Development of potent reversible selective inhibitors of butyrylcholinesterase as fluorescent probes.

Brain butyrylcholinesterase (BChE) is an attractive target for drugs designed for the treatment of Alzheimer's disease (AD) in its advanced stages. It also potentially represents a biomarker for progression of this disease. Based on the crystal structure of previously described highly potent, reversible, and selective BChE inhibitors, we have developed the fluorescent probes that are selective towards human BChE. The most promising probes also maintain their inhibition of BChE in the low nanomolar range with high selectivity over acetylcholinesterase. Kinetic studies of probes reveal a reversible mixed inhibition mechanism, with binding of these fluorescent probes to both the free and acylated enzyme. Probes show environment-sensitive emission, and additionally, one of them also shows significant enhancement of fluorescence intensity upon binding to the active site of BChE. Finally, the crystal structures of probes in complex with human BChE are reported, which offer an excellent base for further development of this library of compounds.

Mass spectrometry based proteomic approach for the screening of butyrylcholinesterase adduct formation with organophosphates.

Organophosphates' toxic effect causes covalent binding to serine-198 in the active site of human plasma butyrylcholinesterase (BChE) with loss of enzymatic function (covalent inhibition). Mass spectrometric detection of modified FGESAGAAS peptide at the active site is a powerful exposure biomarker tool. The aim of this study was to develop mass spectrometry-based method for BChE adduct formation screening, avoiding the use of standard peptides. Immunomagnetic separation of proteins from plasma was optimized. Commercially available anti-butyrylcholinesterase monoclonal antibodies, immobilized on magnetic beads, resulted in stable and reusable affinity sorbent. The method was tested on horse serum BChE and real human plasma from healthy donors, treated with Russian VX (VR). The BChE isolated from blood plasma was digested with pepsin and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The method was evaluated by using synthetic peptides and by comparison to the enzymatic activity Ellman's assay. The minimum concentration of VR exposure, resulting in detectable VR-adduct, was 0.2 ng/mL, which corresponded to the relative BChE inhibition of less than 2%. Adduct formation assessment was performed via monitoring of decrease in non-modified peptide LC-MS/MS signal and increase in VR-modified peptide signal. The designed approach was tested in a pilot study with 5 blood samples from healthy volunteers. Mass spectrometry-based method for BChE adduct formation was found to be in agreement with Ellman's inhibition assay, so the method is applicable for direct BChE inhibition assessment.

Purification of recombinant human butyrylcholinesterase on Hupresin®.

Affinity chromatography on procainamide-Sepharose has been an important step in the purification of butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) since its introduction in 1978. The procainamide affinity gel has limitations. In the present report a new affinity gel called Hupresin® was evaluated for its ability to purify truncated, recombinant human butyrylcholinesterase (rHuBChE) expressed in a stably transfected Chinese Hamster Ovary cell line. We present a detailed example of the purification of rHuBChE secreted into 3940 mL of serum-free culture medium. The starting material contained 13,163 units of BChE activity (20.9 mg). rHuBChE was purified to homogeneity in a single step by passage over 82 mL of Hupresin® eluted with 0.1 M tetramethylammonium bromide in 20 mM TrisCl pH 7.5. The fraction with the highest specific activity of 630 units/mg contained 11 mg of BChE. Hupresin® is superior to procainamide-Sepharose for purification of BChE, but is not suitable for purifying native AChE because Hupresin® binds AChE so tightly that AChE is not released with buffers, but is desorbed with denaturing solvents such as 50% acetonitrile or 1% trifluoroacetic acid. Procainamide-Sepharose will continue to be useful for purification of AChE.

Identification of Carboxylesterase, Butyrylcholinesterase, Acetylcholinesterase, Paraoxonase, and Albumin Pseudoesterase in Guinea Pig Plasma through Nondenaturing Gel Electrophoresis.

