Organophosphorus Neurotoxins Research Articles

Not a Mistake but a Feature: Promiscuous Activity of Enzymes Meeting Mycotoxins

Mycotoxins are dangerous compounds and find multiple routes to enter living bodies of humans and animals. To solve the issue and degrade the toxicants, (bio)catalytic processes look very promising. Hexahistidine-tagged organophosphorus hydrolase (His6-OPH) is a well-studied catalyst for degradation of organophosphorus neurotoxins and lactone-containing quorum-sensing signal molecules. Moreover, the catalytic characteristics in hydrolysis of several mycotoxins (patulin, deoxynivalenol, zearalenone, and sterigmatocystin) were studied in this investigation. The best Michaelis constant and catalytic constant were estimated in the case of sterigmatocystin and patulin, respectively. A possible combination of His6-OPH with inorganic sorbents treated by low-temperature plasma was investigated. Further, enzyme–polyelectrolyte complexes of poly(glutamic acid) with His6-OPH and another enzymatic mycotoxin degrader (thermolysin) were successfully used to modify fiber materials. These catalytically active prototypes of protective materials appear to be useful for preventing surface contact and exposure to mycotoxins and other chemicals that are substrates for the enzymes used.

Sensor Array Chip for Real‐Time Field Detection and Discrimination of Organophosphorus Neurotoxins

AbstractRapid and reliable detection of organophosphorus (OP) threats is essential for facilitating efficient and timely countermeasures. This work describes the design and operation of a miniaturized electrochemical sensor array chip, based on strategic coupling of diverse MOF‐based biomimetic and biocatalytic reagent layers (offering tunable OP target specificities) with different potentiometric, voltametric, and amperometric transducers to generate information‐rich analytical data and robust discrimination power towards rapid field identification of key classes of OP threats. This uniquely integrated 4‐electrode sensor array yields rich analytical data that enables rapid and efficient discrimination of P−F‐ and P−Cl‐type OP nerve agents (NAs) and OP pesticides from coexisting environmental pollutants. The sensor array outputs lead to distinct binary codes for the target OP threats that enable their rapid digital identification. This sensor array chip thus presents a significant advance towards reliable OP threat‐sensing hand‐held devices that meet the urgent demands of different defense surveillance applications.

Advanced Biocatalysts Based on Hexahistidine-Containing organophosphorus Hydrolase for Chemical and Biological Defense

The advanced biocatalysts based on hexahistidine-tagged organophosphorus hydrolase (His6-OPH) were recently developed for the detoxification of various organophosphorus compounds and degradation of N-acyl homoserine lactones. Due to enzyme immobilization, some of obtained biocatalysts are quite stable, easy to use and very effective/active (e.g. tens of millions of substrate solution volumes appeared to be treated with column cartridges containing immobilized His6-OPH). Recently, the possible bioengineering of different stabilized nanocomplexes of His6-OPH due to its non-covalent binding with different compounds (polymers, antioxidants, antimicrobials, etc.) was demonstrated. Firstly, it was realized by computer modeling via molecular docking. Polymers of amino acids (polyglutamic and polyasparctic acids) were established to be the most effective stabilizers of the enzyme that enabled effective preservation of the enzyme activity. Up to 100 %-retention of initial catalytic characteristics of the enzyme was reached in obtained enzymatic complexes. Such nanobiocatalysts were stabilized against inactivating effects of solvents, temperatures and were able to circulate in vivo for at least 25 hours. It appeared that different antioxidants can be applied as partners of the enzyme in the nanocomplexing. Thus, a new set of original enzymatic antidotes were developed possessing dual action: both hydrolytic activity against organophosphorus neurotoxins and improved antioxidant activity. Additionally, it was shown that different organophosphorus compounds and N-acyl homoserine lactones could be molecularly docked directly to the active centers of His6 -OPH dimer, thus allowing to theoretically clarify some new prospective substrates for the enzymatic hydrolysis. It appeared that new type of nanocomplexes of the enzyme with antibiotics also can be prepared. In this case the combination of antibiotics with enzyme quenching the quorum of the pathogenic gram-negative bacteria was performed. The enzyme being stabilized by the various antibiotics (especially those containing β-lactame ring) played the role of a carrier for the antimicrobial compounds significantly improving their efficiency of the action. Such biocatalysts and/or method of their design have a great potential and can be very useful for both chemical and biological defense

