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Biperiden, an antiparkinsonian anticholinergic drug, has been found to inhibit enzymatic hydrolysis of butyrylthiocholine iodide, which is catalyzed by serum cholinesterase. By measuring the difference in the basic and inhibitory hydrolysis reaction rates, in the presence of biperiden as an inhibitor, it is possible to develop a kinetic method for its determination. Both systems, enzyme-substrate-chromogen and enzyme-substrate-chromogen-inhibitor, were characterized by biochemical parameters ( K M = 0.326 – 0.330 mmol dm -3 ; V max = 40 ‑ 99 μmol dm -3 min -1 ), while inhibition was defined as non-competitive with the constant of inhibition K i = 6.142 μmol dm -3 . The reaction conditions have been optimized followed by determination of the calibration curve, the corresponding equation and the limits of detection and quantification yielding 3.84 and 12.80 nmol dm -3 , respectively. Using the calibration chart, it is possible to determine biperiden in different samples in the concentration range of 0.035–35.940 µmol dm -3 . Influence of a number of substances, found in the sample, on the reaction rate was also examined. The optimized method was applied for determination of biperiden in pharmaceutical preparations. Accuracy of the method was tested using the standard addition method. The proposed method has good sensitivity, selectivity, it is simple and fast, and above all easily accessible, and thus applicable in biochemical and pharmaceutical laboratories.

Butyrylcholinesterase (BChE) generally acts as an important plasma biomarker for clinical diagnosis due to its major contribution to human plasma cholinesterase levels, but its current fluorometric assay relying on fluorogenic substrates frequently suffers from the lack of sufficiently fast response time and specific recognition of substrates relative to the traditional Ellman's method. In this work, we report a fluorescent molecular probe for assaying BChE activity based on thiol-triggered fluorescence enhancement via thiol-ene click reactions. A low-temperature experiment and theoretical analysis exclude the possibility of weak fluorescence of the probe caused by an intramolecular photoinduced electron transfer process and support the main cause of an ultraslow radiative rate due to the introduction of two acrylyl groups. This probe has sensitive fluorescence responses to thiols via thiol-ene click chemistry, and it can distinguish between glutathione and cysteine or homocysteine in different emission colors. The rapid reaction kinetics of this probe enables it to monitor hydrolysis reactions catalyzed by butyrylcholinesterase (BChE) in a real-time manner. This probe is used to develop the first fluorometric assay of BChE activity based on fluorescence enhancement triggered by thiol-ene click chemistry using butyrylthiocholine as the substrate. The established BChE assay shows excellent sensitivity, and is capable of avoiding the interference from glutathione and acetylcholinesterase (AChE) in a complex matrix. The inhibition test of tacrine on BChE with this assay substantiates its feasibility in screening potential inhibitors of BChE. This work demonstrates a design strategy of fluorescent probes lighted up by thiol-ene click reactions, reveals the main cause of thiol-triggered fluorescence enhancement by altering the radiative rate, and provides the first fluorometric assay of BChE based on rapid thiol-ene click reactions.

Molecularly imprinted polymers (MIPs) mimic the binding sites of antibodies by substituting the amino acid-scaffold of proteins by synthetic polymers. In this work, the first MIP for the recognition of the diagnostically relevant enzyme butyrylcholinesterase (BuChE) is presented. The MIP was prepared using electropolymerization of the functional monomer o-phenylenediamine and was deposited as a thin film on a glassy carbon electrode by oxidative potentiodynamic polymerization. Rebinding and removal of the template were detected by cyclic voltammetry using ferricyanide as a redox marker. Furthermore, the enzymatic activity of BuChE rebound to the MIP was measured via the anodic oxidation of thiocholine, the reaction product of butyrylthiocholine. The response was linear between 50 pM and 2 nM concentrations of BuChE with a detection limit of 14.7 pM. In addition to the high sensitivity for BuChE, the sensor responded towards pseudo-irreversible inhibitors in the lower mM range., Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe; 1138

The discharge of industrial effluents into nearby water bodies affects the inhabitants including living organisms. The presence of foreign materials such as heavy metals can be a threat to the ecosystem as they are enormously carcinogenic even though in minute concentration. Hence, an economical and time-efficient preliminary screening test is crucial to be developed for the detection of heavy metals, prior to employment of high technology instruments. In this study, cholinesterase (ChE) from Sabah porcupine fish, Diodon hystrix was purified to test for its potential as an alternative biosensor in detecting metal ions. Few enzymatic parameters including specificity of substrate, temperature and pH were applied to determine its optimal enzymatic activity. ChE enzyme was found to be more sensitive towards the presence of substrate, butyrylthiocholine iodide (BTC), in contrast to acetylthiocholine iodide (ATC) and propionylthiocholine iodide (PTC) with the effective coefficient at 7193, 3680.15 and 2965.26 Vmax/Km, respectively. Moreover, the extracted ChE enzyme showed the optimum activity at pH 9 of 0.1 M Tris-HCl and at 25°C to 30°C range of temperature. When subjected to heavy metals, ChE enzyme was significantly inhibited as the enzyme activity was reduced in the sequence of Hg > Ag > Cr > Cu > Cd > Pb ≥ Zn > As. As a conclusion, the partially purified ChE enzyme proved its sensitivity towards metal ion exposure and can be used as an alternative method in screening the level of contamination in the environment.

Introduction Variants of butyrylcholinesterase are frequently associated with prolonged response to suxamethonium or mivacurium. Butyrylcholinesterase (BChE) can be characterized by phenotyping and determination of genotype. Inappropriate timing of blood sampling might interfere with phenotyping methods. However, guidelines regarding delay between exposure to anaesthesia and testing are not clearly defined. In this study, the BChE activity and phenotype in an early (T1) and late (T2) phase were compared and the phenotype/genotype correlation was assessed. Methods Patients with a prolonged paralysis after mivacurium or suxamethonium were selected after ethical committee approval and written consent. BChE activity was based on butyrylthiocholine hydrolysis rate and phenotyping on differential inhibition of BChE activity with dibucaine and fluoride. DNA sequencing allowed genotypic characterization. Results We included the results of 20 patients with prolonged neuromuscular block (NMB) induced by mivacurium or suxamethonium. In these patients, BChE activity was different at T1 and T2 (2120 [1506-2733] U L(-1)and 4055 [2810-5301] U L-1, respectively;P = 0.0014; values are mean [95% CI]). When phenotyping was possible, phenotyping at T1 and T2 yielded identical results. Phenotyping failed to identify one new variant (p.Tyr146Cys) and the K variant in 14 of 16 patients. Conclusion Anaesthesia interfered with BChE activity, but not with phenotyping. Phenotyping can be performed on blood drawn during or immediately after recovery of mivacurium or suxamethonium to screen for clinically relevant variants of BChE. However, accurate diagnosis of BChE deficiency needs further confirmation by determination of genotype.

In this paper, we, for the first time, describe the interaction between the butyrylcholinesterase enzyme and echothiophate, a popular model compound and an analogue of the chemical warfare agents VX and VR, at the atomistic level. Competition between the two echothiophate conformations in the active site was found using molecular modeling techniques. The first one is close to the mode of binding of the substrates of choline series (butyrylcholine and butyrylthiocholine) and is inhibitory, since it is unable to react with the enzyme. The second one is characterized by a significantly worse estimated binding affinity and is reactive. Thus, echothiophate combines the features of two types of inhibitors: competitive and suicidal. This observation will help clarify the kinetic reaction scheme in order to accurately assess the kinetic constants, which is especially important when designing new butyrylcholinesterase variants capable of full-cycle hydrolysis of organophosphorus compounds.