Drugs to protect against nerve agent toxicity are tested in animals. The current preferred small animal model is guinea pigs because their plasma bioscavenging capacity resembles that of NHP. We stained nondenaturing polyacrylamide slab gels with a variety of substrates, inhibitors, and antibodies to identify the esterases in heparinized guinea pig plasma. An intense band of carboxylesterase activity migrated behind albumin. Minor carboxylesterase bands were revealed after background activity from paraoxonase was inhibited by using EDTA. The major butyrylcholinesterase band was a disulfide-linked dimer. Incubation with the antihuman butyrylcholinesterase antibody B2 18-5 shifted the butyrylcholinesterase dimer band to slower migrating complexes. Carboxylesterases were distinguished from butyrylcholinesterase by their sensitivity to inhibition by bis-p-nitrophenyl phosphate. Acetylcholinesterase tetramers formed a complex with the antihuman acetylcholinesterase antibody HR2. Organophosphorus toxicants including cresyl saligenin phosphate, dichlorvos, and chlorpyrifos oxon irreversibly inhibited the serine esterases but not paraoxonase. Albumin pseudoesterase activity was seen in gels stained with α- or ß-naphthyl acetate and fast blue RR. We conclude that guinea pig plasma has 2 types of carboxylesterase, butyrylcholinesterase dimers and 5 minor butyrylcholinesterase forms, a small amount of acetylcholinesterase tetramers, paraoxonase, and albumin pseudoesterase activity. A knockout mouse with no carboxylesterase activity in plasma is available and may prove to be a better model for studies of nerve agent toxicology than guinea pigs.

Characterization of butyrylcholinesterase from porcine milk.

Human butyrylcholinesterase (HuBChE) is under development for use as a pretreatment antidote against nerve agent toxicity. Animals are used to evaluate the efficacy of HuBChE for protection against organophosphorus nerve agents. Pharmacokinetic studies of HuBChE in minipigs showed a mean residence time of 267 h, similar to the half-life of HuBChE in humans, suggesting a high degree of similarity between BChE from 2 sources. Our aim was to compare the biochemical properties of PoBChE purified from porcine milk to HuBChE purified from human plasma. PoBChE hydrolyzed acetylthiocholine slightly faster than butyrylthiocholine, but was sensitive to BChE-specific inhibitors. PoBChE was 50-fold less sensitive to inhibition by DFP than HuBChE and 5-fold slower to reactivate in the presence of 2-PAM. The amino acid sequence of PoBChE determined by liquid chromatography tandem mass spectrometry was 91% identical to HuBChE. Monoclonal antibodies 11D8, mAb2, and 3E8 (HAH 002) recognized both PoBChE and HuBChE. Assembly of 4 identical subunits into tetramers occurred by noncovalent interaction with polyproline-rich peptides in PoBChE as well as in HuBChE, though the set of polyproline-rich peptides in milk-derived PoBChE was different from the set in plasma-derived HuBChE tetramers. It was concluded that the esterase isolated from porcine milk is PoBChE.

Simultaneous Time-concentration Analysis of Soman and VX Adducts to Butyrylcholinesterase and Albumin by LC-MS-MS.

A sensitive method for the purification and determination of two protein adducts, organophosphorus (OP)-BChE and OP-albumin adducts, in a single sample using a simultaneous sample preparation method was developed and validated using liquid chromatography-tandem mass spectrometry. First, we isolated O-ethyl S-2-diisopropylaminoethyl methyl phosphonothiolate (VX) and O-pinacolyl methylphosphonofluoridate (soman, GD)-BChE adducts using an immunomagnetic separation (IMS) method and the HiTrap™ Blue affinity column was subsequently used to isolate and purify VX and GD-albumin adducts from the plasma of rhesus monkeys exposed to nerve agents. Additionally, we examined the time-concentration profiles of two biomarkers, VX and GD-nonapeptides and VX and GD-tyrosines, derived from OP-BChE and OP-albumin adducts up to 8 weeks after exposure. Based on the results, we determined that VX and GD-tyrosine is more suitable than VX and GD-nonapeptide as a biomarker owing to its longevity. This integrated approach is expected to be applicable for the quantification of other OP-BChE and OP-albumin adducts in human plasma, thus serving as a potential generic assay for exposure to nerve agents.

Purification, characterization, and N-glycosylation of recombinant butyrylcholinesterase from transgenic rice cell suspension cultures.

