A Multicomponent Butyrylcholinesterase Preparation for Enzyme Inhibition-Based Assay of Organophosphorus Pesticides

Abstract

A new method of producing butyrylcholinesterase (BChE) preparations, stable in storage and use, has been proposed. The BChE preparation is the enzyme co-immobilized with 0.2 M 5-5′-dithiobis (2-nitrobenzoic acid) in starch or gelatin gel. All experimental preparations retain enzyme activity for at least 300 d. The preparations based on gelatin gel show higher activity but lower sensitivity to the toxicants tested in this study compared to the starch gel-based preparations. A method has been proposed for integrated detection of anti-cholinesterase substances in aqueous solutions using the experimental preparation with immobilized BChE. After the additional incubation of the preparation with the immobilized enzyme in the solution of the analyte, the detection limits of malathion and pirimiphos-methyl determined using the IC₂₀ values were below their maximum allowable concentrations—0.005 µM and 0.03 µM, respectively.

1. Introduction

Pesticides are commonly used in agriculture to enhance crop production and control pests. Many of them have been developed to act upon only a certain enzyme in the target organism [1]. Among the pesticides that have recently become widespread are pesticides based on organophosphorus compounds (OPCs), which inhibit the enzymatic activity of insect acetylcholinesterase and nerve impulse transmission. Although OPCs are intended for inhibiting acetylcholinesterase, they are also inhibitors of butyrylcholinesterase (BChE), sometimes called pseudocholinesterase [2].

The extensive use of pesticides and consequent results have become global issues, and, thus, different countries have established committees regulating and controlling pesticide application. WHO and Food and Agriculture Organization of the United Nations (FAO) issued the “International Code of Conduct on Pesticide Management”—the guidelines for pesticide management by all public and private institutions engaged in pesticide production, regulation, and control (Plant Production and Protection Division: International Code of Conduct on Pesticide Management) [3].

Although OPCs are biodegraded rather quickly [4], pesticide residues may affect non-target species such as humans [5,6]. Therefore, it is important to determine the OPCs in environmental samples. This task can be approached using not only methods of classical analysis but also enzyme inhibition-based assays, which are selective, sensitive, and rapid [7,8]. In these assays, the degree of inhibition of the enzyme is usually correlated with the analyte concentration [9]. This is the basis of the diverse enzyme-based biosensors recently developed, which use enzymes as biorecognition elements. Acetylcholinesterase- and butyrylcholinesterase-based biosensors, which are highly sensitive to organophosphorus and carbamate pesticides, constitute two-thirds of the existing biosensors [2,10].

Although BChE-based biosensors are widespread and diverse, the stability of the enzyme during storage and use remains a challenge. Immobilized enzymes are more stable and more resistant to environmental changes [11]. Various nanoparticles (copper nanoflowers [12], magnetic MnO₂ [13]) or functionalized multi-walled carbon nanotubes [14] can be used as carriers for enzyme immobilization. These methods make it possible to produce enzyme preparations that are stable when working in organic solvents and resistant to high temperatures, providing the possibility of numerous applications in enzymatic catalysis. However, polymers of various origins are more often used to immobilize cholinesterase [14,15,16,17,18]. Starch and gelatin polymer gels produce a stabilizing effect on BChE [19,20]. In addition, these gels have been successfully used to construct stable enzyme preparations and microfluidic chips for integrated analysis of inhibitory substances using a bioluminescence enzyme system of luminescent bacteria [21,22,23,24,25]. The incorporation of enzymes into the polymeric matrix not only increased their storage time but also enhanced their stability when subjected to such environmental factors as temperature, pH, and the ionic strength of the solution [26]. Moreover, because of the relative chemical inertness of starch and gelatin gels, they can be used to fabricate multicomponent preparations, which, in addition to enzymes, contain their substrates, considerably simplifying the use of the immobilized preparations in the assay [23,27]. An important advantage of starch and gelatin is their low cost.

The purpose of the present study was to develop a BChE-based preparation stable in storage and use, which would enable the rapid enzyme inhibition-based assay of OPCs in aqueous solutions.

2. Results

Immobilized BChE-based preparations differing in composition, produced in the present study, are intended for rapid analysis for OPCs in aqueous solutions. Immobilization of the enzyme and co-immobilization of the enzyme and the indicator of thiol groups-5-5′-dithiobis (2-nitrobenzoic acid) (DTNB) were performed by incorporating them into a 3% starch gel and a 1.4% gelatin gel followed by dispensing and drying. The resultant preparations were 6–7 mm diameter dry disks; the dry weights of the starch and gelatin preparations were 1.24 ± 0.03 mg and 1.09 ± 0.03 mg, respectively (Figure 1). One disk was intended for one assay.

BChE incorporated in the starch and gelatin gels retained its activity (Figure 2). The preparation based on gelatin gel showed greater activity compared to the starch gel preparation, with the S-BCh-I hydrolysis rate being 30% higher (Figure 2). Another finding was that DTNB immobilization in the preparations did not result in any significant loss of activity of the preparations based on either starch or gelatin gel (Figure 2). Keeping BChE active indicates the absence of restrictions arising from the diffusion of DTNB from the polymer matrix of the carrier.