The butyrylcholinesterase activity in human blood is an important marker of healthiness. Either level of this enzyme – increased or otherwise – can indicate disease or intoxication. The most popular technique to assess the cholinesterase activity is the detection of thiocholine, a product of enzymatic hydrolysis of butyrylthiocholine. In this work, Ag-paste was used as SERS-substrate and a new technique was created for thiocholine detection. As explained below, the designed technique facilitates also determination of butyrylcholinesterase (BChE) activity both in the buffer and in spike solution with human plasma. Ellman’s assay was used as a reference method, a good correlation between spectrophotometric detection and Raman detection was shown.

To identify and characterize specific esterases from S. mutans with degradative activity toward methacrylate-based resin monomers.Out of several putative esterases, an esterase encoded in an Open Reading Frame as SMU_118c (The National Center for Biotechnology Information, NCBI), was found to have true hydrolase activities. SMU_118c was cloned, expressed, purified and further characterized for its respective hydrolytic activity towards ester-containing nitrophenyl substrates and the universal resin monomers bis-phenyl-glycidyl-dimethacrylate (bisGMA) and triethyleneglycol dimethacrylate (TEGDMA) at neutral (7.0) or cariogenic (5.5) pH. Mass spectrometry (MS) was used to verify the expression of SMU_118c protein in S. mutans UA159.Similar to the whole cell activity of S. mutans, SMU_118c showed the highest affinity toward para-nitrophenyl acetate (pNPA) and para-nitrophenyl butyrate (pNPB) vs. ortho-nitrophenyl butyrate (oNPB) and butyrylthiocholine iodide (BTC) (p 0.05). The esterase retained 60% of its activity after 21 days and hydrolyzed bisGMA at a higher rate than TEGDMA at both neutral and cariogenic pH (p 0.001), similarly to the predominant human salivary esterase degradative activity. MS confirmed that SMU_118c is an intracellular protein in S. mutans UA159 and expressed under pathogenic (pH 5.5) growth conditions.The similarity in the activity profile to the whole S. mutans bacterial cell, the stability over time at cariogenic pH, the preference to hydrolyze bisGMA and confirmed expression profile suggest that SMU_118c could be a significant contributor to the whole bacterial degradative activity of S. mutans toward the degradation of resin composites, adhesives and the restoration-tooth interface, potentially accelerating restoration's failure.The current study builds upon our highly-cited previous study by Bourbia et al., (JDR, 2013) that reported on that the cariogenic bacterium, S. mutans has esterase-like activities that enable the bacterium to degrade dental composites and adhesives. The current submission is the first to report on the isolation and characterization of the specific esterase activity (SMU_118c) from S. mutans that is a significant contributor to the whole bacterial degradative activity toward the hydrolysis of dental resins. This activity compromises the restoration-tooth interface, increases interfacial bacterial microleakage (Kermanshahi et al., JDR 2010), potentially contributing to the pathogenesis of recurrent caries around resin composite restorations. This represent a significant contribution to the field of biomaterials and their clinical performance.

Butyrylcholinesterase (BChE) in human plasma is composed of four molecular forms: C1 (monomer); C2, covalent conjugate between BChE monomer and albumin; C3, dimer; and C4, tetramer, the major form. Catalytic parameters of molecular forms were estimated at high substrate concentration after non-denaturing polyacrylamide gel electrophoresis by activity gel-scanning densitometry, using a chromogenic substrate (butyrylthiocholine (BTC)). Though catalytic parameters Kss (dissociation of enzyme substrate complex at high substrate concentration) and catalytic constants are apparent (phenomenological) parameters, results indicate that the four molecular forms of human BChE do not display significant differences in their catalytic behavior at high BTC concentration.

We previously reported that recombinant human butyrylcholinesterase (rhBChE) complexed with a series of copolymers of poly- l -lysine (PLL) with grafted (polyethylene) glycol (PEG) (i.e., PLL-g-PEG) showed reduced catalytic activity but relatively similar concentration-dependent inactivation of the organophosphorus inhibitor paraoxon. Herein, we compared the kinetics of catalysis (using butyrylthiocholine as the substrate) and inhibition (using four different inhibitors) of free and copolymer-complexed rhBChE. Using scanning electron microscopy, polyionic complexes of rhBChE with three different PLL-g-PEG copolymers (based on PLL size) appeared as spheroid-shaped particles with relatively similar particle sizes (median diameter = 35 nm). Relatively similar particle sizes were also noted using dynamic light scattering (mean = 26–35 nm). The three copolymer-complexed enzymes exhibited reduced kcat (30–33% reduction), but no significant changes in Km. Inhibitory potency (as reflected by the bimolecular rate constant, ki) was similar among the free and copolymer-complexed enzymes when paraoxon was the inhibitor, whereas statistically significant reductions in ki (16–60%) were noted with the other inhibitors. Sensitivity to inactivation by proteases and heat was also compared. Copolymer-complexed enzymes showed lesser time-dependent inactivation by the proteases trypsin and pronase and by heat compared to the free enzyme. Understanding the unique properties of PLL-g-PEG-BChE complexes may lead to enhanced approaches for use of BChE and other protein bioscavengers.

A versatile guest matrix was fabricated from a temperature- and pH-sensitive poly(N-isopropylacrylamide)-co-(3-(N,N-dimethylamino)propylmethacrylamide) microgel (poly(NIPAM-co-DMAPMA), MG) for the gentle incorporation of butyrylcholinesterase (BChE). The microgel/BChE films were built up on a surface of graphite-based screen-printed electrodes (SPEs) premodified with MnO2 nanoparticles via a two-step sequential adsorption under careful temperature and pH control. On this basis, a rather simple amperometric biosensor construct was formed, which uses butyrylthiocholine as BChE substrate with subsequent MnO2-mediated thiocholine oxidation at a graphite-based SPE. The complexation of BChE with the microgel was found to be safe and effective, as confirmed by a high operational and rather good long-term storage stability of the resultant SPE-MnO2/MG/BChE biosensors. The small mesh size of the microgel with respect to the size of BChE results in a predominant outer complexation of BChE within the dangling chains...

Aim: The cholinesterase (ChE) based inhibition studies from fish were investigated and presented here emerged to be one of the great potential biomarkers for heavy metals monitoring. Methodology: In this study, the capability of ChE extracted from the kidney of Lates calcarifer was assessed for of metal. ChE was purified through ammonium sulphate precipitation and ion exchange chromatography. Results: The purified enzyme gave 12 fold purification with the recovery of 12.17% with specific activity of 2.889 U mg-1. The Michaelis-Menten constant (Km) and Vmax value obtained was 0.1426 mM and 0.0217 �mol min-1mg-1, respectively. The enzyme has the ability to hydrolyse acetylthiocholine iodide (ATC) at a faster rate compared to other two synthetic substrates, propionylthiocholine iodide (PTC) and butyrylthiocholine iodide (BTC). ChE gave highest activity at 20-30�C in Tris-HCl buffer pH 8.0. The results showed that cholinesterase from L. calcarifer kidney was very sensitive to sensitive to copper and lead after being tested argentum, arsenic, cadmium, chromium, copper, cobalt, mercury, nickel, lead and zinc. Interpretation: The effect of heavy metals studied on the activity of ChE differed from each other. The result of the study can be used as a tool for further developing a biomarker for the detection of heavy metals in aquatic ecosystems. In addition, the information can also be used for designing a kit, that would give a rapid and accurate result.

