ToxSci Advance Access originally published online on August 13, 2007
Toxicological Sciences 2007 100(1):136-145; doi:10.1093/toxsci/kfm215
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Aging Pathways for Organophosphate-Inhibited Human Butyrylcholinesterase, Including Novel Pathways for Isomalathion, Resolved by Mass Spectrometry
* Eppley Institute and Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805
Centre de Recherches du Service de Santé des Armées, Département de Toxicologie-Unité d'Enzymologie, 24 avenue des Maquis du Grésivaudan-BP87, 38702 La Tronche cedex, France
1 To whom correspondence should be addressed at Eppley Institute, University of Nebraska Medical Center, Box 986805, 600 South 42nd Street, Omaha, NE 68198. Fax: (402) 559-4651. E-mail: olockrid{at}unmc.edu.
Received June 27, 2007; accepted July 24, 2007
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ABSTRACT |
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Some organophosphorus compounds are toxic because they inhibit acetylcholinesterase (AChE) by phosphylation of the active site serine, forming a stable conjugate: Ser–O–P(O)–(Y)–(XR) (where X can be O, N, or S and Y can be methyl, OR, or SR). The inhibited enzyme can undergo an aging process, during which the X–R moiety is dealkylated by breaking either the P–X or the X–R bond depending on the specific compound, leading to a nonreactivatable enzyme. Aging mechanisms have been studied primarily using AChE. However, some recent studies have indicated that organophosphate-inhibited butyrylcholinesterase (BChE) may age through an alternative pathway. Our work utilized matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry to study the aging mechanism of human BChE inhibited by dichlorvos, echothiophate, diisopropylfluorophosphate (DFP), isomalathion, soman, sarin, cyclohexyl sarin, VX, and VR. Inhibited BChE was aged in the presence of H2O18 to allow incorporation of 18O, if cleavage was at the P–X bond. Tryptic-peptide organophosphate conjugates were identified through peptide mass mapping. Our results showed no aging of VX- and VR-treated BChE at 25°C, pH 7.0. However, BChE inhibited by dichlorvos, echothiophate, DFP, soman, sarin, and cyclohexyl sarin aged exclusively through O–C bond cleavage, i.e., the classical X–R scission pathway. In contrast, isomalathion aged through both X–R and P–X pathways; the main aged product resulted from P–S bond cleavage and a minor product resulted from O–C and/or S–C bond cleavage.
Key Words: butyrylcholinesterase; organophosphate; aging; mass spectrometry.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Butyrylcholinesterase (BChE) is a serine hydrolase that catalyzes the hydrolysis of a variety of choline and noncholine esters (Lockridge and Masson, 2000
). BChE is more abundant than acetylcholinesterase (AChE) in mouse and human (Li et al., 2000). BChE reacts with a broad range of toxicants more effectively than AChE. BChE has been suggested to function as a scavenger protein that protects the cholinergic system against anticholinesterase poisons (Lockridge and Masson, 2000).
Organophosphorus (OP) compounds account for a large portion of pesticides and chemical warfare agents. These compounds exert their acute toxicity mainly through inhibition of AChE. OP inhibition of AChE and BChE proceeds in a progressive manner by phosphorylation of the active site Ser, to form a Ser–O–P(O)–(Y)–(XR) adduct (where X can be O, N, or S and Y can be methyl, OR, or SR). The phosphorylated enzyme can be reactivated by treating with strong nucleophilic agents such as oximes. The phosphorylated enzyme can also undergo a spontaneous time-dependent process called "aging" during which the P–X–R component of the OP-serine conjugate is dealkylated, leaving the enzyme irreversibly inhibited (Casida and Quistad, 2004).