Recombinant butyrylcholinesterase produced in a metabolically regulated transgenic rice cell culture (rrBChE) was purified to produce a highly pure (95%), active form of enzyme. The developed downstream process uses common manufacturing friendly operations including tangential flow filtration, anion-exchange chromatography, and affinity chromatography to obtain a process recovery of 42% active rrBChE. The purified rrBChE was then characterized to confirm its comparability to the native human form of the molecule (hBChE). The recombinant and native enzyme demonstrated comparable enzymatic behavior and had an identical amino acid sequence. However, rrBChE differs in that it contains plant-type complex N-glycans, including an α-1,3 linked core fucose, and a ß-1,2 xylose, and lacking a terminal sialic acid. Despite this difference, rrBChE is demonstrated to be an effective stoichiometric bioscavenger for five different organophosphorous nerve agents in vitro. Together, the efficient downstream processing scheme and functionality of rrBChE confirm its promise as a cost-effective alternative to hBChE for prophylactic and therapeutic use.

Simultaneous detection of dual biomarkers from humans exposed to organophosphorus pesticides by combination of immunochromatographic test strip and ellman assay.

A novel sandwich immunoassay based immunochromatographic test strip (ICTS) has been developed for simultaneously measuring both butyrylcholinesterase (BChE) activity and the total amount of BChE (including inhibited and active enzyme) from 70 µLpost-exposure human plasma sample. The principle of this method is based on the BChE monoclonal antibody (MAb) capable of acting as both capture antibody and detection antibody. The BChE MAb which was immobilized on the test line was able to recognize both organophosphorus BChE adducts (OP-BChE) and BChE and provided equal binding affinity, permitting detection of the total enzyme amount in post-exposure human plasma samples. The formed immunocomplexes on the test line can further be excised from the test-strip for subsequent off-line measurement of BChE activity using the Ellman assay. Therefore, dual biomarkers of BChE activity and phosphorylation (OP-BChE) will be obtained simultaneously. The whole sandwich-immunoassay was performed on one ICTS, greatly reducing analytical time. The ICTS sensor showed excellent linear responses for assaying total amount of BChE and active BChE ranging from 0.22 to 3.58nM and 0.22-7.17nM, respectively. Both the signal detection limits are 0.10nM. We validated the practical application of the proposed method to measure 124 human plasma samples from orchard workers and cotton farmers with long-term exposure to organophosphorus pesticides (OPs). The results were in highly agreement with LC/MS/MS which verified our method is extremely accurate. Combining the portability and rapidity of test strip and the compatibility of BChE MAb as both capture antibody and detection antibody, the developed method provides a baseline-free, low-cost and rapid tool for in-field monitoring of OP exposures.

Simultaneous quantification of soman and VX adducts to butyrylcholinesterase, their aged methylphosphonic acid adduct and butyrylcholinesterase in plasma using an off-column procainamide-gel separation method combined with UHPLC-MS/MS.

This work describes a novel and sensitive non-isotope dilution method for simultaneous quantification of organophosphorus nerve agents (OPNAs) soman (GD) and VX adducts to butyrylcholinesterase (BChE), their aged methylphosphonic acid (MeP) adduct and unadducted BChE in plasma exposed to OPNA. OPNA-BChE adducts were isolated with an off-column procainamide-gel separation (PGS) from plasma, and then digested with pepsin into specific adducted FGES*AGAAS nonapeptide (NP) biomarkers. The resulting NPs were detected by UHPLC-MS/MS MRM. The off-column PGS method can capture over 90% of BChE, MeP-BChE, VX-BChE and GD-BChE from their respective plasma materials. One newly designed and easily synthesized phosphorylated BChE nonapeptide with one Gly-to-Ala mutation was successfully reported to serve as internal standard instead of traditional isotopically labeled BChE nonapeptide. The linear range of calibration curves were from 1.00-200ngmL-1 for VX-NP, 2.00-200ngmL-1 for GD-NP and MeP-NP (R2≥0.995), and 3.00-200ngmL-1 for BChE NP (R2≥0.990). The inter-day precision had relative standard deviation (%RSD) of <8.89%, and the accuracy ranged between 88.9-120%. The limit of detection was calculated to be 0.411, 0.750, 0.800 and 1.43ngmL-1 for VX-NP, GD-NP, MeP-NP and BChE NP, respectively. OPNA-exposed quality control plasma samples were characterized as part of method validation. Investigation of plasma samples unexposed to OPNA revealed no baseline values or interferences. Using the off-column PGS method combined with UHPLC-MS/MS, VX-NP and GD-NP adducts can be unambiguously detected with high confidence in 0.10ngmL-1 and 0.50ngmL-1 of exposed human plasma respectively, only requiring 0.1mL of plasma sample and taking about four hours without special sample preparation equipment. These improvements make it a simple, sensitive and robust PGS-UHPLC-MS/MS method, and this method will become an attractive alternative to immunomagnetic separation (IMS) method and a useful diagnostic tool for retrospective detection of OPNA exposure with high confidence. Furthermore, using the developed method, the adducted BChE levels from VX and GD-exposed (0.10-100ngmL-1) plasma samples were completely characterized, and the fact that VX being more active and specific to BChE than GD was re-confirmed.