Cholinesterases (ChE), the enzymes whose primary function is the hydrolysis of choline esters, are widely expressed throughout the nature. Although they have already been found in plants and microorganisms, including ascomycete fungi, this study is the first report of ChE-like activity in fungi of the phylum Basidiomycota. This activity was detected in almost a quarter of the 45 tested aqueous fungal extracts. The ability of these extracts to hydrolyse acetylthiocholine was about ten times stronger than the hydrolytic activity towards butyrylthiocholine and propionylthiocholine. In-gel detection of ChE-like activity with acetylthiocholine indicated a great variability in the characteristics of these enzymes which are not characterized as vertebrate-like based on (i) differences in inhibition by excess substrate, (ii) susceptibility to different vertebrate acetylcholinesterase and butyrylcholinesterase inhibitors, and (iii) a lack of orthologs using phylogenetic analysis. Limited inhibition by single inhibitors and multiple activity bands using in-gel detection indicate the presence of several ChE-like enzymes in these aqueous extracts. We also observed inhibitory activity of the same aqueous mushroom extracts against insect acetylcholinesterase in 10 of the 45 samples tested; activity was independent of the presence of ChE-like activity in extracts. Both ChE-like activities with different substrates and the ability of extracts to inhibit insect acetylcholinesterase were not restricted to any fungal family but were rather present across all included Basidiomycota families. This study can serve as a platform for further research regarding ChE activity in mushrooms.

The study objective was to test the hypothesis that simvastatin and fenofibrate should cause an increase in butyrylcholinesterase (BuChE) activity not only in the plasma and liver but also in the brain of normolipidemic and hyperlipidemic rats. Catalytic enzyme activity was measured using acetylthiocholine (ATCh) and butyrylthiocholine (BTCh) as substrates. Normolipidemic and hyperlipidemic rats were divided in four groups receiving 50 mg/kg of simvastatin a day or 30 mg/kg of fenofibrate a day for three weeks and three control groups receiving saline. Simvastatin and fenofibrate caused an increase in brain BuChE activity in both normo- and hyperlipidemic rats regardless of the substrate. The increase with BTCh as substrate was significant and practically the same in normolipidemic and hyperlipidemic rats after simvastatin treatment (14–17% vs controls). Simvastatin and fenofibrate also increased liver and plasma BuChE activity in both normolipidemic and hyperlipidemic rats regardless of the substrate. In most cases the increase was significant. Considering the important role of BuChE in cholinergic transmission as well as its pharmacological function, it is necessary to continue investigations of the effects of lipid-lowering drugs on BuChE activity., Cilj ispitivanja bio je u normolipidemičnih i hiperlipidemičnih štakora potvrditi pretpostavku da simvastatin (SIMV) I fenofibrat (FENO) ne uzrokuju samo povećanje aktivnosti butirilkolinesteraze (BuChE) u plazmi i jetri nego i povećavaju aktivnosti BuChE u mozgu. Katalitička aktivnost enzima mjerena je upotrebom acetiltiokolina (ATCh) i butiriltiokolina (BTCh) kao supstrata. Normolipidemični i hiperlipidemični štakori raspoređeni su u eksperimentalne skupine, koje su tri tjedna primale SIMV 50 mg/kg na dan ili FENO 30 mg/kg na dan, dok su kontrolne skupine primale fiziološku otopinu. I SIMV i FENO uglavnom su izazvali povećanje aktivnosti BuChE u mozgu obaju sojeva štakora bez obzira na korišteni supstrat. Aktivnosti BuChE mjerene BTCh kao supstratom bile su značajno veće u odnosu na kontrolne vrijednosti u mozgu normolipidemičih štakora nakon primjene SIMV te u mozgu hiperlipidemičnih štakora nakon primjene obaju agensa (14‒17%, p

The purpose of this study was to measure and characterize the enzyme cholinesterase (ChE) in porcine saliva, as well as to evaluate its behavior in experimental stressful conditions. The results of ChE characterization by using different substrates and the selective inhibitors ethopropazine and physostigmine showed that the main enzyme existing in porcine saliva was butyrylcholinesterase (BChE). An automated assay using butyrylthiocholine iodide as substrate was validated providing adequate reproducibility, linearity results and limit of detection. Salivary ChE was measured using the validated assay in two models of acute stress: twenty pigs stressed for 2min with a nasal snare and other twenty pigs subjected to a short-term road transport. Salivary ChE significantly increased after restraint and transport stress in pigs, as well as the ChE to total protein ratio. In conclusion, BChE is the predominant isoenzyme in porcine saliva, it can be measured by the fast, simple and automated method described in this paper and it increases in the models of stress used in this study.

A modified enzyme method is reported for the determination of cholinesterase inhibitors based on the use of a granulated composite carrier with immobilized butyrylcholinesterase. The detection system includes butyrylthiocholine as the substrate and N-(2,3-dimethyl-5-oxo-1-phenyl-3-pyrazoline-4-yl)-2-chloro-5-sulpho-4-iminobenzoquinone as a redox indicator. This method was characterized using carbamate physostigmine and sarin (O-isopropyl methylphosphonofluoridate). The method was also used in a simple tube detector. Evaluation was based on the observation with the naked eye of the decolorization of the redox indicator in comparison to a control time and by tristimulus colorimetry. The limits of detection were 0.5 µg/mL for physostigmine and 7 × 10−4 mg/m3 for sarin visually, or 0.06 µg/mL for physostigmine and 1.1 × 10−4 mg/m3 for sarin by colorimetry.

Fish are ubiquitous organisms that have many features that designate their potential as a biomarker of heavy metals pollution. Thus, an investigation was done to detect the effect of heavy metals on cholinesterase (ChE) activity from Lates calcarifer organs which were gill and muscle. Ammonium sulphate precipitation was performed along with ion exchange chromatography to purify the enzyme. In the substrate specificity study, ChE from L. calcarifer gills was capable of breaking down acetylthiocholine iodide (ATC) at a faster rate compared to the other two synthetic substrates, which are butyrylthiocholine iodide (BTC) and propionylthiocholine iodide (PTC). In contrast, the muscle ChE has a higher affinity towards PTC. The maximum activity of ChE observed at the temperature ranging from 20 to 30 °C in Tris–HCl buffer pH 8. ChE from the two organs of L. calcarifer showed an inhibitive reaction towards heavy metals, but with different effects. ATC from gills showed 50 % inhibition by Cu, Hg and Pb, while PTC from muscle showed 50 % inhibition by Pb. The variation of inhibitory effect that was shown by ChE from L. calcarifer organs can be further studied in designing a biosensor kit that is sensitive towards heavy metal.

The influence of habitat conditions on the activity, the structure of the substrate specificity (the ratio of the substrate hydrolysis rates), and the kinetic parameters of substrate hydrolysis due to the effect of hemolymph cholinesterase of the mussel Crenomytilus grayanus was studied. Mussels were collected from areas that are influenced by seasonal and stationary upwelling, as well as from a polluted area. Upwelling and anthropogenic pressure were shown to alter the structure of hemolymph cholinesterase substrate specificity in mussels, up to complete loss of the ability to catalyze the hydrolysis of propionyland butyrylthiocholine. It was established that during the seasonal upwelling the efficiency of the cholinergic process in mussels is provided by a wide range of effective concentrations of the substrates and by decreasing their affinity to the enzyme. Under the conditions of chronic anthropogenic pollution, the cholinesterase of the mussel hemolymph loses its ability to hydrolyze substrates other than acetylthiocholine.

Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger that protects from the toxicity of nerve agents. Non-human primates are suitable models for toxicity studies that cannot be performed in humans. We evaluated the biochemical properties of native macaque (MaBChE) tetramers, compared to recombinant MaBChE monomers, PEGylated recombinant MaBChE tetramers and monomers, and native HuBChE tetramers. Km and kcat values for butyrylthiocholine were independent of subunit assembly status. The Km for all forms of MaBChE was about 70 μM, compared to 13 μM for HuBChE. The kcat was about 100,000 min−1 for MaBChE and 30,000 min−1 for HuBChE. The reversible inhibitor ethopropazine had similar Ki values of 0.05 μM for all MaBChE forms and HuBChE. The bimolecular rate constant, ki, for inhibition by diisopropylfluorophosphate (DFP), an analog of sarin, was 2.2 to 2.5 × 107 M−1 min−1 for all MaBChE forms and for HuBChE. A major difference between MaBChE and HuBChE was the rate of reactivation by 2-PAM. The second order rate constant for reactivation of DFP-inhibited MaBChE by 2-PAM was 1.4 M−1 min−1, but was 380 fold faster for DFP-inhibited HuBChE (kr 531 M−1 min−1). The acyl pocket of MaBChE has Leu285 in place of Pro285 in HuBChE. The reactivation rate of DFP-inhibited HuBChE mutant P285L by 2-PAM was reduced 5.8-fold (kr 92 M−1 min−1) indicating that P285 determines whether 2-PAM binds in an orientation that favors release of diisopropylphosphate. DFP-inhibited MaBChE treated with 0.2 M 2-PAM recovered 10% of its original activity, whereas DFP-inhibited HuBChE recovered 80% activity. It was concluded that the biochemical properties of MaBChE are similar to those of HuBChE except for the reactivation of DFP-inhibited BChE.