Aging has been studied in detail using AChE as the model enzyme. The generally accepted aging mechanism for alkoxy-OP adducts invokes the catalytic participation of residues from the enzyme. Dealkylation of the OP adduct is facilitated primarily by the protonated histidine of the catalytic triad, a glutamic acid residue adjacent to the catalytic serine, and a nearby tryptophan residue (Shafferman et al., 1996; Viragh et al., 1997). It is agreed that these residues combine to promote the cleavage of the O–C bond with formation of a carbocation on the leaving alkyl group and a negatively charged phospho-oxygen (Shafferman et al., 1996; Viragh et al., 1997). The resultant carbocation is then vulnerable to nucleophilic attack by water. A variety of pH, mutational, crystallographic, and kinetic studies support the catalytic involvement of the amino acid residues (Barak et al., 1997; Harris et al., 1966; Jennings et al., 2003; Michel et al., 1967; Millard et al., 1999; Saxena et al., 1993; Shafferman et al., 1996). Studies on BChE inhibited with diisopropylfluorophosphate (DFP) are also consistent with this mechanism (Masson et al., 1997a).
On the other hand, recent studies have provided evidence for alternative aging pathways. First, studies on aging of tabun-inhibited human AChE suggested a P–N bond cleavage and elimination of the dimethylamine moiety from the tabun-enzyme conjugate (Barak et al., 2000). This result is also corroborated by the x-ray structure of tabun-aged murine AChE (Ekstrom et al., 2006). Second, isomalathion-inhibited equine BChE was shown to age through breaking the P–S bond and releasing either a thiomethyl or a diethylthiosuccinate moiety (Doorn et al., 2001a). Third, a crystal structure study on echothiophate-inhibited human BChE presented the possibility of aging through both P–O scission and O–C scission (Nachon et al., 2005). Given the diversity of aging pathways invoked for various OP compounds and the indication that different aging pathways may be used by BChE and AChE when inhibited by the same OP, systematic investigation of the BChE aging pathway was needed. The use of BChE as an antidote against OP toxicity also calls for a better understanding of its aging mechanism.
In the current study, we investigated the possibility that OP-inhibited human BChE ages through breakage of the P–X bond (where X may be O or S), as opposed to the classical pathway, i.e., breaking the X–C bond. We evaluated the aging pathway by using matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry to determine the mass of the aged product. Including H2O18 in the aging medium enabled us to distinguish between the two pathways. Discrimination was possible because the inhibition step employed OP made with common 16O. If aging occurred via cleavage at P–16O, then the phosphorus would pick up an 18OH from the medium to form P–18O–H. If the cleavage occurred at O–C, then the carbon would pick up the 18OH and the phosphorus would retain the 16O as P–16OH. The net effect would be a 2-Da increase in the mass of the aged adduct if cleavage of the P–O bond occurred. Consequently, the mass of the aged OP-enzyme conjugate revealed which aging pathway had been taken (Fig. 1). The aged enzyme was digested with trypsin, and the resulting tryptic peptides were analyzed by MALDI-TOF mass spectrometry to determine the mass of the OP-labeled, active-site peptide conjugate. Nine OP compounds were tested, including dichlorvos, echothiophate, DFP, soman, sarin, cyclohexyl sarin (GF), VX, VR (Russian VX), and isomalathion (Fig. 2).
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Aging of all the alkoxy adducts followed the classical pathway, with cleavage of the O–C bond. Isomalathion-inhibited BChE, however, gave two aged products. Besides the main aged product resulting from breaking the P–S bond, a minor product resulted from breaking the O–C and/or S–C bond, which had not been reported before.
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MATERIALS AND METHODS |
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Chemicals and reagents.
Dichlorvos and isomalathion were from CIL Cluzeau Info Labo (Sainte-Foy-La-Grande, France). Echothiophate iodide was from Wyeth-Ayerst (Rouses Point, NY). DFP was from Acros (Belgium). Soman, sarin, cyclohexyl sarin, VX, and VR were from Centre d'Etudes du Bouchet (Vert-le-Petit, France). H2O18 (95% pure) was from Aldrich (Milwaukee, WI). Sequence-grade modified trypsin was from Promega (Madison, WI).
-Cyano-4-hydroxycinnamic acid (CHCA) matrix-assisted laser desorption/ionization (MALDI) matrix and external MALDI mass calibration standard mix were purchased from Applied Biosystems (Framingham, MA).
Purification and deglycosylation of human BChE.
