A tunable bifunctional hollow Co3O4/MO3 (M = Mo, W) mixed-metal oxide nanozyme for sensing H2O2 and screening acetylcholinesterase activity and its inhibitor.

A self-templated strategy was adopted to design hollow Co3O4/MO3 (M = Mo, W) mixed-metal oxides via the Mo or W doping of ZIF-67, and subsequent pyrolysis under an atmosphere of air at a low temperature of 450 °C. The hollow Co3O4/MO3 (M = Mo, W) mixed-metal oxides displayed tunable oxidase-like and peroxidase-like activities able to efficiently catalyze the oxidation of TMB to generate a deep blue color in the absence or presence of H2O2. Relative to that of the un-doped Co3O4, the oxidase mimic activity of the Mo-doped Co3O4 increased to 1.3 to 2.1-fold, while its peroxidase mimic activity increased to 7.1 to 19.9-fold, depending on different Mo doping amounts. The oxidase mimic activity of the W-doped Co3O4 increased to 2.1 to 2.3-fold, while its peroxidase mimic activity increased to 4.8 to 5.9-fold, depending on the different W doping amounts. The Mo- and W-doped Co3O4 nanohybrid exhibited both higher O2 and H2O2 activating capability, and their H2O2 activating capacity was superior to the O2 activating capability. Furthermore, the Mo- and W-doped Co3O4 nanohybrids exhibited similar O2 activating abilities, while the Mo-doped one displayed a higher H2O2 activating capability than the W-doped one. The discrepant peroxidase-like nature of Mo- and W-doped Co3O4 nanohybrids is likely attributed to their different catalytic mechanisms. The peroxidase-like activity of Mo-doped Co3O4 is highly related to the ˙OH free radical, while that of W-doped Co3O4 is likely ascribed to the electron transfer between TMB and H2O2. The Km values of Co3O4/MoO3 for TMB and H2O2 were 0.0352 mM and 0.134 mM, which were 3.2- and 1.9-fold lower than that of pure Co3O4, respectively. A Co3O4/MoO3-based colorimetric platform was developed for the determination of H2O2 in the 0.1-200 µM range, with a limit of detection of 0.08 µM (3σ). Based on the thiocholine (TCh) inhibition of the excellent peroxidase-like activity of Co3O4/MoO3 and the TCh generation via acetylcholinesterase (AChE) catalyzed hydrolysis of acetylthiocholine chloride (ATCh), the colorimetric platform was extended to screen AChE activity and its inhibitor.

Acetylcholine esterase enzyme doped multiwalled carbon nanotubes for the detection of organophosphorus pesticide using cyclic voltammetry.

Use of immobilized acetylcholine esterase (AChE) for detecting organophosphorus pesticides in water sources and body fluids can bring down the detection costs dramatically. In the present study, AChE was directly doped on multiwalled carbon nanotube (MWCNT) surface modified with carboxylic groups through amide bond and used for organophosphorus pesticide detection. Amide bond formation between MWCNTs and the enzyme molecules avoid use of any intermediate membranes, cross-linkers or binding materials. This strategy overcomes the hindrance to electron transfer posed by membranes or cross-linkers and increases the sensitivity of detection. MWCNTs carrying carboxyl groups were deposited on glassy carbon electrode and were subsequently immobilized with AChE. The activity of AChE was monitored by cyclic voltammetry after immobilization. Scanning electron microscopy and atomic force microscopy were used to characterize the electrode surface. FT-IR spectra were taken to characterize enzyme-MWCNT complex. Under optimized parameters, the electrode showed linear range between 10 and 50 nM, which is promising for detection of trace amounts of the pesticide. The lower and higher detection limits of the sensor are 0.1 nM and 500 nM respectively. The stability and reusability of the electrode were determined. Finally, successful detection of organophosphorus compounds in real samples established it as reliable for sensor applications.

Paper-based amperometric sensor for determination of acetylcholinesterase using screen-printed graphene electrode.