A new spectrofluorimetric method more sensitive than the Ellman method was developed for determination of both acetylcholinesterase and butyrylcholinesterase activity and for kinetic analysis of these enzymes and their mutants. Two selected mutants of human butyrylcholinesterase (E197Q and E197G) were included in this work. As for the Ellman's method, substrates are thiocholine esters, but the chromogenic reagent, DTNB (dithio-bisnitro benzoic acid) is replaced by a fluorogenic probe, “Calbiochem Probe IV”, (3-(7-Hydroxy-2-oxo-2H-chromen-3-ylcarbamoyl)acrylic acid methylester). Compared to the classical Ellman's method, the sensitivity of this new spectrofluorimetric assay is 2 orders of magnitude higher. The method allows measurement of activity in media containing

Herein, we report a first example of a rapid and easy to use paper-based printed sensor embedded in a 3D printing device to measure the butyrylcholinesterase activity in serum samples. The innovative device relies on a paper-based sensor fabricated using wax printing and screen-printing technologies, combined with a printed holder made by stereolithography using 3D printing technology. To measure the butyrylcholinesterase activity, butyrylthiocholine was used as enzymatic substrate and the by-product thiocholine was detected with a paper-based electrochemical sensor modified with a Carbon Black and Prussian Blue nanocomposite. The presence of this nanocomposite allowed to perform the butyrylcholinesterase activity measurement at low applied potential (+300 mV vs. Ag/AgCl pseudoreference), since it is able to electrocatalyse the thiocholine oxidation, overcoming the fouling problem occurring at a bare electrode. To obtain a “reagent free” sensor, butyrylthiocholine was loaded on the filter paper for a reliable stand-alone measurement, requiring only the sample addition. This device, after the calibration with standard solutions, was tested in serum samples, achieving a linear range up to 12 UI/mL and a detection limit of 0.5 IU/mL. To evaluate the accuracy of the device, recovery studies were performed in serum samples, demonstrating the suitability as a point of care for screening analysis of BChE activity measurement, taking into account that the normal value range is comprised between 5.9 and 13.2 IU/mL.

Nowadays extensive volumes of pesticides are employed for agricultural and environmental practices, but they have negative effects on human health. The levels of pesticides are necessarily restricted by international regulatory agencies, thus rapid, cost-effective and in-field analysis of pesticides is an important issue. In the present work, we propose a butyrylcholinesterase (BChE)-based biosensor embedded in a flow system for organophosphorus pesticide detection. The BChE was immobilized by cross-linking on a screen-printed electrode modified with Prussian Blue Nanoparticles. The detection of paraoxon (an organophosphorus pesticide) was carried out evaluating its inhibitory effect on BChE, and quantifying the enzymatic hydrolysis of butyrylthiocholine before and after the exposure of the biosensor to paraoxon, by measuring the thiocholine product at a working voltage of +200 mV. The operating conditions of the flow system were optimized. A flow rate of 0.25 mL/min was exploited for inhibition steps, while a 0.12 mL/min flow rate was used for substrate measurement. A substrate concentration of 5 mM and an incubation time of 10 min allowed a detection limit of 1 ppb of paraoxon (corresponding to 10% inhibition). The stability of the probe in working conditions was investigated for at least eight measurements, and the storage stability was evaluated up to 60 days at room temperature in dry condition. The analytical system was then challenged in drinking, river and lake water samples. Matrix effect was minimized by using a dilution step (1:4 v/v) in flow analysis. This biosensor, embedded in a flow system, showed the possibility to detect paraoxon at ppb level using an automatable and cost-effective bioanalytical system.

Mouse butyrylcholinesterase (mBChE) and an mBChE-based cocaine hydrolase (mCocH, i.e. the A199S/S227A/S287G/A328W/Y332G mutant) have been characterized for their catalytic activities against cocaine, i.e. naturally occurring (−)-cocaine, in comparison with the corresponding human BChE (hBChE) and an hBChE-based cocaine hydrolase (hCocH, i.e. the A199S/F227A/S287G/A328W/Y332G mutant). It has been demonstrated that mCocH and hCocH have improved the catalytic efficiency of mBChE and hBChE against (−)-cocaine by ~8- and ~2000-fold respectively, although the catalytic efficiencies of mCocH and hCocH against other substrates, including acetylcholine (ACh) and butyrylthiocholine (BTC), are close to those of the corresponding wild-type enzymes mBChE and hBChE. According to the kinetic data, the catalytic efficiency ( k cat /K M) of mBChE against (−)-cocaine is comparable with that of hBChE, but the catalytic efficiency of mCocH against (−)-cocaine is remarkably lower than that of hCocH by ~250-fold. The remarkable difference in the catalytic activity between mCocH and hCocH is consistent with the difference between the enzyme–(−)-cocaine binding modes obtained from molecular modelling. Further, both mBChE and hBChE demonstrated substrate activation for all of the examined substrates [(−)-cocaine, ACh and BTC] at high concentrations, whereas both mCocH and hCocH showed substrate inhibition for all three substrates at high concentrations. The amino-acid mutations have remarkably converted substrate activation of the enzymes into substrate inhibition, implying that the rate-determining step of the reaction in mCocH and hCocH might be different from that in mBChE and hBChE. Abbreviations: ACh, acetylcholine; AChE, acetylcholinesterase; BChE, butyrylcholinesterase; BTC, butyrylthiocholine; CHO, Chinese hamster ovary; CIP, calf intestinal alkaline phospahtase; CocH, cocaine hydrolases; hBChE, human butyrylcholinesterase; hCocH, human-butyrylcholinesterase-based cocaine hydrolase; mBChE, mouse butyrylcholinesterase; mCocH, mouse butyrylcholinesterase-based cocaine hydrolase; MD, molecular dynamics; TS, transition state

The application of traditional ion-selective electrodes for comparative enzymatic analysis was demonstrated for the first time in this study. A kinetic-potentiometric method based on the monitoring of the concentration of the ionic substrate involved in the enzymatic reaction catalyzed by different cholinesterases is used for this purpose. A comparative study was performed comprising both enzymatic assays using different ionic substrates and the corresponding inhibited reactions in presence of neostigmine (a synthetic anticholinesterase). The developed approach is used to obtain valuable comparative results through calculation of kinetic parameters, such as Michaelis and inhibition constants. Interesting results were obtained for acetylcholinesterase and butyrylcholinesterase enzymes, which were selected as proof-of-concept: (i) the binding affinity that these enzymes have for their natural substrates showed to be higher (acetylcholine and butyrylcholine respectively) than for their corresponding thiol derivatives (acetylthiocholine and butyrylthiocholine), which are traditionally used in spectrophotometric enzymatic assays; (ii) as expected, the maximum hydrolysis rate found in the assays of each enzyme was independent of the substrate used; (iii) acetylcholinesterase enzyme inhibition due to neostigmine was found to be higher (higher inhibition constant). Advantageously, the use of ion-selective electrodes permits to perform cholinesterases’ enzymatic assays using their natural substrates and under physiological conditions, unlike the traditional spectrophotometric methods used in routine enzymatic assays. Importantly, while well-known enzymes are use throughout this work, this approach can be extended to other types of enzymatic assays as a tangible alternative to traditional spectrophotometric methods.