Human BChE (gi:116353; Swiss protein P06276
[GenBank]
) was purified from plasma as previously described (Lockridge et al., 2005). Recombinant GST-PNGaseF was expressed and purified as previously described (Grueninger-Leitch et al., 1996). Two milligrams of purified human BChE was deglycosylated by treating with 3.2 mg of GST-PNGaseF for 5 min at 37°C in 6.66 ml of 100mM Tris-HCl, pH 7.5. GST-PNGaseF was removed by loading the sample onto GSH-agarose (Sigma, St Louis, MO) packed in a 5-ml column, equilibrated with 100mM Tris-HCl, pH 8.0. The flow through containing human BChE was concentrated to 4.5 mg/ml in a Centricon PM 10 microconcentrator (Millipore, Bedford, MA). BChE was deglycosylated because the deglycosylated protein released the active-site tryptic peptide upon digestion with trypsin. By contrast, when the BChE was not deglycoslyated, the active-site peptide was not available to trypsin unless the protein was first denatured and its disulfide bonds reduced and alkylated.
Organophosphate treatment of BChE.
A 10-µl aliquot of deglycosylated human BChE stock at a concentration of 53µM enzyme (4.5 mg/ml) was diluted with 90 µl of H2O16 or H2O18 and 1 µl of 1M sodium phosphate pH 7.0. Each 100-µl diluted BChE sample received 1 µl of dichlorvos (6.5M stock), echothiophate (10mM stock), DFP (50mM stock), soman (27.4mM stock), sarin (35.6mM stock), cyclohexyl sarin (27.7mM stock), VX (18.6mM stock), VR (18.6mM stock), or isomalathion (32mM stock). The final concentration of OP is at least 20 times higher than the BChE concentration in the reaction system, ensuring that the inhibition and aging reaction can reach completion under the experimental conditions. The excess OP should not affect the aging reaction pathways. The H2O18 level in the final reaction mixture was at least 85%. Enzyme inhibition and aging proceeded at room temperature for 4 days or more. The BChE used in this study was purified from sterilized human plasma, and we kept the sterilization condition throughout the OP treatment to prevent the microbial contamination. Samples were stored at – 80°C.
Tryptic digestion of BChE.
To remove excess OP, H2O18, and salts, 50 µl of OP-treated BChE was subjected to buffer exchange using Millipore Microcon YM-3 centrifuge filters with 3000 MW cutoff. The protein was diluted with 25mM ammonium bicarbonate pH 8.3, concentrated to 50 µl, rediluted, and reconcentrated 5 times before digestion. The final H2O18 level was no more than 0.5% of the total volume. The BChE was incubated with modified porcine trypsin at a trypsin:BChE ratio of 1:30, at room temperature, overnight. To wash off salts from the digestion that may interfere with mass spectrometric analysis, trypsinized BChE was adsorbed onto a C18 ZipTip (Millipore) and washed with 0.1% trifluoroacetic acid (TFA). Peptides were eluted from the ZipTip with 15 µl of 30% acetonitrile, 0.1% TFA, followed by 15 µl of 60% acetonitrile, 0.1% TFA. The active-site peptide was eluted with 60% acetonitrile, 0.1% TFA.
MALDI-TOF mass spectrometry.
All MALDI-TOF mass spectrometry experiments were performed on an Applied Biosystems Voyager DE-PRO workstation equipped with a 337-nm pulsed nitrogen laser. The OP-labeled, trypsinized, BChE active-site peptide that had been adsorbed to a reverse phase C18 ZipTip was eluted into a microcentrifuge tube with 15 µl of 60% acetonitrile, 0.1% TFA. One microliter of eluant was mixed 1:1 (vol/vol) with the matrix solution CHCA (10 mg/ml in 50% acetonitrile, 0.3% TFA) on the MALDI target plate and allowed to dry at room temperature.