A simple and highly effective catalytic nanozyme scavenger for organophosphorus neurotoxins

A simple and highly efficient catalytic scavenger of poisonous organophosphorus compounds, based on organophosphorus hydrolase (OPH, EC 3.1.8.1), is produced in aqueous solution by electrostatic coupling of the hexahistidine tagged OPH (His6-OPH) and poly(ethylene glycol)-b-poly(l-glutamic acid) diblock copolymer. The resulting polyion complex, termed nano-OPH, has a spherical morphology and a diameter from 25nm to 100nm. Incorporation of His6-OPH in nano-OPH preserves catalytic activity and increases stability of the enzyme allowing its storage in aqueous solution for over a year. It also decreases the immune and inflammatory responses to His6-OPH in vivo as determined by anti-OPH IgG and cytokines formation in Sprague Dawley rats and Balb/c mice, respectively. The nano-OPH pharmacokinetic parameters are improved compared to the naked enzyme suggesting longer blood circulation after intravenous (iv) administrations in rats. Moreover, nano-OPH is bioavailable after intramuscular (im), intraperitoneal (ip) and even transbuccal (tb) administration, and has shown ability to protect animals from exposure to a pesticide, paraoxon and a warfare agent, VX. In particular, a complete protection against the lethal doses of paraoxon was observed with nano-OPH administered iv and ip as much as 17h, im 5.5h and tb 2h before the intoxication. Further evaluation of nano-OPH as a catalytic bioscavenger countermeasure against organophosphorus chemical warfare agents and pesticides is warranted.

Discriminative Detection of Neurotoxins by a Layer-by-Layer Based Carbon Nanotube/bi-enzyme Biosensor

Multi-enzyme systems have attracted considerable interests due to the versatility for multiplexed detection of different analytes. In this study, a novel layer-by-layer (LbL) assembled multi-enzyme biosensor incorporating acetylcholinesterase (AChE) and organophosphate hydrolase (OPH) was proposed for discriminative detection between organophosphorus (OP) and non-organophosphorus (non-OP) neurotoxins. This was achieved through LbL assembly of electrostatically interacted enzyme armored carbon nanotubes (CNT-OPH and CNT-AChE) with a set of cushioning bilayers consisting of CNT-polyethyleneimine (PEI) and CNT-DNA on glassy carbon electrode with electrochemical detection. OPH catalyzes the hydrolysis of OPs (e.g. paraoxon) and AChE can be used for detection of both OPs and non-OPs (e.g. carbamate) through inhibition assays, and thus makes it possible for detection of OP neurotoxins from multi-analytes samples, and determination of the non-OP neurotoxins to their true concentration with AChE inhibition. Optimization of the LbL architecture was initially conducted with penetration analyses including position of active layer, number of total layers and impedance analyses with respect to different concentration of CNTs. Surface morphology and chemical analyses were characterized with Scanning Electron Microscopy and Raman spectroscopy to have a better understanding of the layered structure. The simple and inexpensive LbL approach can be further extended to a wide variety of enzymes in a multi-detection biosensing system, wherein each analyte could be “fingerprinted” with respect to its catalytic response to different enzymes.

Fe3O4 Magnetic Nanoparticle Peroxidase Mimetic-Based Colorimetric Assay for the Rapid Detection of Organophosphorus Pesticide and Nerve Agent