A simple paper-based sensor with electrochemical detection was first developed for rapid, selective, and sensitive determination of acetylcholinesterase (AChE). The screen-printed graphene electrode was used as working electrode providing sensitivity for the sensor. The amperometric detection of AChE is based on the determination of thiocholine (TCh) produced from hydrolysis of acetylthiocholine chloride (ATCh) by AChE. To detect TCh, the ATCh immobilized sheet was stacked onto the detection sheet using double adhesive tape, then samples of AChE were dropped on the back side of an ATCh immobilized sheet with only 1min of incubation time. To avoid interference, glutathione (GSH), the potential of 0.5V vs. Ag/AgCl was applied onto a graphene electrode and the current, which depends on AChE concentration, was measured. Under optimized conditions, the limit of detection (LOD) from the experiment of AChE determination was 0.1U/mL with AChE concentration in range of 0.1-15U/mL. The data correlated well with the data obtained using spectrophotometric method. The developed sensor had been successfully applied to detect AChE in blood samples.

An investigation into the butyrylcholinesterase-catalyzed hydrolysis of formylthiocholine using heavy atom kinetic isotope effects.

Heavy atom kinetic isotope effects (KIEs) were determined for the butyrylcholinesterase-catalyzed hydrolysis of formylthiocholine (FTC). The leaving-S, carbonyl-C, and carbonyl-O KIEs are (34)k=0.994±0.004, (13)k=1.0148±0.0007, and (18)k=0.999±0.002, respectively. The observed KIEs support a mechanism for both acylation and deacylation where the steps up to and including the formation of the tetrahedral intermediate are at least partially rate determining. These results, in contrast to previous studies with acetylthiocholine, suggest that the decomposition of a tetrahedral intermediate is not rate-determining for FTC hydrolysis. Structural differences between the two substrates are likely responsible for the observed mechanism change with FTC.

Microfluidic Device for Coulometric Detection of Organophosphate Pesticides.

A microdevice for coulometric detection of organophosphate pesticides (OPs) was fabricated based on the measurement of the inhibition of an enzyme, acetylcholinesterase (AChE), by OPs. Thiocholine (TCh) produced in the enzymatic reaction of AChE with acetylthiocholine (ATCh) as a substrate was oxidized on a microelectrode array formed in a main flow channel. Volumes of plugs of necessary solutions were measured using a structure consisting of a row of rhombuses formed in an auxiliary flow channel. The plugs were merged and solution components were mixed at a T-junction formed with the main and auxiliary flow channels. A linear relationship was confirmed between the generated charge and the logarithm of the OP (malathion) concentration in a concentration range between 10(-6) and 10(-3) M with a correlation coefficient of 0.951. The lower limit of detection was 412 nM.

Toxicological and biochemical characterizations of AChE in phosalone-susceptible and resistant populations of the common pistachio psyllid, Agonoscena pistaciae.

The toxicological and biochemical characteristics of acetylcholinesterases (AChE) in nine populations of the common pistachio psyllid, Agonoscena pistaciae Burckhardt and Lauterer (Hemiptera: Psyllidae), were investigated in Kerman Province, Iran. Nine A. pistaciae populations were collected from pistachio orchards, Pistacia vera L. (Sapindales: Anacardiaceae), located in Rafsanjan, Anar, Bam, Kerman, Shahrbabak, Herat, Sirjan, Pariz, and Paghaleh regions of Kerman province. The previous bioassay results showed these populations were susceptible or resistant to phosalone, and the Rafsanjan population was most resistant, with a resistance ratio of 11.3. The specific activity of AChE in the Rafsanjan population was significantly higher than in the susceptible population (Bam). The affinity (K(M)) and hydrolyzing efficiency (Vmax) of AChE on acetylthiocholine iodide, butyrylthiocholine iodide, and propionylthiocholine odide as artificial substrates were clearly lower in the Bam population than that in the Rafsanjan population. These results indicated that the AChE of the Rafsanjan population had lower affinity to these substrates than that of the susceptible population. The higher Vmax value in the Rafsanjan population compared to the susceptible population suggests a possible over expression of AChE in the Rafsanjan population. The in vitro inhibitory effect of several organophosphates and carbamates on AChE of the Rafsanjan and Bam populations was determined. Based on I50, the results showed that the ratios of AChE insensitivity of the resistant to susceptible populations were 23 and 21.7-fold to monocrotophos and phosphamidon, respectively. Whereas, the insensitivity ratios for Rafsanjan population were 0.86, 0.8, 0.78, 0.46, and 0.43 for carbaryl, eserine, propoxur, m-tolyl methyl carbamate, and carbofuran, respectively, suggesting negatively correlated sensitivity to organophosphate-insensitive AChE. Therefore, AChE from the Rafsanjan population showed negatively correlated sensitivity, being insensitive to phosphamidon and monocrotophos and sensitive to N-methyl carbamates.