Synthesis and study on activity in vitro of the high purity human butyrylcholinesterase conjugated with gold nanoparticles.

The aim of this research was to design a method of immobilization of high-purity human butyrylcholinesterase on the surface of gold nanoparticles preserving the activity of the enzyme. In order to achieve this aim, the method of fractionation and purification of human butyrylcholinesterase from plasma was modified. The synthesis of 15-nm gold nanoparticles was carried out by citrated method. A method of conjugation of the high-purity butyrylcholinesterase with gold nanoparticles was developed. It was found that the Immobilization of butyrylcholinesterase on the surface of gold nanoparticles resulted in a significant (to 23%) increase in the specific activity of the enzyme.

Comparative chemical profiling, cholinesterase inhibitions and anti-radicals properties of essential oils from Polygonum hydropiper L: a preliminary anti- Alzheimer's study.

BACKGROUND: Cholinesterase inhibition is a vital target for the development of novel and mechanism based inhibitors, owing to their role in the breakdown of acetylcholine (ACh) neurotransmitter to treat various neurological disorders including Alzheimer's disease (AD). Similarly, free radicals are implicated in the progression of various diseases like neurodegenerative disorders. Due to lipid solubility and potential to easily cross blood brain barrier, this study was designed to investigate the anticholinesterase and antioxidant potentials of the standardized essential oils from the leaves and flowers of Polygonum hydropiper. METHODS: Essential oils from the leaves (Ph.LO) and flowers (Ph.FO) of P. hdropiper were isolated using Clevenger apparatus. Oil samples were analyzed by GC-MS to identify major components and to attribute the antioxidant and anticholinesterase activity to specific components. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory potentials of the samples were determined following Ellman's assay. Antioxidant assays were performed using 1,1-diphenyl,2-picrylhydrazyl (DPPH), 2,2-azinobis[3-ethylbenzthiazoline]-6-sulfonic acid (ABTS) and hydrogen peroxide (H2O2) free radical scavenging assays. RESULTS: In the GC-MS analysis 141 and 122 compounds were indentified in Ph.LO and Ph.FO respectively. Caryophylene oxide (41.42 %) was the major component in Ph.FO while decahydronaphthalene (38.29 %) was prominent in Ph.LO. In AChE inhibition, Ph.LO and Ph.FO exhibited 87.00** and 79.66***% inhibitions at 1000 µg/ml with IC50 of 120 and 220 µg/ml respectively. The IC50 value for galanthamine was 15 µg/ml. In BChE inhibitory assay, Ph.LO and Ph.FO caused 82.66*** (IC50 130 µg/ml) and 77.50***% (IC50 225 µg/ml) inhibitions respectively at 1000 µg/ml concentration. In DPPH free radical scavenging assay, Ph.LO and Ph.FO exhibited IC50 of 20 and 200 µg/ml respectively. The calculated IC50s were 180 & 60 µg/ml for Ph.LO, and 45 & 50 µg/ml for Ph.FO in scavenging of ABTS and H2O2 free radicals respectively. CONCLUSIONS: In the current study, essential oils from leaves and flowers of P. hydropiper exhibited dose dependent anticholinesterase and antioxidant activities. Leaves essential oil were more effective and can be subjected to further in-vitro and in-vivo anti-Alzheimer's studies.

Comparison of 5 monoclonal antibodies for immunopurification of human butyrylcholinesterase on Dynabeads: KD values, binding pairs, and amino acid sequences.

Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger of nerve agents and organophosphorus pesticides. Mass spectrometry methods detect stable nerve agent adducts on the active site serine of HuBChE. The first step in sample preparation is immunopurification of HuBChE from plasma. Our goal was to identify monoclonal antibodies that could be used to immunopurify HuBChE on Dynabeads Protein G. Mouse anti-HuBChE monoclonal antibodies were obtained in the form of ascites fluid, dead hybridoma cells stored frozen at -80 °C for 30 years, or recently frozen hybridoma cells. RNA from 4 hybridoma cell lines was amplified by PCR for determination of their nucleotide and amino acid sequences. Full-length light and heavy chains were expressed, and the antibodies purified from culture medium. A fifth monoclonal was purchased. The 5 monoclonal antibodies were compared for ability to capture HuBChE from human plasma on Dynabeads Protein G. In addition, they were evaluated for binding affinity by Biacore and ELISA. Epitope mapping by pairing analysis was performed on the Octet Red96 instrument. The 5 monoclonal antibodies, B2 12-1, B2 18-5, 3E8, mAb2, and 11D8, had similar KD values of 10(-9) M for HuBChE. Monoclonal B2 18-5 outperformed the others in the Dynabeads Protein G assay where it captured 97% of the HuBChE in 0.5 ml plasma. Pairing analysis showed that 3E8 and B2 12-1 share the same epitope, 11D8 and B2 18-5 share the same epitope, but mAb2 and B2 12-1 or mAb2 and 3E8 bind to different epitopes on HuBChE. B2 18-5 was selected for establishment of a stable CHO cell line for production of mouse anti-HuBChE monoclonal.

Small-scale purification of butyrylcholinesterase from human plasma and implementation of a µLC-UV/ESI MS/MS method to detect its organophosphorus adducts.

Human butyrylcholinesterase (hBChE) is a serine hydrolase (EC 3.1.1.8) present in all mammalian tissues and the bloodstream. Similar to acetylcholinesterase, the enzyme reacts with organophosphorus compounds (OP) like nerve agents or pesticides that cause enzyme inhibition (BChE adducts). These adducts represent valuable biomarkers for analytical verification of OP exposure. For establishment of these mass spectrometry based methods sufficient amounts of hBChE in high purity are required. Unfortunately, commercial lots are of inappropriate purity thus favouring in-house isolation. Therefore, we developed a small scale procedure to isolate hBChE from citrate plasma. After precipitation by polyethylene glycol (8% w/v and 20% w/v PEG 6000) hBChE was purified from plasma by four consecutive chromatographic steps including anion exchange, affinity extraction and size exclusion. Protein elution was monitored on-line by UV-absorbance (280 nm) followed by continuous fractionation for off-line analysis of (1) hBChE enzyme activity by Ellman assay, (2) protein purity by gel electrophoresis, and (3) protein identity by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Numerous major impurities separated from hBChE were identified. The purified material was used for in vitro incubation with diverse OP to establish a µ-liquid chromatography-ultra violet detection/electrospray ionization tandem-mass spectrometric method (µLC-UV/ESI MS/MS) for detection of hBChE adducts suitable for verification analysis. Analytical data for diverse OP pesticides including deuterated analogues as well as G- and V-type nerve agents and their precursor are summarized. This method was successfully applied to plasma samples provided by the Organisation for the Prohibition of Chemical Weapons (OPCW) for the 4th Biomedical Exercise.

Discovery of butyrylcholinesterase inhibitors among derivatives of azaphenothiazines.

The study presents the discovery of novel butyrylcholinesterase (BuChE) inhibitors among derivatives of azaphenothiazines by application of in silico and in vitro screening methods. From an in-house library of compounds, 143 heterocyclic molecules derived from the azaphenothiazine scaffold were chosen for virtual screening. Based on results of the docking procedure, 15 compounds were identified as exhibiting the best fit for the two screening complexes (ligand - AChE and ligand - BuChE). Five compounds displayed moderate AChE and good BuChE inhibitory activity at screening concentrations of 10 µM. The IC50 values for active BuChE inhibitors were in the 11.8-122.2 nM range. Three of the most active inhibitors are tetra- or pentacyclic derivatives of azaphenothiazines with the same N-methyl-2-piperidinethyl substituent.

Oligomerization status influences subcellular deposition and glycosylation of recombinant butyrylcholinesterase in Nicotiana benthamiana.