Mass spectra were acquired in positive ion, linear, or reflector mode under delayed extraction conditions, using an acceleration voltage of 20 kV. Laser intensity was adjusted so that the strongest ion intensity in a spectrum did not exceed 80% of the maximum, saturated intensity value. Laser positioning on the sample spot was monitored with a video camera. Spectra shown are the average of 500 laser shots collected from multiple locations on the target spot. Calibration for the mass spectra was performed both externally using adrenocorticotropic hormone peptides (amino acid residues 1–17, 18–39, and 7–38) and internally by reference to BChE tryptic fragments other than the labeled, active-site peptide. The sequence of human BChE (accession number: P06276 [GenBank] ) was obtained from the SwissProt database (http://ca.expasy.org/cgi-bin/sprot-search-ful). The reference masses of the tryptic BChE peptides were obtained using the MS-Digest feature of ProteinProspector version 4.0.6 (http://prospector.ucsf.edu/), a comprehensive proteomic data analysis software package. The MS-Fit feature of ProteinProspector was used to identify human BChE from MALDI peptide mass data, where the mass tolerance was set at 100 ppm.
Half-life of aging.
To determine the aging half-life of echothiophate-inhibited BChE, a time course experiment was conducted. Twenty-five microliters of deglycosylated human BChE (150µM enzyme in 15mM 2-(N-morpholino)ethanesulfonic acid buffer, pH 6.5) was mixed with 25 µl of 100mM Tris-Cl, pH 8.6 to give a final pH of 8.4. Five microliters of enzyme solution were taken immediately after mixing and diluted into 100 µl of 25mM ammonium bicarbonate, pH 8.3. This enzyme fraction served as the 0-min time point and was stored in the – 80°C freezer until trypsin digestion. After the 0-min time point had been taken, a 2.2-fold molar excess of echothiophate (1 µl of 7.5mM echothiophate iodide dissolved in water) was added to the enzyme solution. The OP inhibition and aging process proceeded in a 37°C water bath. A total of nine time points were taken (5 min, 1 h, 2 h, 4 h, 8 h, 12 h, 16 h, 23 h and 28 h). For each time point, a 5-µl aliquot was taken from the reaction solution and diluted to 100 µl with 25mM ammonium bicarbonate. Excess OP was removed from the mixture by gel filtration on a spin column (Performa SR gel filtration cartridge, Edge BioSystems, Gaithersburg, MD). Samples were stored at –80°C until trypsin digestion.
When all times points had been collected, the samples were thawed, subjected to tryptic digestion (as described under Tryptic digestion of BChE), and prepared for MALDI-TOF mass spectrometry (as described under MALDI-TOF mass spectrometry).
The Data Explorer software for the Voyager DE-PRO mass spectrometer provided the area of the peptide peaks for the initial and aged adduct in each spectrum. The aging half-life was calculated from a semilog plot of the normalized peak area for unaged OP peptide against time. The normalized peak area was obtained by taking the ratio of the unaged peak area to the sum of the areas for the aged and unaged peak areas, at each time point. This ratio compensated for the variation in MALDI peak intensities from shot-to-shot and sample-to-sample. It was assumed that the signal response of the aged and unaged peptides would be essentially the same in the same sample (i.e., each individual time point) because there is only a small difference between the structures of the two peptides (i.e., an ethoxy phosphate in the unaged form is converted to a hydroxy in the aged form). SigmaPlot (SigmaPlot for Windows version 10.0, Systat Software, Inc.) was used to generate the equation describing the plot and to run statistic tests on the data. The data set passed the Durbin-Watson test, the normality test, and the constant variance test.
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RESULTS |
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Identification of the BChE Tryptic Peptide Containing the Catalytic Serine
MALDI mass spectra of trypsinized BChE were obtained in both reflector mode for high peak resolution and in linear mode for maximum signal intensity. In reflector mode, the isotopic envelope of each peptide can be resolved and the observed peak values represent the mass of each isotope of a peptide. Peak values observed in linear mode are the average mass of the isotopic variants of the peptides. For a molecule that has a monoisotopic mass around 3000 Da, as the OP-BChE peptides in this study, the average mass of the molecule will be about 2 Da heavier than the monoisotopic mass.
Amino acid numbers are for the mature secreted protein, which has no signal peptide and is therefore shorter by 28 amino acids than the sequence in accession number P06276. Tryptic digestion of BChE generates a 29 amino acid, active-site peptide extending from Ser 191 to Arg 219, where Ser 198 (in bold) is the catalytic Ser: 191SVTLFGESAGAASVSLHLLSPGSHSLFTR219. In the reflector mode spectrum, 11 peaks can be assigned to the tryptic peptides of BChE (Fig. 3A). The isotopic peaks for each peptide are well resolved. The theoretical monoisotopic m/z for the singly protonated [M+H]+ active-site peptide is 2928.5, the observed m/z is 2928.4. In the linear mode spectrum (Fig. 3B), 13 peaks can be assigned to tryptic peptides of human BChE, including the active-site peptide. The average mass of the active-site peptide is 2930.3 m/z.