Optimal detection of cholinesterase activity in biological samples: modifications to the standard Ellman's assay.

Ellman's assay is the most commonly used method to measure cholinesterase activity. It is cheap, fast, and reliable, but it has limitations when used for biological samples. The problems arise from 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), which is unstable, interacts with free sulfhydryl groups in the sample, and may affect cholinesterase activity. We report that DTNB is more stable in 0.09 M Hepes with 0.05 M sodium phosphate buffer than in 0.1M sodium phosphate buffer, thereby notably reducing background. Using enzyme-linked immunosorbent assay (ELISA) to enrich tissue homogenates for cholinesterase while depleting the sample of sulfhydryl groups eliminates unwanted interactions with DTNB, making it possible to measure low cholinesterase activity in biological samples. To eliminate possible interference of DTNB with enzyme hydrolysis, we introduce a modification of the standard Ellman's assay. First, thioesters are hydrolyzed by cholinesterase to produce thiocholine in the absence of DTNB. Then, the reaction is stopped by a cholinesterase inhibitor and the produced thiocholine is revealed by DTNB and quantified at 412 nm. Indeed, this modification of Ellman's method increases butyrylcholinesterase activity by 20 to 25%. Moreover, high stability of thiocholine enables separation of the two reactions of the Ellman's method into two successive steps that may be convenient for some applications.

Acetylcholinesterase biosensor for carbamate drugs based on tetrathiafulvalene-tetracyanoquinodimethane/ionic liquid conductive gels.

A highly sensitive acetylcholinesterase biosensor was developed for detection of carbamate drugs based on TTF-TCNQ-ionic liquid gel thiocholine sensor. The TTF-TCNQ-ionic/ionic liquid gel was characterized by FT-IR and scanning electron microscopy. The electrocatalytic behavior of TTF-TCNQ-ionic liquid gels toward oxidation of thiocholine was thoroughly investigated. 1-Ethyl-3-methylimidazolium tetracyanoborate gel based sensor allowed amperometric detection of thiocholine at +400 mV vs. Ag/AgCl with a high sensitivity of 55.9±1.2 µA mM(-1)cm(-2) and a low detection limit equal to 7.6 µM. The catalytic rate constant and diffusion constant of thiocholine were estimated from chronoamperometric data. The proposed biosensor based on AChE immobilized in sol-gel matrix was used for the detection of two carbamate therapeutic drugs. Very low detection limits of 26 pM eserine and 0.3 nM neostigmine were achieved. The analysis of spiked tap water proved the biosensor capability to be used as a screening method for detection of carbamate drugs in wastewaters.

Bivalve esterases as biomarker: identification and characterization in European cockles (Cerastoderma edule).

This study characterized esterase activity in Cerastoderma edule tissues using different substrates and specific inhibitors and identified the tissue distribution of esterases in this species. Synthetic thiocholines and thioacetate esters and specific inhibitors (eserine, BW284C51 and iso-OMPA) were used to identify and quantify cholines and carboxyl esterases. The results demonstrated the presence of a non-specific propionyl thiocholine (PrSCh)-cleaving cholinesterase (ChE) and a large amount of carboxylesterases (CaE). For further studies using C. edule esterases as biomarkers, our results suggest that the adductor muscle, with PrSCh (5 mM) as substrate should be used to analyze ChE, and for CaE analyses, phenyl thioacetate should be used in digestive gland extracts (PSA, 5 mM).

A Pseudomonas aeruginosa PAO1 acetylcholinesterase is encoded by the PA4921 gene and belongs to the SGNH hydrolase family.

Through the use of molecular and biochemical experiments and bioinformatic tools, this work demonstrates that the PA4921 gene of the Pseudomonas aeruginosa PAO1 genome is a gene responsible for cholinesterase (ChoE) activity. Similar to the acetylcholinesterase (AchE) of Zea mays, this ChoE belongs to the SGNH hydrolase family. In mature ChoE, i.e., without a signal peptide, (18)Ser, (78)Gly, (127)N, and (268)H are conserved aminoacyl residues. Acetylthiocholine (ATC) and propionylthiocholine (PTC) are substrates of this enzyme, but butyrylcholine is an inhibitor. The enzyme also catalyzes the hydrolysis of the artificial esters p-nitrophenyl propionate (pNPP) and p-nitrophenyl butyrate (pNPB) but with lower catalytic efficiency with respect to ATC or PTC. The second difference is that pNPP and pNPB did not produce inhibition at high substrate concentrations, as occurred with ATC and PTC. These differences plus preliminary biochemical and kinetic studies with alkylammonium compounds led us to propose that this enzyme is an acetylcholinesterase (AchE) or propionylcholinesterase. Studies performed with the purified recombinant enzyme indicated that the substrate saturation curves and the catalytic mechanism are similar to those properties described for mammalian AchEs. Therefore, the results of this work suggest that the P. aeruginosa ChoE is an AchE that may also be found in Pseudomonas fluorescens.