Plants have a proven track record for the expression of biopharmaceutically interesting proteins. Importantly, plants and mammals share a highly conserved secretory pathway that allows similar folding, assembly and posttranslational modifications of proteins. Human butyrylcholinesterase (BChE) is a highly sialylated, tetrameric serum protein, investigated as a bioscavenger for organophosphorous nerve agents. Expression of recombinant BChE (rBChE) in Nicotiana benthamiana results in accumulation of both monomers as well as assembled oligomers. In particular, we show here that co-expression of BChE with a novel gene-stacking vector, carrying six mammalian genes necessary for in planta protein sialylation, resulted in the generation of rBChE decorated with sialylated N-glycans. The N-glycosylation profile of monomeric rBChE secreted to the apoplast largely resembles the plasma-derived orthologue. In contrast, rBChE purified from total soluble protein extracts was decorated with a significant portion of ER-typical oligomannosidic structures. Biochemical analyses and live-cell imaging experiments indicated that impaired N-glycan processing is due to aberrant deposition of rBChE oligomers in the endoplasmic reticulum or endoplasmic-reticulum-derived compartments. In summary, we show the assembly of rBChE multimers, however, also points to the need for in-depth studies to explain the unexpected subcellular targeting of oligomeric BChE in plants.

Human butyrylcholinesterase produced in insect cells: huprine-based affinity purification and crystal structure.

Butyrylcholinesterase (BChE) is a serine hydrolase that is present in all mammalian tissues. It can accommodate larger substrates or inhibitors than acetylcholinesterase (AChE), the enzyme responsible for hydrolysis of the neurotransmitter acetylcholine in the central nervous system and neuromuscular junctions. AChE is the specific target of organophosphorous pesticides and warfare nerve agents, and BChE is a stoichiometric bioscavenger. Conversion of BChE into a catalytic bioscavenger by rational design or designing reactivators specific to BChE required structural data obtained using a recombinant low-glycosylated human BChE expressed in Chinese hamster ovary cells. This expression system yields ≈ 1 mg of pure enzyme per litre of cell culture. Here, we report an improved expression system using insect cells with a fourfold higher yield for truncated human BChE with all glycosylation sites present. We developed a fast purification protocol for the recombinant protein using huprine-based affinity chromatography, which is superior to the classical procainamide-based affinity. The purified BChE crystallized under different conditions and space group than the recombinant low-glycosylated protein produced in Chinese hamster ovary cells. The crystals diffracted to 2.5 Å. The overall monomer structure is similar to the low-glycosylated structure except for the presence of the additional glycans. Remarkably, the carboxylic acid molecule systematically bound to the catalytic serine in the low-glycosylated structure is also present in this new structure, despite the different expression system, purification protocol and crystallization conditions.

Identification of phosphorylated butyrylcholinesterase in human plasma using immunoaffinity purification and mass spectrometry.

Paraoxon (diethyl 4-nitrophenyl phosphate) is an active metabolite of the common insecticide parathion and is acutely toxic due to the inhibition of cholinesterase (ChE) activity in the nervous systems. The inhibition of butyrylcholinesterase (BChE) activity by paraoxon is due to the formation of phosphorylated BChE adduct, and the detection of the phosphorylated BChE adduct in human plasma can serve as an exposure biomarker of organophosphate pesticides and nerve agents. In this study, we developed an immunoaffinity purification and liquid chromatography-mass spectrometry (LC-MS) strategy for identifying phosphorylated BChE in human plasma treated by paraoxon. BChE was captured by biotinylated anti-BChE polyclonal antibodies conjugated to streptavidin magnetic beads. Western blot analysis showed that the antibody was effective to recognize both native and modified BChE with high specificity. Using a purified BChE protein, we initially identified the exact phosphorylation site on the serine residue (S198) with a 108 Da modification by both MS/MS and accurately measured parent ion masses and quantified the extent of phosphorylation on S198 following paraoxon treatment to be >99.9%. Then, the phosphorylated BChE peptide in paraoxon-treated human plasma following immunoaffinity purification was successfully identified based on the accurate measured mass and retention time information initially obtained from the purified BChE protein. Thus, immunoaffinity purification combined with LC-MS represents a viable approach for the detection and quantification of phosphorylated BChE as an exposure biomarker of organophosphates and nerve agents.