To analyze the stability of the preparations during storage, changes in their activities were monitored for two months. The activity of the enzyme in the starch or gelatin gel stored less than two months changed insignificantly (Figure 3a), suggesting effective stabilization of the enzyme by these carriers.

The preparations with the co-immobilized enzyme and DTNB retained their activity during long-term storage. For example, the activity of the preparation containing 0.11 U of BChE and 0.2 mM DTNB did not change during ten-month storage (Figure 3b).

The current study showed that preparations with the immobilized enzyme remained active not only in the buffer but also in distilled water (Figure 4). In distilled water, the activity of the preparations decreased by a factor of 2 or 3, depending on the type of the gel. However, the activity of preparations in distilled water was high enough to make it possible to use distilled water as a control solution in the analysis of the sample for anti-cholinesterase substances.

Then, we determined the sensitivity of BChE co-immobilized with DTNB to the organophosphorus substances—pirimiphos-methyl and malathion. Sensitivity of the preparations to inhibitors was described using parameters IC50 and IC20—concentrations of the active ingredients decreasing BChE activity by 50% and 20%, respectively. These parameters correspond to parameter EC50, commonly used in bioassays, which represents effective concentration of an active ingredient causing a 50% decrease in a vital function parameter of a test organism [28]. IC20 corresponds to the OPC detection limit for the preparation with the immobilized enzyme.

Preparations based on gelatin gel were found to be less sensitive to OPCs compared to the preparations based on starch gel. The activity of BChE immobilized in the gelatin carrier did not change in the presence of 0.5 µM of malathion—the amount 10 times higher than the MAC of this pesticide (Figure 5). This could be caused by the high degree of stabilization of the enzyme by the gelatin carrier. At the same time, preparations based on starch gel showed a 74% reduction in activity in the 0.05 µM malathion solution (malathion MAC).

As the BChE preparations based on gelatin gel showed low sensitivity to OPCs and, thus, were less effective in detecting residual OPCs, further research was performed with the preparations based on starch gel. The OPCs used in this study (pirimiphos-methyl, glyphosate, and malathion) differed in the strength of their effect on the activities of preparations. Pirimiphos-methyl decreased the activity of immobilized BChE, and IC50 was 50 µM, which was 1500 times higher than the maximum allowable concentration (MAC) of that pesticide. However, malathion IC50 was 0.02 µM, which was below its MAC.

To increase the detection limit of OPCs, we added the stage of preliminary incubation of the preparation in the tested pesticide solution. Preliminary incubation of the preparation in the 0.055 mM pirimiphos-methyl solution for 5 min resulted in a decrease in the residual activity of the preparation by a factor of 2 (Figure 6); a similar result was obtained for a soluble enzyme (Figure 6, green columns). However, preliminary incubation of the preparation in the 10 mM glyphosate solution did not cause any change in that parameter. For soluble BChE in the presence of the 10 mM glyphosate solution, a complete loss of activity was observed regardless of the incubation time. The observed increase in the detection limit in this case is most likely due to the occurrence of serious diffusion and/or steric limitations in the polymer matrix of the starch gel. Thus, the addition of this stage can enhance the sensitivity of the assay of some OPCs in aqueous solutions. For instance, the resultant pirimiphos-methyl IC20 was 0.03 µM.

Thus, the current study proposes a technique of producing BChE preparations stable in storage and use and a method for the integrated analysis of aqueous solutions for OPCs. The method includes incubation of the BChE-based preparation in the tested solution (sample) for at least 5 min followed by adding the S-BCh-I substrate. Then, the rate of change in the absorbance of the resultant solution, which is proportional to the enzyme activity, is measured at a 412 nm absorption wavelength. Concentrations of the anti-cholinesterase substances in the solution are estimated from a decrease in the activity of BChE preparations in the solution of the sample relative to the activity of the preparations in the control solution. A 0.05 M potassium phosphate buffer (pH 8) or distilled water can be used as a control solution.

3. Discussion

The immobilization of cholinesterases is a widespread approach to increasing enzyme stability. For example, the immobilization of acetylcholinesterase from electric eel in photocrosslinkable PVA-SbQ (8%) [29] enhanced the stability of the enzyme in the presence of organic solvents; that was a very important finding, as most OPCs are not water soluble. That polymer, though, has a rather high cost; moreover, precautions should be observed while working with it, as the toxicological properties of that compound are not known. Another method is the production of polyurethane-based ion-selective membranes with immobilized BChE [17]. However, storage of these membranes for four weeks resulted in the loss of one-third of the initial enzyme activity.

The present study demonstrated the method of producing multicomponent preparations of BChE co-immobilized with DTNB in gels of natural polymers—starch and gelatin. The definite advantages of the method include not only the low cost of the carrier (starch and gelatin gels) but also the biological safety of these carriers, which makes the production and use of the enzyme-based preparations harmless to human health. Moreover, the incorporation of DTNB simplifies the use of the preparation in the assay for anti-cholinesterase substances, as the solvents of the analytical system need not be preliminarily diluted. This is a clear advantage of the proposed method over many other methods used to detect anti-cholinesterase substances such as the immobilized enzyme reactor based on silica-encapsulated equine BChE [30] or the use of cholinesterases immobilized in dry films composed of poly(vinyl pyrollidone) (PVP) [31]. In addition, the process of BChE immobilization proposed in the current study enables the production of dosage forms intended for a single measurement, which also simplifies the analysis.