Rapid and sensitive detection methods are in urgent demand for the screening of extensively used organophosphorus pesticides and highly toxic nerve agents for their neurotoxicity. In this study, we developed a novel Fe(3)O(4) magnetic nanoparticle (MNP) peroxidase mimetic-based colorimetric method for the rapid detection of organophosphorus pesticides and nerve agents. The detection assay is composed of MNPs, acetylcholinesterase (AChE), and choline oxidase (CHO). The enzymes AChE and CHO catalyze the formation of H(2)O(2) in the presence of acetylcholine, which then activates MNPs to catalyze the oxidation of colorimetric substrates to produce a color reaction. After incubation with the organophosphorus neurotoxins, the enzymatic activity of AChE was inhibited and produced less H(2)O(2), resulting in a decreased catalytic oxidation of colorimetric substrates over MNP peroxidase mimetics, accompanied by a drop in color intensity. Three organophosphorus compounds were tested on the assay: acephate and methyl-paraoxon as representative organophosphorus pesticides and the nerve agent Sarin. The novel assay displayed substantial color change after incubation in organophosphorus neurotoxins in a concentration-dependent manner. As low as 1 nM Sarin, 10 nM methyl-paraoxon, and 5 μM acephate are easily detected by the novel assay. In conclusion, by employing the peroxidase-mimicking activity of MNPs, the developed colorimetric assay has the potential of becoming a screening tool for the rapid and sensitive assessment of the neurotoxicity of an overwhelming number of organophosphate compounds.

Enzyme-based intravascular defense against organophosphorus neurotoxins: Synergism of dendritic-enzyme complexes with 2-PAM and atropine

Novel, enzyme-complexed, nano-delivery systems have been developed to antagonize the lethal effects of organophosphorus (OP) molecules such as diisopropylfluorophosphate and paraoxon. Polymeric nanocapsules can be used to deliver metabolizing enzymes to the circulation, often increasing the enzyme's efficacy by extending their circulatory life and, in some cases, enhancing their specific activity. The bacterial enzymes organophosphorus hydrolase (OPH) and organophosphorus anhydrolase (OPAA) were encapsulated within a nanocapsule, polyoxazoline-based dendritic polymer carrier and employed in combination with the OP antagonists pralidoxime (2-PAM) and atropine. The effective doses for OPH and OPAA, respectively, were 500–550 and 1500–1650 units/kg mice; the size of the entire complex is approximately 200 nm in diameter. These studies compare the efficacy of the two enzymes as prophylactic systems encapsulated within the dendritic polymer. When used in combination with 2-PAM and atropine, the dendritic encapsuled OPAA provided a 25×LD50 protection against DFP intoxication, while the similarly constructed OPH complex showed a more dramatic protection (780×LD50) against paraoxon intoxication in Balb/c mice. The studies demonstrate a synergistic enhancement of the antagonist, since the antidotal protection of 2-PAM+atropine against DFP and paraoxon is approximately 8 and 60×LD50, respectively.

Nanoparticle-based optical biosensors for the direct detection of organophosphate chemical warfare agents and pesticides

Neurotoxic organophosphates (OP) have found widespread use in the environment for insect control. In addition, there is the increasing threat of use of OP based chemical warfare agents in both ground based warfare and terrorist attacks. Together, these trends necessitate the development of simple and specific methods for discriminative detection of ultra low quantities of OP neurotoxins. In our previous investigations a new biosensor for the direct detection of organophosphorus neurotoxins was pioneered. In this system, the enzymatic hydrolysis of OP neurotoxins by organophosphate hydrolase (OPH) generated two protons in each hydrolytic turnover through reactions in which P–X bonds are cleaved. The sensitivity of this biosensor was limited due to the potentiometric method of detection. Recently, it was reported that a change in fluorescence properties of a fluorophore in the vicinity of gold nanoparticles might be used for detection of nanomolar concentrations of DNA oligonucleotides. The detection strategy was based on the fact that an enhancement or quenching of fluorescence intensity is a function of the distances between the gold nanoparticle and fluorophore. While these reports have demonstrated the use of nanoparticle-based sensors for the detection of target DNA, we observed that the specificity of enzyme–substrate interactions could be exploited in similar systems. To test the feasibility of this approach, OPH-gold nanoparticle conjugates were prepared, then incubated with a fluorescent enzyme inhibitor or decoy. The fluorescence intensity of the decoy was sensitive to the proximity of the gold nanoparticle, and thus could be used to indicate that the decoy was bound to the OPH. Then different paraoxon concentrations were introduced to the OPH–nanoparticle–conjugate–decoy mixtures, and normalized ratio of fluorescence intensities were measured. The greatest sensitivity to paraoxon was obtained when decoys and OPH–gold nanoparticle conjugates were present at near equimolar levels. The change in fluorescence intensity was correlated with concentration of paraoxon presented in the solution.