His-tag truncated butyrylcholinesterase as a useful construct for in vitro characterization of wild-type and variant butyrylcholinesterases.

Human butyrylcholinesterase (BChE) can scavenge and thereby provide protection against various toxic esters, including organophosphate-based chemical warfare agents and the recreational drug cocaine. It is currently being used in molecular evolution studies to generate novel enzymes with improved ability to hydrolyze toxic ester compounds. Currently, the most commonly used purification strategies for recombinant BChE enzymes involve using affinity resins based on small molecule interactions with the enzyme's substrate binding site. However, as BChE variants are discovered and developed, a generic purification protocol that is insensitive to amino acid substitutions is necessary. In the current manuscript, an expression vector encoding a C-terminal truncation and a His6-tag was designed for BChE and used to express recombinant "wild-type" enzyme and two variants (i.e., G117H BChE and G117H/E197Q BChE). All the three His6-tagged enzymes were successfully purified via metal-affinity columns using similar procedures with good recovery. Steady-state kinetic parameters were determined for each enzyme, and values were compared to those obtained with the corresponding non-truncated non-His6-tagged enzymes. Rates of inhibition by echothiophate, a model compound for organophosphate-based pesticides, and rates of oxime-mediated reactivation after inhibition with a nerve agent model compound were also determined for selected enzymes. Rates of spontaneous reactivation from ETP inhibition were determined for the G117H variants. In all instances examined, truncation of the C-terminus of BChE and introduction of a His6-tag had no significant effects on the observed kinetic parameters, making this a highly useful construct for in vitro characterization of wild-type and variant BChEs.

Exposure to tri-o-cresyl phosphate detected in jet airplane passengers.

The aircraft cabin and flight deck ventilation are supplied from partially compressed unfiltered bleed air directly from the engine. Worn or defective engine seals can result in the release of engine oil into the cabin air supply. Aircrew and passengers have complained of illness following such "fume events". Adverse health effects are hypothesized to result from exposure to tricresyl phosphate mixed esters, a chemical added to jet engine oil and hydraulic fluid for its anti-wear properties. Our goal was to develop a laboratory test for exposure to tricresyl phosphate. The assay was based on the fact that the active-site serine of butyrylcholinesterase reacts with the active metabolite of tri-o-cresyl phosphate, cresyl saligenin phosphate, to make a stable phosphorylated adduct with an added mass of 80 Da. No other organophosphorus agent makes this adduct in vivo on butyrylcholinesterase. Blood samples from jet airplane passengers were obtained 24-48 h after completing a flight. Butyrylcholinesterase was partially purified from 25 ml serum or plasma, digested with pepsin, enriched for phosphorylated peptides by binding to titanium oxide, and analyzed by mass spectrometry. Of 12 jet airplane passengers tested, 6 were positive for exposure to tri-o-cresyl phosphate that is, they had detectable amounts of the phosphorylated peptide FGEpSAGAAS. The level of exposure was very low. No more than 0.05 to 3% of plasma butyrylcholinesterase was modified. None of the subjects had toxic symptoms. Four of the positive subjects were retested 3 to 7 months following their last airplane trip and were found to be negative for phosphorylated butyrylcholinesterase. In conclusion, this is the first report of an assay that detects exposure to tri-o-cresyl phosphate in jet airplane travelers.

Biochemical, molecular and preclinical characterization of a double-virus-reduced human butyrylcholinesterase preparation designed for clinical use.

BACKGROUND AND OBJECTIVES: A human plasma-derived butyrylcholinesterase preparation manufactured on the industrial scale is described. MATERIAL AND METHODS: The human butyrylcholinesterase (hBChE) product was extensively investigated for its purity using immunological and electrophoretic methods and characterized by thorough glycoproteomic approaches. A comprehensive preclinical testing programme addressing safety and pharmacokinetic parameters supplemented the biochemical characterization. RESULTS: The high-purity hBChE preparation is tetrameric and has high specific activity and molecular integrity of the protein backbone. Acute toxicity studies and in vivo thrombogenicity studies provided evidence of a sufficient safety margin for use in humans. CONCLUSION: Extensive preclinical safety and pharmacokinetic testing confirmed that this hBChE preparation can be used for further efficacy testing as a bioscavenger for toxic organophosphate compounds in appropriate animal models and ultimately in humans.