Visual detection of organophosphorus pesticides represented by mathamidophos using Au nanoparticles as colorimetric probe.

With citrate-coated Au nanoparticles as colorimetric probe, a novel visual method for rapid assay of organophosphorus pesticides has been developed. The assay principle is based on catalytic hydrolysis of acetylthiocholine into thiocholine by acetylcholinesterase, which induces the aggregation of Au nanoparticles and the color change from claret-red to purple or even grey. The original plasmon absorption of Au nanoparticles at 522 nm decreases, and simultaneously, a new absorption band appears at 675 nm. The irreversible inhibition of organophosphorus pesticides on acetylcholinesterase prevents aggregation of Au nanoparticles. Under optimum conditions, the absorbance at 522 nm of Au nanoparticles is related linearly to the concentration of mathamidophos in the range of 0.02-1.42 µg/mL with a detection limit of 1.40 ng/mL. This colorimetric method has been successfully utilized to detect mathamidophos in vegetables with satisfactory results. The proposed colorimetric assay exhibits good reproducibility and accuracy, providing a simple and rapid method for the analysis of organophosphorus pesticides.

Surface-enhanced Raman scattering detection of cholinesterase inhibitors.

A new sensitive surface-enhanced Raman scattering (SERS) assay for detection of cholinesterase inhibitors such as organophosphorous pesticides using silver colloidal nanoparticles was developed and optimized. Acetylcholinesterase (AChE) mediated the hydrolysis of acetylthiocholine to produce thiocholine, which interacted with the silver nanoparticles to give a specific SERS spectrum. Variation in enzyme activity due to inhibition was measured from changes in intensity of a characteristic peak (772 cm(-1)) of the SERS spectrum that was directly correlated with the concentration of produced thiocholine. The method was demonstrated for the detection of paraoxon as reference AChE inhibitor. Limit of detection of paraoxon for 5 min incubation at 25 °C was 1.8×10(-8) M. This assay can be utilized for the detection of trace amounts of any AChE inhibitor.

TiO2-decorated graphene nanohybrids for fabricating an amperometric acetylcholinesterase biosensor.

This work describes a highly sensitive and rapid amperometric biosensor for organophosphate compounds (OPs) based on immobilization of acetylcholinesterase (AChE) on a novel TiO(2)-decorated graphene (TiO(2)-G) nanohybrid, which was constructed by in situ growth of TiO(2) nanoparticles (NPs) on the graphene sheet. The well-dispersed TiO(2) NPs eliminated the restacking of TiO(2)-G nanohybrids. Due to the integrating of TiO(2)-G nanohybrids, the as-prepared biosensor showed high affinity to acetylthiocholine (ATCl) with a Michaelis-Menten constant (K(m)) value of 0.22 mM, and rapid inhibition time (3 min). Further, based on the inhibition of OPs on the enzymatic activity of the immobilized AChE, and using carbaryl as a model compound, the inhibition of carbaryl was proportional to its concentration ranging from 0.001 to 0.015 and 0.015 to 2 µg mL(-1) with a detection limit of 0.3 ng mL(-1) (S/N = 3). The developed biosensor exhibited a good performance for organophosphate pesticide detection, including good reproducibility and acceptable stability, which provided a new and promising tool for the analysis of enzyme inhibitors.

Long route or shortcut? A molecular dynamics study of traffic of thiocholine within the active-site gorge of acetylcholinesterase.