Design of physostigmine-loaded polymeric microparticles for pretreatment against exposure to organophosphate agents

Physostigmine is an anti-cholinesterase used for the pretreatment of a poisoning caused by highly toxic organophosphorus neurotoxins. The aim of this study is to design a polymeric microparticle system for sustained release of physostigmine. In this paper, we have attempted to encapsulate physostigmine in microparticles made from poly( d, l-lactide-co-glycolide) (PLGA) with various contents of glycolide and poly( d, l-lactide) (PLA) using spray-drying and single emulsion techniques. It was found that during the single emulsion process, most of the physostigmine molecules were lost in the external aqueous phase. However, more than 90% encapsulation efficiency of physostigmine was obtained using the spray-drying technique. SEM micrographs revealed that spherical microparticles containing physostigmine with a smooth surface were yielded with PLA, PLGA 50:50, RG 502 (PLGA 50:50 with a lower molecular weight) and PLGA 65:35 but PLGA 85:15, PLGA 75:25 and PLGA 50:50 with a high concentration produced microparticles with irregular shapes. An increased inlet temperature yielded a higher physostigmine release rate from the PLA microparticles. Physostigmine release from the microparticles showed a biphasic pattern, characterized by an initial burst release followed by a sustained release for PLGA 65:35, PLGA 50:50 and RG 502 or a non-detectable release for PLGA 85:15, PLGA 75:25 and PLA. A sustained-release of physostigmine with a low initial burst over 1 week was achieved from RG 502 microparticles, which would be used as an injectable dosage form in our further animal studies.

Dual amperometric–potentiometric biosensor detection system for monitoring organophosphorus neurotoxins

A dual-transducer flow-injection biosensor detection system for monitoring organophosphorus (OP) neurotoxins is described. Such simultaneous use of different physical transducers in connection to the same (organophosphorous hydrolase (OPH)) enzyme enhances the information content and provides discrimination between various subclasses of OP compounds. While the potentiometric biosensor responds favorably to all OP compounds, reflecting the pH changes associated with the OPH activity, the amperometric device displays well-defined signals only towards OP substrates (pesticides) liberating the oxidizable p-nitrophenol product. The potentiometric detection has been accomplished with a silicon-based pH-sensitive electrolyte-insulator-semiconductor (EIS) transducer, operated in the constant-capacitance (ConCap) mode. Both transducers are prepared by a thin-film fabrication technology, and respond rapidly and independently to sudden changes in the level of the corresponding OP compound, with no apparent cross reactivity. Relevant experimental variables were evaluated and optimized. Such development holds great promise for field screening of OP neurotoxins in connection to various defense and environmental scenarios. The multiple-transduction concept could be extended for increasing the information content of other ‘class-enzyme’ biosensor systems. © 2002 Elsevier Science B.V. All rights reserved.

Chemical and biological safety: Biosensors and nanotechnological methods for the detection and monitoring of chemical and biological agents

Abstract The elaboration of highly sensitive and express methods for quantitative and qualitative detection and monitoring of chemical warfare agents (CWA), organophosphate and carbamate pesticides, compounds with delayed neurotoxicity, and pathogenic microorganisms and viruses is discussed. The application of potentiometric and amperometric biosensors, automatic biosensors discriminating the neurotoxins of different classes, is performed. The information about biosensors detecting the compounds with delayed neurotoxicity through the evaluation of “neurotoxic esterase”activity in the blood is presented. The use of immunochip technology for the detection of pathogenic microorganisms and viruses is demonstrated. The enzymatic methods of destruction of organophosphorus neurotoxins are considered as the base of new defense technology.