Highly efficient and rapid lead compound evaluation for estimation of inhibition parameters and type of inhibition is proposed. This is based on a single progress-curve measurement in the presence of each candidate compound, followed by the simultaneous analysis of all of these curves using the ENZO enzyme kinetics suite, which can be implemented as a web application. In the first step, all of the candidate ligands are tested as competitive inhibitors. Where the theoretical curves do not correspond to the experimental data, minimal additional measurements are added, with subsequent processing according to modified reaction mechanisms.

Paper-based microfluidic devices are gaining large popularity because of their uncontested advantages of simplicity, cost-effectiveness, limited necessity of laboratory infrastructure and skilled personnel. Moreover, these devices require only small volumes of reagents and samples, provide rapid analysis, and are portable and disposable. Their combination with electrochemical detection offers additional benefits of high sensitivity, selectivity, simplicity of instrumentation, portability, and low cost of the total system. Herein, we present the first example of an integrated paper-based screen-printed electrochemical biosensor device able to quantify nerve agents. The principle of this approach is based on dual electrochemical measurements, in parallel, of butyrylcholinesterase (BChE) enzyme activity towards butyrylthiocholine with and without exposure to contaminated samples. The sensitivity of this device is largely improved using a carbon black/Prussian Blue nanocomposite as a working electrode modifier. The proposed device allows an entirely reagent-free analysis. A strip of a nitrocellulose membrane, that contains the substrate, is integrated with a paper-based test area that holds a screen-printed electrode and BChE. Paraoxon, chosen as nerve agent simulant, is linearly detected down to 3µg/L. The use of extremely affordable manufacturing techniques provides a rapid, sensitive, reproducible, and inexpensive tool for in situ assessment of nerve agent contamination. This represents a powerful approach for use by non-specialists, that can be easily broadened to other (bio)systems.

Butyrylcholinesterase deficiency is characterized by prolonged apnea after the use of muscle relaxants (suxamethonium or mivarcurium) in patients who have mutations in the BCHE gene. Here, we report a case of prolonged neuromuscular block after administration of mivacurium leading to the discovery of a novel BCHE variant (c.185C>T, p.Ala34Val). Inhibition studies, kinetic analysis and molecular dynamics were undertaken to understand how this mutation remote from the active center determines the “silent” phenotype. Low activity of patient plasma butyrylcholinesterase with butyrylthiocholine (BTC) and benzoylcholine, and values of dibucaine and fluoride numbers fit with a heterozygous enzyme of type atypical/silent. Kinetic analysis with succinyldithiocholine (SCdTC) as the substrate showed that Ala34Val BChE was inactive against this substrate. However, with BTC, the mutant enzyme was active, displaying an unexpected activation by excess substrate. Competitive inhibition of BTC by mivacurium gave a K i = 1.35 mM consistent with the lack of activity with the related substrate SCdTC, and with the clinical data. Molecular dynamic simulations revealed the mechanism by which mutation Ala34Val determines the silent phenotype: a chain of intramolecular events leads to disruption of the catalytic triad, so that His438 no longer interacts with Ser198, but instead forms hydrogen bonds either with residues Glu197 and Trp82, or peripheral site residue Tyr332. However, at high BTC concentration, initial binding of substrate to the peripheral site triggers restoration of a functional catalytic triad, and activity with BTC.

Human butyrylcholinesterase (hBChE) is currently being developed as a detoxication enzyme for stoichiometric binding and/or catalytic hydrolysis of organophosphates. Herein, we describe the use of a molecular evolution method to develop novel hBChE variants with increased resistance to stereochemically defined nerve agent model compounds of soman, sarin, and cyclosarin. Novel hBChE variants (Y332S, D340H, and Y332S/D340H) were identified with an increased resistance to nerve agent model compounds that retained robust intrinsic catalytic efficiency. Molecular dynamics simulations of these variants revealed insights into the mechanism by which these structural changes conferred nerve agent model compound resistance.

Sericostoma vittatum is a caddisfly species, endemic to the Iberian Peninsula, proposed as a biomonitor species for lotic ecosystems. Since inhibition of cholinesterases[U+05F3] (ChE) activity has been used to evaluate the exposure of macroinvertebrates to organophosphates and carbamate pesticides, this work intended to characterize the ChE present in this species so their activity can be used as a potential biomarker of exposure. Biochemical and pharmacological properties of ChE were characterized in this caddisfly species using different substrates (acetylthiocholine iodide, propionylthiocholine iodide, and butyrylthiocholine iodide) and selective inhibitors (eserine sulfate, BW284c51, and iso-OMPA). Also, the in vitro effects of two insecticides (carbaryl and chlorantraniliprole) and two psychiatric drugs (fluoxetine and carbamazepine) on ChE activity were investigated. The results suggest that S. vittatum possess mainly AChE able to hydrolyze both substrates acetylthiocholine and propionylthiocholine since: (1) it hydrolyzes the substrate acetylthiocholine and propionylcholine at similar rates and butyrylthiocholine at a much lower rate; (2) it is highly sensitive to eserine sulfate and BW284c51, but not to iso-OMPA; and (3) its activity is inhibited by excess of substrate, a characteristic of typical AChE. in vitro inhibitions were observed only for carbaryl exposure while exposure to chlorantraniliprole and to relevant environmental concentrations of psychiatric drugs did not cause any significant effect on AChE activity. This study suggests that AChE activity in caddisflies can indeed be used to discriminate the effects of specific insecticides in monitoring programs. The use of non-target species such as caddisflies in ecotoxicological research in lotic ecosystems is also discussed.

Several studies indicate that more than one cholinesterase form may be present in the blood of mammals. In this study the predominant plasma cholinesterase activity, the physiological cholinesterase activity as well as cholinesterase sex-dependent changes in non-exposed individuals of rabbit have been established. Plasma cholinesterase was characterized using three substrates (acetylthiocholine iodide, propionylthiocholine iodide, and S-butyrylthiocholine iodide) and three cholinesterase inhibitors (eserine sulfate, BW284C51 and iso-OMPA). The results indicated that propionylthiocholine was the preferred substrate by plasma cholinesterase followed by acetylthiocholine and butyrylthiocholine, and the predominant enzymatic activity was acetylcholinesterase. Physiological plasma cholinesterase activity was 198.9±5.8 nmol/min/ml for male and 205.2±5.0 nmol/min/ml for female using acetylthiocholine as substrate. Thus, sex had no significant effect on the physiological cholinesterase activity (p>0.05). In addition, the in vivo and in vitro sensitivity of plasma cholinesterase to diazinon was also investigated. In rabbits exposed to single doses of diazinon (25 or 125 mg/kg) the higher inhibitions of plasma cholinesterase were reached 9 h after oral administration (53% and 87% inhibition, respectively). Cholinesterase activity significantly recovered up to values similar to pre-administration between 3 and 7 d depending on the administered dose and sex of the animals. Plasma cholinesterase activity decreased to 24%, 53% and 74% of the initial activity at 9 h of in vitro exposure to 1.25, 3.13 and 6.25 mg/l of diazinon, respectively, and it remained steadily depressed throughout the experimental period (10 d). This study has demonstrated the sensitivity of cholinesterase activity in plasma of rabbits following both in vivo and in vitro exposure to sub-lethal concentrations of diazinon.