The principal role of acetylcholinesterase is termination of nerve impulse transmission at cholinergic synapses, by rapid hydrolysis of the neurotransmitter acetylcholine to acetate and choline. Its active site is buried at the bottom of a deep and narrow gorge, at the rim of which is found a second anionic site, the peripheral anionic site. The fact that the active site is so deeply buried has raised cogent questions as to how rapid traffic of substrate and products occurs in such a confined environment. Various theoretical and experimental approaches have been used to solve this problem. Here, multiple conventional molecular dynamics simulations have been performed to investigate the clearance of the product, thiocholine, from the active-site gorge of acetylcholinesterase. Our results indicate that thiocholine is released from the peripheral anionic site via random pathways, while three exit routes appear to be favored for its release from the active site, namely, along the axis of the active-site gorge, and through putative back- and side-doors. The back-door pathway is that via which thiocholine exits most frequently. Our results are in good agreement with kinetic and kinetic-crystallography studies. We propose the use of multiple molecular dynamics simulations as a fast yet accurate complementary tool in structural studies of enzymatic trafficking.

Screen-printed poly(3,4-ethylenedioxythiophene) (PEDOT): A new electrochemical mediator for acetylcholinesterase-based biosensors.

This work describes the use of a PEDOT:PSS-based conductive polymer for designing AChE-based biosensors. The transducers were obtained directly by screen-printing a PEDOT:PSS suspension on the surface of thick film carbon electrodes. The obtained working electrodes showed a high conductivity when compared with electrodes modified with conventional mediators like cobalt phthalocyanine or tetracyanoquinodimethane. The PEDOT:PSS polymer was shown to be suitable for thiocholine oxidation, allowing the measurement of AChE activity at 100 mV vs Ag/AgCl. The high conductivity of PEDOT:PSS allowed the accurate detection of the organophosphate insecticide chlorpyrifos-oxon at concentrations as low as 4x10(-9)M, corresponding to an inhibition ratio of 5%.

Direct detection of enzyme-catalyzed products by FET sensor with ferrocene-modified electrode.

An FET-based biosensor with a ferrocene-modified gold electrode detects the enzyme-produced electrons by using mediators that transfer the electrons from the enzyme to the sensor. Since an extended-gate FET sensor with a light-shielding mask can be operated without a light-shielding box, a small portable instrument will soon be realised. However, when the FET sensor detected enzyme-catalyzed products with the mediators under light conditions, measurements fluctuated due to photo-reduction of the mediators, resulting in decreased sensitivity. To improve sensitivity by reducing the fluctuation, we developed a procedure for directly detecting enzyme-catalyzed products without using the mediators. The key technique used in this procedure was a measurement technique using our developed potential-keeping method, in which the modified electrode of the FET sensor was oxidised by ferricyanide solution to make its surface the same high potential every time, and this high potential was kept until measurement because of the high input impedance of the FET structure. After this method was applied, the interfacial potential of the gold electrode decreased depending on the amount of enzyme-catalyzed products due to the ferrocene molecules immobilised on the gold electrode directly reacting with the products. The results obtained in light conditions indicated that model compounds of the products were detected from 10 µM to 10 mM with the Nernstian response of 59.2 mV/decade. Also, this method was applied to pesticide detection by using the enzyme inhibition by pesticide, and 5 ppb of diazinon was successfully detected by using only a sensor chip.

Development of Au nanoparticles dispersed carbon nanotube-based biosensor for the detection of paraoxon.

A disposable and sensitive biosensor has been fabricated for the detection of the organophosphorous (OP) compound paraoxon using an amperometric technique. For the measurements, gold nanoparticles dispersed on the outer surface of multiwalled carbon nanotubes (Au-MWNTs) has been used as the electrode material, as it possesses high electron transfer rates and provides large immobilization sites for the bioenzymes, which combines with the high electrocatalytic activity of MWNTs for thiocholine oxidation at low potential. Au-MWNTs have been synthesized by chemically reducing Au salt over functionalized MWNTs, and the same has been characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM) and high resolution transmission electron microscopic (HRTEM) techniques. The ability of the Au-MWNTs nanocomposite-based biosensor has been demonstrated to reliably measure the concentration of paraoxon in the nanomolar range.

Direct detection of the hydrolysis of nerve agent model compounds using a fluorescent probe.