Enzyme-based biosensor for the direct detection of fluorine-containing organophosphates

The ability of the enzyme organophosphorus acid anhydrolase (OPAA) to selectively hydrolyze the PF bond of fluorine containing organophosphates has been used to develop a biosensor for specific detection of these compounds. Hydrolysis rate of diisopropyl fluorophosphate (DFP), paraoxon and demeton-S, by soluble and immobilized OPAA was measured. These compounds were selected as representative substrates of OPAA hydrolysis of PF, PO and PS bonds, respectively. Results indicate that hydrolysis of phosphofluoridates such as DFP is dominant while hydrolysis of phosphotriesters such as paraoxon or of phosphothiolates such as demeton-S, is negligible. Two experimental approaches were used for biosensor development. In the first, OPAA was covalently immobilized on silica gel and used in batch-mode measurements with flat glass pH electrode to detect pH changes due to PX bond cleavage. In the second approach, the enzyme was covalently immobilized to the porous silica modified gate insulator of a pH-sensitive field effect transistor (pH-FET) and changes in pH relative to a second non-enzyme coated pH-FET were measured in stop-flow mode. Concentrations of DFP down to 25 μM with the glass electrode and 20 μM with the pH-FET were readily detected. No sensor response was observed with paraoxon or demeton-S indicating that such OPAA-based biosensors could be useful for direct and discriminative detection of fluorine containing organophosphorus neurotoxins (such as the G-type chemical warfare agents sarin GB and soman GD) in samples also containing multiple organophosphate pesticides.

Immobilization and characterization of sol–gel-encapsulated acetylcholinesterase fiber-optic biosensor

A rapid approach for preparing sol–gel based fiber-optic biosensor with encapsulated pH-sensitive fluorophore-acetylcholinesterase conjugates is developed for the determination of acetylcholine. The sensor consists of a pH-sensitive fluorescent indicator encapsulated with enzyme in a sol–gel network on a glass cap that can be fixed on an optical fiber and then integrated with a flow-through reactor for continuous monitoring. The sol–gel preparation procedure has been modified for maintaining a stable pH value and enzyme activity. Among the nine fluorescent indicators tested, FITC-dextran was the most suitable candidate for biosensor. The optimized ratio of TMOS:HCl:H 2O and enzyme-dye:sol solution for the rapid preparation of sol–gel film were 1:3.6×10 −5:2 and 3:5, respectively. The 1 mM pH 8.5 Tris–buffer had sufficient buffer capacity for acetylcholine preparation and the substrate concentration must be below 50 mM to obtain a stable pH value. The response of the developed biosensor to acetylcholine was highly reproducible (R.S.D.=3.5%, n=8). A good linearity of acetylcholine in the range from 0.5 to 20 mM was also obtained ( R 2=0.98). Furthermore, a 30% inhibition can be achieved within 30 min when 0.54 μM (152 ppb) paraoxon was added into the system. This indicates that the developed biosensor is suitable for the analysis of acetylcholine and organophosphorus neurotoxins.

Cycloplatinated aryl ketoximes as efficient biomimicking catalysts for hydrolysis of esters of phosphorothioic acid

Cyclometallated aryl ketoximes are introduced as catalysts for hydrolysis of organophosphorus neurotoxins. Platinum-containing catalysts exhibit the highest activity and selectivity with respect to O-alkyl phosphorothioates (parathion, methyl parathion, coumaphos) and efficiently promote the hydrolysis of S-alkyl phosphorothioates and -dithioates (demeton-S, malathion) at the P—S bonds.

Biocatalytic nerve agent detoxification in fire fighting foams.

Current events across the globe necessitate rapid technological advances to combat the epidemic of nerve agent chemical weapons. Biocatalysis has emerged as a viable tool in the detoxification of organophosphorus neurotoxins, such as the chemical weapons VX and sarin. Efficient detoxification of contaminated equipment, machinery, and soils are of principal concern. This study describes the incorporation of a biocatalyst (organophosphorus hydrolase, E.C. 3.1.8.1) into conventional formulations of fire fighting foam. The capacity of fire fighting foams to decrease volatilization of contained contaminants, increase surface wettability, and control the rate of enzyme delivery to large areas makes them useful vehicles for enzyme application at surfaces. The performance of enzyme containing foams has been shown to be not only reproducible but also predictable. An empirical model provides reasonable estimations for the amounts of achievable surface decontamination as a function of the important parameters of the system. Theoretical modeling illustrates that the enzyme-containing foam is capable of extracting agent from the surface and is catalytically active at the foam-surface interface and throughout the foam itself. Biocatalytic foam has proven to be an effective, "environmentally friendly" means of surface and soil decontamination.