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The peptide mass data acquired in both linear and reflector mode were submitted to the protein identification program MS-Fit to validate the peptide assignments. The Homo sapiens subset of the SwissProt database (2005.01.06 released version) was selected for the search. Human BChE was successfully identified as the first hit using either monoisotopic or average peptide mass.
Aging of Echothiophate-, DFP-, Dichlorvos-, Soman-, Sarin-, and Cyclohexyl Sarin–Inhibited BChE Occurs via O–C Bond Breakage
Aging of echothiophate-treated BChE was allowed to occur in 18O water. The active-site tryptic peptide was analyzed by MALDI-TOF. It was calculated that if aging resulted from P–O bond breakage (predicted monoisotopic mass 3038.5 Da), the active-site peptide would be 2 Da heavier than if aging resulted from O–C bond breakage (predicted monnoisotopic mass 3036.5 Da) (Table 1). As shown in Figure 4, a peak with m/z of 3036.3 can be matched to the theoretical monoisotopic m/z of the echothiophate peptide conjugate, indicating O–C bond scission. Neither peak 2928.5 (monoisotopic m/z of unlabeled BChE) nor peak 3064.5 (for unaged BChE) were detected, indicating that all the BChE had been inhibited and that the aging was complete. The observed isotopic distribution is exactly that expected for the aged, active-site conjugate (data not shown). The absence of any trace of a peak at 3038.5 m/z indicates that for echothiophate-inhibited human BChE, aging follows the O–C bond breaking pathway exclusively, under the applied experimental conditions.
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To rule out the possibility that peak 3036.3 observed in the spectrum from echothiophate-treated BChE (Fig. 4) may be from an unknown peptide which happens to have the same m/z value as the aged echothiophate BChE peptide conjugate, we monitored the disappearance of unaged echothiophate peptide conjugate (average m/z: 3066.4, Table 1) and the appearance of aged echothiophate peptide conjugate (average m/z: 3038.3, Table 1) as a function of time. To avoid confusion, the 3038.3 m/z mass observed in this experiment is the average mass for the aged, active-site peptide; the monoisotopic mass for this peptide is 3036.3 m/z, as described in Figure 3. A total of nine time points were taken after adding echothiophate into the enzyme solution. A MALDI mass spectrum was acquired for each time point, as shown in Figure 5. The predominant peak after 5 min incubation with OP was peak 3066.4, corresponding to the unaged echothiophate BChE peptide conjugate. Peak 3066.4 diminished while peak 3038.3 grew, as the incubation time became longer. After 28 h incubation, peak 3066.4 almost completely disappeared from the spectrum, indicating that aging was essentially complete. This result confirmed that peak 3038.3 was the echothiophate active-site peptide conjugate resulting from O–C bond breakage during aging. The aging half-life of echothiophate-inhibited human BChE was calculated to be 7.2 ± 0.7 h (Fig. 6) under the experimental conditions, which is consistent with previous studies on BChE inhibited by OP with diethoxy moieties on the phosphorus atom (Mason et al., 1993
, Masson et al., 1997b).
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where "a" is a proportionality constant = 0.92 ± 0.039 and "b" is the apparent rate constant = 0.086 ± 0.0090/h. Aging half-life was measured from the plot as 7.2 ± 0.7 h, representing the time when there are equal amounts of aged and unaged OP-peptide conjugates, i.e., the time when the area ratio equals 0.5 on the plot.DFP-treated BChE was analyzed the same way as echothiophate-treated BChE in Figure 4. The peptide mass spectrum of aged, DFP-inhibited BChE revealed a new monoisotopic peak at 3050.4 Da, indicting that aging of this OP-enzyme conjugate is through breaking the O–C bond of one of the isopropoxy groups (Fig. 7, Table 1). The observed isotopic distribution was consistent with that expected for the 3050.4 Da peptide.