Nerve agents are highly toxic organophosphorus compounds (OPs) that are used as chemical warfare agents. Developing a catalytic bioscavenger to efficiently detoxify nerve agents in the bloodstream of affected individuals has been recognized as an attractive approach to prevent nerve agent toxicity. However, the search for nerve agent catalysts has been hindered by the lack of efficient direct assays for nerve agent hydrolysis. In addition, authentic nerve agents are restricted and access to use for experiments by the general research community is prohibited. Herein we report development of a method that combines use of novel nerve agent model compounds possessing a thiocholine leaving group that reacts with the fluorescent thio-detection probe, BES-Thio, to afford detection of sub-micromolar amounts of nerve agent model compounds hydrolysis products. The detection sensitivity of BES-Thio assay was approximately 10 times better than the Ellman assay. This developed method is useful as a direct, sensitive screening method for evaluating OP hydrolysis efficiency from catalytic cholinesterases. When the assay was assembled in the presence of oxime, OP-inhibited cholinesterases that were able to be reactivated by specific oxime showed oxime-assisted enzyme-mediated OP hydrolysis. Therefore, this method is also useful to screen oxime analogs to identify novel agents that can reactivate OP-inhibited cholinesterases or to screen various enzymes to identify pseudo-catalytic bioscavengers that can be readily reactivated by clinically approved oximes.

Cholinesterase activities as potential biomarkers: characterization in two freshwater snails, Potamopyrgus antipodarum (Mollusca, Hydrobiidae, Smith 1889) and Valvata piscinalis (Mollusca, Valvatidae, Müller 1774).

Anti-cholinesterase insecticides constitute a major portion of modern synthetic pesticides and the assessment of cholinesterase (ChE) inhibition is widely used as a specific biomarker for evaluating the exposure of non-target organisms to these pollutants. However, most studies on this biomarker were developed on vertebrates and among invertebrates, gastropod mollusks are rarely used. Gastropods are important members of aquatic habitats and therefore present a high ecological relevance for freshwater ecosystems. In this context, ChE activities were characterized in two freshwater gastropod mollusks, Potamopyrgus antipodarum and Valvata piscinalis, in order to ascertain their value as sentinel species. Firstly, characterization of ChE activities was performed using different substrates (acetylcholine iodide, butyrylcholine iodide and propionylcholine iodide) and specific inhibitors (eserine, iso-OMPA and BW284c51). Secondly, in vivo effect of a widely used organophosphate insecticide, chlorpyrifos, was tested on ChE activity in both species. Results suggested that P. antipodarum possesses two isoforms of cholinesterases, one isoform which properties are intermediate between an acetyl and a propionyl ChE, and one minor isoform which correspond to a butyryl ChE, while V. piscinalis seems to possess only one isoform which displays typical properties of an acetyl ChE. Chlorpyrifos induced no effect on V. piscinalis ChE. In contrast, P. antipodarum activity was significantly decreased by environmental realistic chlorpyrifos concentrations (2.86 and 14.2 nM) after seven days of contact. The present study suggests that P. antipodarum may be employed as a biological indicator for assessing pesticide contamination.

Characterization of trimeric acetylcholinesterase from a legume plant, Macroptilium atropurpureum Urb.

We recently identified plant acetylcholinesterases (E.C.3.1.1.7; AChEs) homologous to the AChE purified from a monocotyledon, maize, that are distinct from the animal AChE family. In this study, we purified, cloned and characterized an AChE from a dicotyledon, siratro. The full-length cDNA of siratro AChE is 1,441 nucleotides, encoding a 382-residue protein that includes a signal peptide. This AChE is a disulfide-linked 125-kDa homotrimer consisting of 41-42 kDa subunits, in contrast to the maize AChE, which exists as a mixture of disulfide and non-covalently linked 88-kDa homodimers. The plant AChEs apparently consist of various quaternary structures, depending on the plant species, similar to the animal AChEs. We compared the enzymatic properties of the dimeric maize and trimeric siratro AChEs. Similar to electric eel AChE, both plant AChEs hydrolyzed acetylthiocholine (or acetylcholine) and propionylthiocholine (or propionylcholine), but not butyrylthiocholine (or butyrylcholine), and their specificity constant was highest against acetylcholine. There was no significant difference between the enzymatic properties of trimeric and dimeric AChEs, although two plant AChEs had low substrate turnover numbers compared with electric eel AChE. The two plant AChE activities were not inhibited by excess substrate concentrations. Thus, similar to some plant AChEs, siratro and maize AChEs showed enzymatic properties of both animal AChE and animal BChE. On the other hand, both siratro and maize AChEs exhibited low sensitivity to the AChE-specific inhibitor neostigmine bromide, dissimilar to other plant AChEs. These differences in enzymatic properties of plant AChEs may reflect the phylogenetic evolution of AChEs.