Overwhelming amount of heavy metals discharged due to industrialization is a serious global concern. Thus, an investigation was done on acetylcholinesterase (AChE) from Asian swamp eel,Monopterus albus, as an alternative biosensor to detect various metal ions. AChE from the brain ofM. albuswas purified through ammonium sulphate precipitation and procainamide affinity chromatography. Enzyme recovery was obtained at 38.73% with the specific activity of 1847 U μg−1. The Michaelis constant (Km) value and maximal velocity (Vmax) were determined at 8.910 mM and 29.44 μmol min−1mg−1, respectively for acetylthiocholine iodide (ATC). Based on effective coefficient ratio, AChE fromM. albusbrain showed higher affinity to ATC compared to butyrylthiocholine iodide (BTC) and propionylthiocholine iodide (PTC) at the value of 3.304, 1.515, 2.965 Vmax.Km−1respectively. Optimum activity of AChE was obtained at 40°C and incubated in 0.1M Tris HCl buffer pH 9.0. Inhibition study performed on 10 heavy metals resulted in this descending order of inhibition mercury

Competing substrate kinetic analysis of human butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) from the time-course of enzyme-catalyzed substrate hydrolysis, using spectrophotometric assays is described. This study is based on the use of a chromogenic reporter “visible” substrate (substrate A), whose complete hydrolysis time course is retarded by a competing “invisible” substrate (substrate B). For BChE, four visible substrates were used, two thiocholine esters, benzoylthiocholine and butyrylthiocholine, and two aryl-acylamides, o-nitro trifluoro acetaminide and 3-(acetamido)-N,N,N-trimethylanilinium. Three different competing invisible substrates were used, phenyl acetate, acetylcholine and butyrylcholine. For AChE, two visible substrates were used, acetylthiocholine and 3-(acetamido)-N,N,N-trimethylanilinium. For AChE, acetylcholine was competing with visible substrates. The ratio (R) of bimolecular rate constants, kcat/Km, for all couples of substrates, invisible/visible (B/A) covered all possible limit situations, R ≪ 1, R ≈ 1 and R ≫ 1. The kinetic approach, based on the method developed by Golicnik and Masson allowed determination of binding and catalytic parameters of cholinesterases for both visible and invisible substrates. This analysis was applied to michaelian and non-michaelian catalytic behaviors (activation and inhibition by excess substrate). Reevaluation of catalytic parameters obtained for acetylcholine and butyrylcholine more than 50 years ago was made. The method is fast, reliable, and particularly suitable for poorly soluble substrates and for substrates B when no direct spectrophotometric assays exist. Moreover, replacing substrate B by a reversible inhibitor, mechanism of cholinesterase inhibition was possible to study. It is therefore, useful for screening libraries of new substrates and inhibitors, and/or screening of new cholinesterase mutants. This method can be applied to any other enzymes.

Due to their significant value in both economy and ecology, Daphnia had long been employed to investigate in vivo response of cholinesterase (ChE) in anticholinesterase exposures, whereas the type constitution and property of the enzyme remained unclear. A type of ChE was purified from Daphnia magna using a three-step procedure, i.e., Triton X-100 extraction, ammonium sulfate precipitation, and diethylaminoethyl (DEAE)-Sepharose™-Fast-Flow chromatography. According to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), molecular mass of the purified ChE was estimated to be 84 kDa. Based on substrate studies, the purified enzyme preferred butyrylthiocholine iodide (BTCh) [with maximum velocity (V max)/Michaelis constant (K m)=8.428 L/(min·mg protein)] to acetylthiocholine iodide (ATCh) [with V max/K m=5.346 L/(min·mg protein)] as its substrate. Activity of the purified enzyme was suppressed by high concentrations of either ATCh or BTCh. Inhibitor studies showed that the purified enzyme was more sensitive towards inhibition by tetraisopropylpyrophosphoramide (iso-OMPA) than by 1,5-bis(4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284C51). Result of the study suggested that the purified ChE was more like a type of pseudocholinesterase, and it also suggested that Daphnia magna contained multiple types of ChE in their bodies.

Summary Equipment for fast and accurate detection of organophosphate nerve agents is developed and tested. The method is based on the spectrophotometric monitoring of the enzyme activity of butyrylcholinesterase after its contact with air in a special absorption unit (a “scrubber”) developed for the purpose. The scrubber was made from a glass tube filled with glass beads (diam. 3 mm) and filled with approx. 5 ml of butyrylcholinesterase in a phosphate buffer of pH 7.4. The air sample was bubbled through this solution for 20 s at a flow rate of 80 l hour −1 . Thereafter 8 μl of the enzyme solution were aspirated into the micro-SIA-LOV analyzer and the activity of the enzymes were evaluated by using Ellman's reagent, i.e. 2.5 mmol l −1 butyrylthiocholine iodide and 0.25 mmol 5,5′-dithiobis (2-nitrobenzoic acid). The absorbance of the coloured reaction product was measured at 412 nm after the reaction time of 60 s. The residue of the absorption liquid was washed away from the absorber and the system was washed with the enzyme solution prior to next analysis. The contaminated air caused partial inhibition of the enzyme activity of the absorption liquid. The activity of the contaminated sample was compared with the activity of the unaffected enzyme (blank measurement). The analysis was controlled by two PCs. The effect of the concentration of analyte in the absorption liquid on the enzyme activity was tested for 10 −5 –10 −9 mol l −1 sarin. A single analysis (including the absorption step) took

Butyrylcholinesterase (BChE) is an enzyme presented in quite high level in blood plasma where it participates in detoxification reactions. Due to fact that the enzyme is constituted in livers, it is a marker of liver parenchyma function. It can be used for diagnosis of poisoning for e.g., nerve agents or carbofuran and intoxication by some drugs such as rivastigmine. The present experiment is devoted for the creation of new spectrophotometric tests for assay of BChE activity in biological samples. Standard Ellman's method was compared with use of 2,6-dichloroindophenol acetate and indoxylacetate as chromogenic substrates. Maximal velocities and Michaelis constants were calculated for the substrates. Considering calibration, 2,6-dichloroindophenol acetate provided the lowest limit of detection: 1.20×10 −9 kat and a long linear range. All methods were verified using pooled human plasma samples and tested for potential interferents. 2,6-dichloroindophenol acetate is recommended as suitable substrate for BChE assay in clinical diagnostics.

Organophosphate insecticides (OPs) continue to be an important class of agrochemicals used in modern agriculture worldwide. Even though these pesticides persist in the environment for a relatively short time, they show a high acute toxicity that may represent a serious hazard for wildlife. Sub-lethal effects on non-target species are a focus in pest management programs and should be used as biomarkers. Cholinesterases (ChEs) are the most used biomarker of OP exposure in vertebrate and invertebrate species. However, the combined monitoring of ChE and carboxylesterase (CE) activities may provide a more useful indication of exposure and effect of the organisms. The objective of the present work was to find the most sensitive combination of enzyme, substrate, tissue and capacity to recovery of B-esterases in the freshwater gastropod Planorbarius corneus exposed to the OP azinphos-methyl. For this purpose, ChE and CE activities in different tissues of P. corneus (head-foot, pulmonary region, digestive gland, gonads and whole organism soft tissue) were studied. Measurements of ChE activity were performed using three substrates: acetylthiocholine, propionylthiocholine and butyrylthiocholine and CE activity using four different substrates: p-nitrophenyl acetate, p-nitrophenyl butyrate, 1-naphthyl acetate, and 2-naphthyl acetate in control and exposed organisms. Finally, the recovery rates of ChE and CE activities following 48 h exposure to azinphos-methyl were analyzed. Our results show a preference for acetylthiocholine as substrate, a high inhibition with eserine (a selective ChE inhibitor) and inhibition with excess of substrate in all the analyzed tissues. The highest ChE and CE activity was found in the pulmonary region and in the digestive gland, respectively. The highest CE Vmax was obtained with 1 and 2-naphthyl acetate in all the tissues. CEs were more sensitive than ChE to azinphos-methyl exposure. The highest sensitivity was found using p-nitrophenyl acetate and butyrate as substrates. On the other hand, CEs of the digestive gland and the pulmonary region were more sensitive than CEs of the whole organism soft tissue. Regarding the recovery of enzyme activities after 48 h exposure, ChE and CEs with p-nitrophenyl butyrate reached control values after 14 days in the digestive gland and after 21 days in the pulmonary region. Our results show marked differences in P. corneus basal ChE and CE activities depending on substrates and the tissue. Also, both tissue-dependent and substrate-dependent variations in sensitivity to azinphos-methyl exposure and recovery were obtained. CEs measured with p-nitrophenyl butyrate in the pulmonary region were the best combination to be used as biomarker of exposure to azinphos-methyl due to their sensitivity and low recovery capacity. Environmental concentrations of azinphos-methyl inhibited CE activity so they could be used as effective biomarkers of aquatic contamination. Fil: Otero, Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Kristoff, Gisela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina

Since several reports have indicated that cholinesterases (ChE) type and distribution is species specific and that in some species there is a relationship among gender, size and ChE activities, characterization has been suggested. The aim of the present study was to characterize the ChE present in head and muscle of Gambusia yucatana (using selective substrates and inhibitors) and to find its relationship with total length or gender. Results indicated that the ChE present in G. yucatana is an acetylcholinesterase (AChE) with high sensitivity to BW284C51 and an atypical smaller Km with butyrylthiocholine. Scatterplots indicated that there is no linearity between total length and AChE in male or female wild mosquitofish. There were no sex differences in AChE activities. Results indicated significant differences between a single collection site in the Yucatan peninsula and depurated organisms. This study emphasized the importance of characterizing ChE before usage in biomonitoring.

Pesticide exposure has been associated with different adverse health effects which may be modulated to some extent by paraoxonase-1 (PON1) activity and genetic polymorphisms. This study assessed seasonal variations in PON1 activity (using paraoxon –POase–, phenylacetate –AREase–, diazoxon –DZOase– and dihydrocoumarin –DHCase– as substrates), erythrocyte acetylcholinesterase (AChE) and plasma cholinesterase (using butyrylthiocholine –BuChE– and benzoylcholine –BeChE– as substrates. The study population consisted of intensive agriculture workers regularly exposed to pesticides other than organophosphates and non-exposed controls from Almeria (Southeastern Spain). The effect of common genetic polymorphisms of PON1 and BCHE on paraoxonase-1 and cholinesterase activities toward different substrates was also assessed. Linear mixed models were used to compare esterase activities in agricultural workers and control subjects over the two study periods (high and low exposure to pesticides). The significant decrease in AChE and increase in BuChE and BeChE activities observed in workers with respect to control subjects was attributed to pesticide exposure. Workers also had higher levels of AREase, DZOase and, to a lesser extent, of POase, but showed decreased DHCase activity. While PON1 Q192R and PON1 -108C/T gene polymorphisms were significantly associated with all PON1 activities, PON1 L55M showed a significant association with AREase, DZOase and DHCase. BCHE-K (Karlow variant) was significantly associated with lower BeChE activity (but not with BuChE) and BCHE-A (atypical variant) showed no significant association with any cholinesterase activity. These findings suggest that increased PON1, BuChE and BeChE activities in exposed workers might result from an adaptive response against pesticide exposure to compensate for adverse effects at the biochemical level. This response appears to be modulated by PON1 and BCHE gene polymorphisms.

Cholinesterase are often taken as biomarkers of exposure and effect. Then acetylhydrolase isozymes and acylcholine acylhydrolase were examined for their suitability as biomarkers of pesticide exposure. The study of effects of embryos of the rabbit is already an accepted tool in monitor food waste levels, but effects of various groups of substances to the rabbit embryo remain to be studied. A 43% inhibition of acetylhydrolase enzyme activities was reached by 250 mg/kg malathion, while 35% inhibition of acylcholine acylhydrolase enzyme activities was reached by 500 mg/kg malathion indicating the possible use of these enzymes as biomarkers of exposure. There are found some significant specific activities of the acetylhydrolase ranged from 1.5-7.1 U mg−1 protein. The specific activity of acylcholine acylhydrolase in the range of 0.9-4.5 U mg−1 protein in the respective developmental stages. Substratespecificity was analyzed using specific inhibitors (iso-OMPA and BW284c51). The results showed that the observed cholinesterase’s activities in the whole embryo may be attributed mainly to acetylhydrolase with a partial capability to use butyrylthiocholine (BuSCh) as an additional substrate.

In this study, the cholinesterase (ChE) and carboxylesterase (CES) activities present in whole organism homogenates from Planorbarius corneus and their in vitro sensitivity to organophosphorous (OP) pesticides were studied. Firstly, a characterization of ChE and CES activities using different substrates and selective inhibitors was performed. Secondly, the effects of azinphos-methyl oxon (AZM-oxon) and chlorpyrifos oxon (CPF-oxon), the active oxygen analogs of the OP insecticides AZM and CPF, on ChE and CES activities were evaluated. Finally, it was analyzed whether binary mixtures of the pesticide oxons cause additive, antagonistic or synergistic ChE inhibition in P. corneus homogenates. The results showed that the extracts of P. corneus preferentially hydrolyzed acetylthiocholine (AcSCh) over propionylthiocholine (PrSCh) and butyrylthiocholine (BuSCh). Besides, AcSCh hydrolyzing activity was inhibited by low concentrations of BW284c51, a selective inhibitor of AChE activity, and also by high concentrations of substrate. These facts suggest the presence of a typical AChE activity in this species. However, the different dose-response curves observed with BW284c51 when using PrSCh or BuSCh instead of AcSCh suggest the presence of at least another ChE activity. This would probably correspond to an atypical BuChE. Regarding CES activity, the highest specific activity was obtained when using 2-naphthyl acetate (2-NA), followed by 1-naphthyl acetate (1-NA); p-nitrophenyl acetate (p-NPA), and p-nitrophenyl butyrate (p-NPB). The comparison of the IC50 values revealed that, regardless of the substrate used, CES activity was approximately one order of magnitude more sensitive to AZM-oxon than ChE activity. Although ChE activity was very sensitive to CPF-oxon, CES activity measured with 1-NA, 2-NA, and p-NPA was poorly inhibited by this pesticide. In contrast, CES activity measured with p-NPB was equally sensitive to CPF-oxon than ChE activity. Several specific binary combinations of AZM-oxon and CPF-oxon caused a synergistic effect on the ChE inhibition in P. corneus homogenates. The degree of synergism tended to increase as the ratio of AZM-oxon to CPF-oxon decreased. These results suggest that synergism is likely to occur in P. corneus snails exposed in vivo to binary mixtures of the OPs AZM and CPF. Fil: Cacciatore, Luis Claudio. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Kristoff, Gisela. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Verrengia Guerrero, Noemí Rosario. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Cochon, Adriana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Cholinesterases and carboxylesterases belong to the group of B-esterases, the serine superfamily of esterases that are inhibited by organophosphorus compounds. It is now generally accepted that before using the B-esterases as biomarkers of exposure to organophosphorus and carbamates in a given species, the biochemical characteristics of these enzymes should be carefully studied. In this study, the enzyme/s and the tissue/s to be selected as sensitive biomarkers of organophosphorus exposition in the freshwater gastropod Biomphalaria glabrata were investigated. Firstly, the substrate dependence of cholinesterase and carboxylesterase activities in whole organism soft tissue and in different tissues of the snail (head-foot, pulmonary region, digestive gland, and gonads) was analyzed. Measurements of cholinesterase activity were performed using three substrates: acetylthiocholine (AcSCh), propionylthiocholine (PrSCh), and butyrylthiocholine (BuSCh). Carboxylesterase activity was determined using four different substrates: 1-naphthyl acetate (1-NA), 2-naphthyl acetate (2-NA), p-nitrophenyl acetate (p-NPA), and p-nitrophenyl butyrate (p-NPB). Regardless of the tissue analyzed, the highest specific activity was obtained when using AcSCh, followed by PrSCh. Cholinesterase activity measured with BuSCh was very low in all cases. On the other hand, the highest cholinesterase activity was measured in head-foot and in pulmonary region, representing in the case of AcSCh hydrolysis 196% and 180% of the activity measured in whole organism soft tissue, respectively. In contrast, AcSCh hydrolysis in digestive gland and gonads was 28% and 50% of that measured in whole organism soft tissue. Regarding carboxylesterase activity, although all tissues hydrolyzed the four substrates assayed, substrate preferences varied among tissues. In particular, digestive glands showed higher carboxylesterase activity than the other tissues (299%, 359% and 137% of whole organism soft tissue activity) when measured with 1-NA, 2-NA and p-NPA as substrates, respectively. In contrast, with p-NPB as substrate, the highest carboxylesterase activity was observed in pulmonary region. Exposure of the snails for 48 h to azinphos-methyl concentrations in the range of 0.05-2.5 mg L⁻¹ resulted in different degrees of inhibition of cholinesterase and carboxylesterase activities, depending on the enzyme, pesticide concentration, the substrate, and the tissue analyzed. In general, carboxylesterase activity measured with p-NPA and p-NPB was much more sensitive to azinphos-methyl inhibition than cholinesterase activity. The results also showed that while B-esterase activities in whole organism soft tissue and pulmonary region recovered completely within 14 days, carboxylesterase activity in digestive glands remained highly inhibited. On the whole, the results of the present study emphasize how important it is to characterize and measure cholinesterase and carboxylesterase activities jointly to make a proper assessment of the impact of organophosphorus pesticides in non-target species.

