxaphosphetane complexation with lithium during the Wittig reaction. Int. J. Quantum Chem. 108, 1052−1058. (27) Audley, B. G., Archer, B. L., and Mann, N. P. (1973) Decomposition of 2-chloroethylphosphonic acid in stems and leaves of Hevea brasiliensis. Phytochemistry 12, 1535−1538. (28) Audley, B. G., Archer, B. L., and Carruthers, I. B. (1976) Metabolism of ethephon (2-chloroethylphosphonic acid) and related compounds in Hevea brasiliensis. Arch. Environ. Contam. Toxicol. 4, 183−200. (29) Audley, B. G., and Archer, B. L. (1973) Decomposition of 2chloroethylphosphonic acid in aqueous solution: formation of 2hydroxyethylphosphonic acid. Chem. Ind. London, 634. (30) Barthel, W. F., Alexander, B. H., Giang, P. A., and Hall, S. A. (1955) Insecticidal phosphates obtained by a new rearrangement reaction. J. Am. Chem. Soc. 77, 2424−2427. (31) Lorenz, W., Henglein, A., and Schrader, G. (1955) The new insecticide O,O-dimethyl 2,2,2-trichloro-1-hydroxyethylphosphonate. J. Am. Chem. Soc. 77, 2554−2556. (32) Reiner, E., Krauthacker, B., Simeon, V., and Skrinjaric-Spoljar, M. (1975) Mechanism of inhibition in vitro of mammalian acetylcholinesterase and cholinesterase in solutions of 0,0-dimethyl 2,2,2-trichloro-1-hydroxyethyl phosphonate (Trichlorphon). Biochem. Pharmacol. 24, 717−722. (33) Arthur, B. W., and Casida, J. E. (1957) Metabolism and selectivity of O,O-dimethyl 2,2,2-trichloro-1-hydroxyethyl phosphonate and its acetyl and vinyl derivatives. J. Agric. Food Chem. 5, 186− 192. (34) Schopfer, L. M., Voelker, T., Bartels, C. F., Thompson, C. M., and Lockridge, O. (2005) Reaction kinetics of biotinylated organo-

Graduate studies for M.S.L. were supported by a Fulbright Russia student grant and a fellowship from the Department of Environmental, Agricultural and Occupational Health, College of Public Health, University of Nebraska Medical Center. Contract 200-2012-M-53381 was provided to O.L. by the Center for Disease Control and Prevention. Graduate studies for S.R.L. were supported by an EPA STAR fellowship (FP917139). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Mass spectra were obtained with the support of the Mass Spectrometry and Proteomics core facility at the University of Nebraska Medical Center.

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ABBREVIATIONS BChE, butyrylcholinesterase; DHB, 2,5-dihydroxybenzoic acid; MALDI-TOF/TOF, matrix-assisted laser desorption/ionization tandem mass spectrometer with dual time-of-flight analyzers; MS/MS, mass spectrum of collision-induced dissociation fragments; TFA, trifluoroacetic acid

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REFERENCES

(1) MacBean, C. The Pesticide Manual, 16th ed.; British Crop Protection Council: Farnham, Surrey, U.K., 2012. (2) Gianessi, L. P., and Marcelli, M. B. (2000) Pesticide Use in U.S. Crop Production: 1997. National Summary Report. National Center for Food and Agricultural Policy, 1−101. (3) Hennighausen, G., and Tiefenbach, B. (1978) Mechanism of acute toxic effects of chlorocholine chloride and 2-chloroethyl phosphonic acid (Ethephon). Arch. Exp. Vet. Med. 32, 609−621. (4) Johnson, S. L. (1997) Rhone-Poulenc Ag Company; pesticide tolerance petition filing with the Environmental Protection Agency. Fed. Reg. 62, 2149−2154. (5) Hennighausen, G., Tiefenbach, B., and Lohs, K. (1977) The effect of 2-chlorethylphosphonic acid (ethephon) on the activity of cholinesterases in vivo and in vitro with reference to its acute toxicity. Pharmazie 32, 181−182. (6) Haux, J. E., Lockridge, O., and Casida, J. E. (2002) Specificity of ethephon as a butyrylcholinesterase inhibitor and phosphorylating agent. Chem. Res. Toxicol. 15, 1527−1533. (7) Haux, J. E., Quistad, G. B., and Casida, J. E. (2000) Phosphobutyrylcholinesterase: phosphorylation of the esteratic site of butyrylcholinesterase by ethephon [(2-chloroethyl)phosphonic acid] dianion. Chem. Res. Toxicol. 13, 646−651. (8) Segall, Y., Grendell, R. L., Toia, R. F., and Casida, J. E. (1991) Composition of technical ethephon [(2-chloroethyl)phosphonic acid] and some analogues relative to their reactivity and biological activity. J. Agric. Food Chem. 39, 380−385. (9) Zhang, N., and Casida, J. E. (2002) Novel irreversible butyrylcholinesterase inhibitors: 2-chloro-1-(substituted-phenyl)ethylphosphonic acids. Bioorg. Med. Chem. 10, 1281−1290. (10) Lockridge, O., Schopfer, L. M., Winger, G., and Woods, J. H. (2005) Large scale purification of butyrylcholinesterase from human plasma suitable for injection into monkeys; a potential new therapeutic for protection against cocaine and nerve agent toxicity. J. Med. CBR Def. 3, online publication. (11) Ellman, G. L., Courtney, K. D., Andres, V., Jr., and Featherstone, R. M. (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88−95. (12) Masson, P., and Lockridge, O. (2010) Butyrylcholinesterase for protection from organophosphorus poisons: catalytic complexities and hysteretic behavior. Arch. Biochem. Biophys. 494, 107−120. 430

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phosphorus toxicant, FP-biotin, with human acetylcholinesterase and human butyrylcholinesterase. Chem. Res. Toxicol. 18, 747−754. (35) Schopfer, L. M., Furlong, C. E., and Lockridge, O. (2010) Development of diagnostics in the search for an explanation of aerotoxic syndrome. Anal. Biochem. 404, 64−74. (36) Liyasova, M., Li, B., Schopfer, L. M., Nachon, F., Masson, P., Furlong, C. E., and Lockridge, O. (2011) Exposure to tri-o-cresyl phosphate detected in jet airplane passengers. Toxicol. Appl. Pharmacol. 256, 337−347. (37) Kaschani, F., Gu, C., Niessen, S., Hoover, H., Cravatt, B. F., and van der Hoorn, R. A. (2009) Diversity of serine hydrolase activities of unchallenged and botrytis-infected Arabidopsis thaliana. Mol. Cell. Proteomics 8, 1082−1093.

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