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Similarly, the O–C bond was cleaved during aging of dichlorvos-inhibited BChE (spectrum not shown).
We also investigated the aging pathways for sarin- (Fig. 8, Table 1), cyclohexyl sarin–, and soman- (Table 1, spectra not shown) inhibited BChE. Aging of these three compounds involved only O–C bond cleavage of the alkoxy groups. Peak 3048.4 in Figure 8 and peak 3088.6 (see Table 1, spectrum not shown) represent the monoisotopic m/z of unaged sarin and cyclohexyl sarin BChE conjugate peptides, respectively, indicating that the aging of BChE treated with these two compounds was not complete under the experimental conditions applied in this study.
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Aging of VX-inhibited human AChE and human BChE has been previously found to be slow. Seventy percent of the human erythrocyte AChE activity can be restored by incubation with 2-pralidoxime (2-PAM) 48 h after VX inhibition (Sidell and Groff, 1974
). Aging half-life of VX- and VR-inhibited human erythrocyte AChE has recently been measured to range between 36 and 138 h, respectively (Aurbek et al., 2006). Aging of VX-inhibited BChE is so slow that BChE spontaneously reactivates to 50% of its original activity, without any need of 2-PAM treatment (van der Schans et al., 2004). In our experiment, the expected peak of the aged VX active-site peptide conjugate with monoisotopic m/z of 3006.5 was absent from the sample spectrum, whereas peak 3034.7 matched the unaged VX active-site peptide conjugate. This result indicated that aging of VX-inhibited BChE did not occur under our experimental conditions (Fig. 9, Table 1). Similarly, the VR-inhibited BChE did not age (spectrum not shown).
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Trypsin Proteolysis of Echothiophate-Inhibited BChE in H2O18 Medium
These experiments are controls to prove that a two mass unit shift can be detected in linear mode. The mass spectra in Figures 3A,4, and 7–9 had been acquired in reflector mode. However, signal intensities for isomalathion- and tabun-inhibited BChE were not high enough to support data collection in reflector mode. Mass spectra were therefore acquired using linear mode settings because linear mode data have better signal intensities than those acquired using reflector mode settings. However, the linear mode spectrum is less well resolved than a reflector mode spectrum, and the observed mass represents the average mass of the isotopes of a peptide rather than the monoisotopic mass. For a molecule that has a monoisotopic mass around 3000 Da, as the OP-BChE peptide conjugates in this study, the average mass of the molecule will be 2 Da heavier than the monoisotopic mass (as shown in Figure 3 for BChE active-site peptide and in Table 1 for echothiophate BChE peptide conjugates). To prove that a two mass unit shift, occurring as a consequence of adding the hydroxyl group from H2O18 to a peptide, can be detected from linear mode spectra, we carried out the tryptic digestion of echothiophate-treated BChE without removing the H2O18 from the reaction medium. The hydroxyl group from water is added to the C-terminus of a tryptic peptide when the amide bond is cleaved. Thus, peptides created in H2O18 will be two mass units heavier than comparable peptides made in H2O16.
The echothiophate-treated BChE was digested with trypsin in 85% H2O18. Thus 85% of each tryptic peptide, including the echothiophate active-site peptide conjugate, will carry an 18O hydroxyl group at the C-terminus. The observed mass of a peptide from this H2O18 digestion medium should be 2 Da heavier than the same peptide digested in H2O16 medium. We observed a peak with an m/z of 3040.2 from this sample (spectrum not shown), which is two units heavier than the mass of the aged, echothiophate-labeled, active-site peptide conjugate resulting from tryptic digestion in an H2O16 medium. Seven other peaks in the spectrum from the H2O18 medium were found to be 2 Da heavier than the theoretical masses of the same peptides digested in H2O16 medium. This result demonstrates that MALDI data collected in linear mode are capable of discerning a two mass unit shift resulting from incorporation of an 18O hydroxyl group into a peptide.