Cationic triarylmethane dyes (TAM(+))s which are used as colorants in industry and as frequent tools and reagents in analytical, cell biological and biomedical research have been recently characterized as reversible inhibitors of human butyrylcholinesterase. In this study, the inhibitory effects of two TAM(+)s, malachite green (MG) and methyl green (MeG) on five human BChE mutants (A277V, P285L, H77L, A328F and F329A) were studied spectrophotometrically at 25 degrees C in 50 mM MOPS buffer pH 8, using butyrylthiocholine as substrate. The kinetic results obtained with mutant enzymes were compared to those obtained with recombinant wild type BChE. MG and MeG were found to act as competitive/linear mixed inhibitors of recombinant wild type BChE and all BChE mutants except the F329A mutant. Both dyes caused complex nonlinear inhibition of F329A mutant, pointing to multisite binding. K-i values for MG and MeG, estimated by nonlinear regression analysis, were 3.8 and 27 mu M, respectively, as compared to the 50- to 150-fold lower values observed with recombinant wild type BChE. The observed significant differences in kinetic pattern and K-i values between recombinant wild type BChE and F329A mutant suggest that phenylalanine at position 329 in human BChE is a critical residue in MG and MeG binding to enzyme. (C) 2011 Elsevier Inc. All rights reserved.

Triatoma patagonica Del Ponte (Hemiptera: Reduviidae), a vector of Chagas' disease, is widely distributed in Argentina and is found in sylvatic and peridomiciliary ecotopes, as well as occasionally in human dwellings after the chemical control of Triatoma infestans. Anti-cholinesteratic products can be applied in peridomiciliary areas and thus knowledge of cholinesterase activity during embryonic development in this species might contribute further information relevant to effective chemical control. Cholinesterase activity was characterized by reactions to eserine 10 -5m, to increasing concentrations of substrate and to varying centrifugal speeds. Acetylcholinesterase activity was detected on day 4 and was significant from day 5. A reduction in cholinesterase activity towards acetylthiocholine (ATC) was observed on days 9 and 10 of development. Cholinesterase activity towards ATC and butyrylthiocholine (BTC) in homogenates of eggs was inhibited by eserine 10 -5m. The shape of the curve indicating levels of inhibition at different concentrations of ATC was typical of acetylcholinesterase. Activity towards BTC did not appear to be inhibited by excess substrate, which parallels the behaviour of butyrylcholinesterases. Cholinesterase activity towards ATC was reduced in supernatant centrifuged at 15 000 g compared with supernatant centrifuged at 1100 g. The cholinesterase system that hydrolyzes mainly ATC seems to belong to the nervous system, as indicated by its behaviour towards the substrates assayed, its greater insolubility and the fact that it evolves parallel to the development of the nervous system. Knowledge of biochemical changes associated with the development and maturation of the nervous system during embryonic development would contribute to the better understanding of anti-cholinesteratic compounds with ovicidal action that might be used in control campaigns against vectors of Chagas' disease. Fil: Visciarelli, Elena Concepcion. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina Fil: Chopa, Alicia Beatriz. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina Fil: Picollo, Maria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina Fil: Ferrero, Adriana Alicia. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina

An integration strategy was investigated to measure initial rates of horse butyrylcholinesterase (BChE) at 50.0 µmol L−1 butyrylthiocholine (BTCh) for detecting irreversible inhibitors as pollutants in environment and foods with dimethoate as a model. In this integration strategy: (a) if BTCh consumption within 5.0 min was >60%, BChE initial rates were derived from maximal reaction rates, estimated by an improved integrated method, according to Michaelies-Menten kinetics at 47.0 µmol L−1 BTCh and Michaelis-Menten constant at 94.0 µmol L−1; (b) or else initial rates were determined by the classical initial rate method. Thus, the differences in BChE initial rates without and after dimethoate treatment indexed final dimethoate contents in reaction mixtures to treat BChE. Results supported that this integration strategy determined BChE activities with a linear range about two magnitudes and an upper limit about twice that by the classical initial rate method alone at 2.0 mmol L−1 BTCh. The coefficient of vari...

The kinetic behavior of cholinesterases is unconventional. While their activities are higher than expected by classical Michaelis-Menten reaction mechanisms, at intermediate substrate concentrations they show strong inhibition by excess of substrate. To date, the main explanations used for all of their kinetic peculiarities include hindrance of product exit, entropically improved water orientation by a second substrate molecule, and complete blockade of the fully occupied active site. However, with the hydrolysis of butyryl(thio)choline by vertebrate acetylcholinesterase, there are time-dependent and substrate-concentration-dependent decreases in catalytic activity. As the substrate depletion results in the expected downwardly concave shape of the progress curves for product formation at low substrate concentrations, this cannot be the reason for the bending of the linear progress curves at higher substrate concentrations. A good theoretical and practical explanation was reached by including the time-dependent appearance of a non-productive enzyme-substrate complex in the reaction scheme. The slow establishment of this complex appears to be a rare occurrence of incorrect substrate orientation at the bottom of the active site, with this blocked by a second substrate molecule.

Human serum Butyrylcholinesterase (BChE) is an important enzyme in detoxification with its capacity for hydrolyzing esters. The inhibitory effect of cisplatin (CDDP) and cyclophosphamide (CY) on BChE is characterized. Time dependent inhibition of BChE with both chemotherapeutics was rapid, reversible. CY was found as non-competitive inhibitor with Ki of 503.6 +/- 50.4 microM. Time dependent CDDP studies displayed progressive inhibition. The constants for apparent dissociation (Ka), first order constant for the break down of the Michaelis complex (k + 2), and bimolecular rate (ka) were calculated as 6.38 x 10(-5) M(-1) min(-1), 0.063 min(-1), and 9.83 x 10(-4) M, respectively. Enzyme protection could be achieved with moderate butyrylthiocholine concentrations (0.3 mM) but higher concentrations increased CDDP inhibition. Apparent Ki value for CDDP was 191.8 +/- 71.2 microM. These results suggest that used in combination therapy, CY and CDDP cause considerable BChE inhibition and may aggravate conditions observed during chemotherapy.