Modification of near active site residues in organophosphorus hydrolase reduces metal stoichiometry and alters substrate specificity.

Organophosphorus hydrolase (OPH, EC 8.1.3.1) is a dimeric, bacterial enzyme that detoxifies many organophosphorus neurotoxins by hydrolyzing a variety of phosphonate bonds. The histidinyl residues at amino acid positions 254 and 257 are located near the bimetallic active site present in each monomer. It has been proposed that these residues influence catalysis by interacting with active site residues and the substrate in the binding pocket. We replaced the histidine at position 254 with arginine (H254R) and the one at position 257 with leucine (H257L) independently to form the single-site-modified enzymes. The double modification was also constructed to incorporate both changes (H254R/H257L). Although native OPH has two metals at each active site (four per dimer), all three of these altered enzymes possessed only two metals per dimer while retaining considerable enzymatic activity for the preferred phosphotriester (P-O bond) substrate, paraoxon (5-100% kcat). The three altered enzymes achieved a 2-30-fold increase in substrate specificity (kcat/Km) for demeton S (P-S bond), an analogue for the chemical warfare agent VX. In contrast, the substrate specificity for diisopropyl fluorophosphonate (P-F bond) was substantially decreased for each of these enzymes. In addition, H257L and H254R/H257L showed an 11- and 18-fold increase, respectively, in specificity for NPPMP, the analogue for the chemical warfare agent soman. These results demonstrate the ability to significantly enhance the specificity of OPH for various substrates by site-specific modifications, and it is suggested that changes in metal requirements may affect these improved catalytic characteristics by enhancing structural flexibility and improving access of larger substrates to the active site, while simultaneously decreasing the catalytic efficiency for smaller substrates.

Neurotoxic Organophosphate Degradation with Polyvinyl Alcohol Gel-Immobilized Microbial Cells

A genetically engineered strain of Escherichia coli that expresses organophosphorus hydrolase (OPH) was immobilized in a polyvinyl alcohol (PVA) cryogel to form a porous biocatalyst that successfully degrades organophosphorus (OP) neurotoxins. The impacts of both diffusion and reaction on biocatalyst efficiency were determined to enable prediction and optimization of the biocatalyst performance. The kinetic rate parameters and activation energies of pure OPH, free cell suspensions, and the immobilized cell biocatalyst were compared. Diffusion was a determining factor for paraoxon hydrolysis because of the very rapid OPH kinetics for its model substrate. Both the paraoxon diffusion through the PVA matrix and the diffusion associated with microbial transport of paraoxon were shown to impact the biocatalyst reaction. However, the enhancement in storage stability resulting from diffusional limitations provides an advantage to diffusion-limited operation. This research may serve as a guide to define the influence of diffusion in biological reaction systems. The broad substrate specificity and hydrolytic efficiency of OPH coupled with the ability to genetically engineer the enzyme for specific target OP neurotoxins enhance the suitability of OPH-based technologies for detoxification of these compounds. Cryoimmobilization provides a suitable vehicle as a cost-effective, efficient technology for bioremediation of environmental media contaminated with OP compounds.