Aging of Isomalathion-Inhibited BChE Occurs via both P–S and O–C/S–C Bond Breakage
Previous studies showed that stereoisomers of isomalathion take different inhibitory and aging pathways when reacting with BChE, depending on the stereo configuration of the inhibitor. Inhibition of the enzyme with (1R)-isomalathion proceeds with loss of diethylthiosuccinate as the primary leaving group, resulting in an O,S-dimethyl phosphate adduct; aging of this adduct occurs through breaking the P–S bond and loss of the thiomethyl moiety. On the other hand, the primary leaving group for (1S)-isomalathion inhibition of the enzyme is the thiomethyl group. Inhibition is followed by a quick aging reaction, which also involves breaking a P–S bond. In this case, the diethylthiosuccinate moiety is released (Doorn et al., 2001a,b). In both cases, aging of isomalathion-inhibited BChE occurs via the P–X bond scission pathway.
The isomalathion used in the present study was a mixture of all stereoisomers. The chemical structures and corresponding theoretical masses after inhibition of BChE are summarized in Table 2. The mass spectrum of tryptic peptides of isomalathion-treated BChE (Fig. 10) does not have peaks matching the unaged isomalathion active-site peptide conjugates, regardless of the primary leaving group (theoretical m/z should be 3054.4 when diethylthiosuccinate is the primary leaving group or 3198.5 when thiomethyl is the primary leaving group). This indicates that aging is complete. Peak 3026.6 in the spectrum matches the theoretical m/z of an aged isomalathion peptide conjugate formed as a result of a P–S bond scission. Formation of this fragment can occur from either primary adduct (see Table 2). This result is consistent with the reports in the literature (Doorn et al., 2001a,b).
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In addition, a comparatively minor peak with m/z of 3040.6 is present, which matches the theoretical m/z of the aged isomalathion peptide conjugate resulting from O–C or S–C bond scission. An O–C cleavage pathway would generate this mass only from a primary adduct that was formed by elimination of the diethylthiosuccinate moiety, whereas the 3040.6 mass is consistent with an S–C cleavage starting with either primary adduct. This result indicates that isomalathion-inhibited human BChE can age not only via the P–X pathway, as indicated in previous studies, but also via the X–R pathway. The relative intensities of peaks 3026.6 and 3040.6 suggest that the P–X pathway is the predominant aging pathway under the experimental conditions of this study.
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DISCUSSION |
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Direct Evidence Supporting the X–R Scission Pathway for Aging of Human BChE Inhibited by Alkoxy-OP
Classical aging theory for alkoxy-OP–inhibited cholinesterases states that aging involves activation of the alkoxy oxygen, O–C bond scission, and formation of a carbonium ion (Shafferman et al., 1996
; Viragh et al., 1997). Numerous studies involving a wide range of alkoxy-type OP have demonstrated that aging results in the net loss of an alkyl group (Doorn et al., 2001b; Michel et al., 1967; Millard et al., 1999; Nachon et al., 2005; Viragh et al., 1999). However, cleavage at either the P–O or the O–C bond could account for these observations. Only in the case of soman-inhibited AChE has cleavage of the O–C bond been conclusively demonstrated (Michel et al., 1967; Viragh et al., 1999).
To test whether aging of other alkoxy-OP adducts proceeds via an O–C cleavage, we studied the aging of human BChE inhibited by dichlorvos, echothiophate, DFP, soman, sarin, cyclohexyl sarin, VX, and VR. Our results showed that for all of these OP except VX and VR (in which aging did not occur), aging did indeed occur via cleavage of the O–C bond, i.e., they aged through the X–R scission pathway (see Figs. 4,7,8, and related text). Based on these observations, it seems fair to predict that whenever BChE is inhibited by an OP that leaves an alkoxy group in the inhibited adduct, aging will proceed through the X–R scission pathway. Although AChE demonstrates different kinetic characteristics from BChE upon OP treatment (Giacobini, 2003), it is reasonable to predict that AChE conjugated with an OP retaining an alkoxy group after inhibition will age through the same pathway.
Isomalathion-Inhibited Human BChE Ages via both X–R and P–X Scission Pathways
The two asymmetric centers of isomalathion, one at the phosphorus and the other at the -carbon of the diethylthiosuccinate group, yield four stereoisomers for this compound. Among the four stereoisomers, two belong to the (1R)-stereoisomer group and the other two belong to the (1S)-stereoisomer group, based on the phosphorus asymmetric center (Berkman et al., 1993b). Previous studies have demonstrated that (1R)- and (1S)-isomers have different inhibition and aging mechanisms when reacting with cholinesterases (Berkman et al., 1993a; Doorn et al., 2001a,b; Jianmongkol et al., 1999). For (1R) isomers, the primary leaving group upon conjugation with the catalytic serine is the diethylthiosuccinate, yielding an O,S-dimethyl phosphate adduct; aging of this adduct appears to proceed through a P–X scission reaction where the P–S bond is cleaved and the end product is the o-methyl phosphate adduct (refer to Table 2). For (1S)-isomers, the inhibitory reaction is believed to proceed with loss of the thiomethyl. The aging reaction follows quickly with release of the bulky diethylthiosuccinate, through breakage of the P–S bond, another P–X scission reaction. These reaction pathways have been investigated in rat and bovine AChE and horse BChE (Berkman et al., 1993a; Doorn et al., 2001a,b; Jianmongkol et al., 1999).
We have reexamined this reaction with human BChE. The isomalathion used in our study was from a commercial source and was a mixture of stereoisomers. It is thus necessary for us to consider all the possible OP-BChE conjugates that can result from the primary inhibition and aging (see Table 2). The OP was added to human BChE at room temperature, and the reaction proceeded for at least 4 days. Due to this relatively long incubation time, we did not see peaks for the unaged isomalathion conjugates (3054.4 and 3198.5 m/z), and there was no unmodified active-site peptide in the spectrum (2930.3 m/z), indicating that the BChE was completely inhibited and had completely aged (see Fig. 10). A peak with m/z of 3026.5, for the o-methyl phosphate adduct containing O18H, appeared in the spectrum as expected for a P–X bond scission. This confirmed the results from previous studies.
More interestingly, a minor peak with m/z corresponding to the aged OP adduct resulting from either O–C or S–C bond scission (3040.4 m/z) appeared in the spectrum. This minor peak was not observed in tryptic peptide spectra of isomalathion-treated equine BChE (Doorn et al., 2001a). We suggest that this new aged product is due to subtle differences in the geometry of the active-site gorge between human and equine BChE (e.g., Phe398 in human BChE is an Ile in equine BChE at the equivalent position, which may affect the conformation stability of the catalytic histidine) that render either O–C or S–C bond cleavage possible for human BChE but not for equine BChE.
It is noteworthy that a consistent difference was found in the reactivation rates of human BChE inhibited with the (1S, 3R) or the (1S, 3S) isomers of isomalathion (Doorn et al., 2001b). The (1S, 3S)-isomer–inhibited enzyme cannot be reactivated at all, whereas a small portion of the (1S, 3R)-isomer–inhibited enzyme was reactivated after adding 2-PAM. This suggests that there is something different about the two 1S adducts. Possibly, one of the (1S)-isomers ages via the P–S bond scission, which proceeds very fast after initial enzyme inhibition, thus leaves no opportunity for reactivation; and the other ages via the relatively slow S–C bond scission, so a partial reactivation was observed.
In conclusion, the aging pathways for BChE inhibited by nine different organophosphates were studied. The organophosphates which were chosen included nerve agents such as soman, sarin, and cyclohexyl sarin. The toxicity of the nerve agents is generally considered to be due to their rapid rate of aging, which results in irreversible inhibition of AChE. BChE is being considered for use as a prophylactic against nerve agent exposure. The nature of the aging pathways for these compounds, as described herein, may help in designing a mutant BChE that has better resistance to aging and thus serves as a more potent anti-organophosphate intoxication drug.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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U.S. Army Medical Research and Materiel Command Contract (W81XWH-06-1-0102); Edgewood Biological Chemical Center Contract (W911SR-04-C-0019); Eppley Cancer Center grant (P30CA36727); National Institute of Health (1 U01 NS058056-01); and grants DGA/DSP/STTC-PEA 010807 and EMA/LR 06 from France.
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Mass spectra were obtained with the support of the Protein Structure Core Facility at the University of Nebraska Medical Center.
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