Enzymatic Hydrolysis of the Chemical Warfare Agent VX and its Neurotoxic Analogues by Organophosphorus Hydrolase

Organophosphorus hydrolase (OPH) is a bacterial enzyme that hydrolyzes a variety of organophosphorus (OP) neurotoxins, including many widely used pesticides and chemical warfare agents containing P-O, P-F, P-CN and P-S bonds. It has extremely high efficiency in hydrolysis of many different phosphotriester and phosphothiolester pesticides (P-O bond) such as paraoxon (kcat3800s−1) and coumaphos (kcat = 800s−1) or phosphonate (P-F) neurotoxins such as DFP (kcat = 350s−1) and the chemical warfare agent sarin (kcat = 56s−1). In contrast, the enzyme has much lower catalytic capabilities for phosphonothioate neurotoxins such as acephate (kcat = 2.8 s−1) or the chemical warfare agent VX [O-ethyl S-(2-diisopropyl-aminoethyl) methylphosphonothioate] (kcat = 0.3s−1). This lower specificity for VX and its analogues are reflected by the specificity constants (kcat/Km values) for VX = 0.75 × 103 M−1 s−1 compared to 5.5 × 107M−1 s−1 for paraoxon. Different metal-associated forms of the enzyme demonstrated significantly varying hydrolytic capabilities for VX and its analogues, and the activity of OPH (Co+2) was consistently higher than that of OPH (Zn+2) by five to twenty fold. Hydrolysis of the P-S bonds was determined by monitoring the formation of free -SH groups, and the specific cleavage of the P-S bond was verified by 31P NMR analysis.

The development of a new biosensor based on recombinant E. coli for the direct detection of organophosphorus neurotoxins

A new biosensor for the direct detection of organophosphorus (OP) neurtoxins has been developed utilizing cryoimmobilized, recombinant E. coli cells capable of hydrolyzing a wide spectrum of OP pesticides and chemical warfare agents. The biological transducer was provided by the enzymatic hydrolysis of OP neurotoxins by organophosphate hydrolase which generates two protons through a reaction in which PO, PF, PS or PCN bonds are cleaved, and the proton release corresponded with the quantity of organophosphate hydrolyzed. This stoichiometric relationship permitted the creation of a potentiometric biosensor for detection of OP neurotoxins and a pH-based assay was developed as a direct function of the concentration of OP neurotoxins and the immobilized biomass. In these studies utilizing paraoxon as the substrate, neurotoxin concentration was determined with two different types of measuring units containing immobilized cells: (1) a stirred batch reactor; and (2) a flow-through column minireactor. A pH glass electrode was used as the physical transducer. The linear detection range for paraoxon spanned a concentration range of 0.25–250 ppm (0.001–1.0 mM). The response times were 10 min for the batch reactors and 20 min for the flow-through systems. It was possible to use the same biocatalyst repetitively for 25 analyses with a 10 min intermediate washing of the biocatalyst required for reestablishing the starting conditions. The cryoimmobilized E. coli cells exhibited stable hydrolytic activity for over 2 months under storage in 50 mM potassium-phosphate buffer at +4°C and provide the potential for the development of a stable biotransducer for detecting various OP neurotoxins.

Characterization of P-S Bond Hydrolysis in Organophosphorothioate Pesticides by Organophosphorus Hydrolase

The extensive use of organophosphorothioate insecticides in agriculture has resulted in the risk of environmental contamination with a variety of broadly based neurotoxins that inhibit the acetylcholinesterases of many different animal species. Organophosphorus hydrolase (OPH, EC 3.1.8.1) is a broad-spectrum phosphotriesterase that is capable of detoxifying a variety of organophosphorus neurotoxins by hydrolyzing various phosphorus-ester bonds (P-O, P-F, P-CN, and PS) between the phosphorus center and an electrophilic leaving group. OPH is capable of hydrolyzing the P-X bond of various organophosphorus compounds at quite different catalytic rates: P-O bonds (kcat = 67-5000 s−1), P-F bonds (kcat = 0.01-500 s−1), and P-S bonds s (kcat = 0.0067 to 167 s−1). P-S bond cleavage was readily demonstrated and characterized in these studies by quantifying the released free thiol groups using 5,5′-dithio-bis-2-nitrobenzoic acid or by monitoring an upheld shift of approximately 31 ppm by 31P NMR. A decrease in the toxicity of hydrolyzed products was demonstrated by directly quantifying the loss of inhibition of acetylcholinesterase activity. Phosphorothiolate esters, such as demeton-S, provided noncompetitive inhibition for paraoxon (a P-O triester) hydrolysis, suggesting that the binding of these two different classes of substrates was not identical.