A study by Zeman et al. [32] described the production of detectors containing carriers in the form of pellets with immobilized butyrylcholinesterase, substrate, and detection reagent, but the enzyme activity dropped by a factor of 2 over 12 months of storage. The immobilization of BChE in the starch and gelatin gels results in a considerable increase in the time of storage of the preparations with immobilized BChE. The likely reasons for the enhanced stability of the enzyme molecule are the suppression of unfolding processes within the polymeric matrix of the natural gel and establishment of the conditions preventing aggregation of the enzyme molecules [33].

The stabilizing effects of starch and gelatin on BChE differ in strength. The decrease in the activity of the enzyme immobilized in starch gel may be associated with the greater ability of starch, compared to gelatin, to bind water, which is the substrate in the reaction catalyzed by BChE. Thus, the osmotic effect may occur inside the starch carrier, resulting in a reduction in the enzyme activity. In addition, the effects of starch and gelatin polymer networks on the rate of diffusion of the substrate and/or inhibitor to the enzyme may differ in strength. Starch is the more appropriate enzyme carrier for the practice of analytical control of OPCs in environmental samples. In the presence of starch, the inhibitory effect of the anti-cholinesterase substances tested in this study was retained at a level comparable with the sensitivity of soluble cholinesterases or cholinesterases immobilized in other carriers such as poly(vinyl pyrollidone) (PVP) [34]. Similar results were obtained for other enzymes. For instance, the coupled enzyme system NADH-FMN-oxidoreductase+luciferase of luminescent bacteria co-immobilized in starch gel with the substrates retained its sensitivity to salts of heavy metals, phenols, and quinones [21].

The conventional method of assessing the toxicity of aqueous solutions is based on determining how many dilutions of the sample with distilled water are needed to minimize the toxic effect on the test system (enzyme). The present study shows that BChE immobilized in gels retains a sufficient level of activity in distilled water used as the control solution. The gels are prepared using the buffer solution, and, when dried, the salts necessary to maintain the buffer properties of the solution stay within the polymeric matrix of the carrier. During the assay with the immobilized enzyme preparations, the ions diffuse into the solution, ensuring the stability of the analytical signal of the enzyme system. For the future use of BChE preparations in bioassays, the fact that they remain active in distilled water is very important, suggesting that these preparations can be used in sample analysis by the conventional method, i.e., by determining the safe level of sample dilution.

4. Materials and Methods

Freeze-dried BChE from equine serum 900 U/mg (“Sigma”, Saint Louis, MO, USA) was used in the study. Other reagents used were butyrylthiocholine iodide (≥99.0%), 5-5′-dithiobis (2-nitrobenzoic acid) (≥98%), starch, and gelatin (“Sigma”, Germany). Malathion, pirimiphos-methyl, and glyphosate (“Sigma”, Germany) were used as inhibitors. Solutions of BChE, starch, and gelatin were prepared using a 0.05 M potassium phosphate buffer, pH 8. All measurements were conducted at 25 °C.

BChE immobilization into gels was performed as follows. Starch suspension was slowly heated and boiled for 5 min with constant stirring. Gelatin suspension was slowly heated to 80 °C. The resultant gels were cooled to 25 °C, and then, an enzyme solution and a solution of 5-5′-dithiobis (2-nitrobenzoic acid) were added sequentially to the gels. The resultant mixtures were stirred thoroughly. Then, using an epMotion 5075 automated liquid handling system (“Eppendorf”, Leipzig, Germany), 25 µL samples of the gel containing the system components were transferred onto the fluoropolymer film and dried at a temperature of 8 °C for 24 h. The produced preparations were stored at 4 °C and 45–50% relative humidity.

BChE activity was analyzed using Ellman’s method [35]; 2 mM butyrylthiocholine iodide was used as substrate. BChE activity was determined by measuring the absorbance of the reaction solution at a 412 nm absorption wavelength. The effect of each inhibitor was estimated by the change in the activity in its presence (A) relative to the activity in the control solution (distilled water (A₀)). To measure the activity of soluble BChE, 1.75 mL of 0.05 M potassium phosphate buffer pH 8 (or distilled water), 50 µL of the BChE solution with 0.11 U, 100 µL 4 mM DTNB, and 100 µL 2 mM S-BCh-I were sequentially added to the spectrophotometer cuvette. To measure the activity of immobilized BChE, 1 disk of the enzyme preparation, 1.9 mL of 0.05 M potassium phosphate buffer pH 8 (or distilled water, or an inhibitor solution), and 100 µL 2 mM S-BCh-I were added to the spectrophotometer cuvette.

5. Conclusions

The study proposed a method of producing preparations of BChE immobilized in the starch or gelatin gel, which can be used in enzyme inhibition-based assays for detecting OPC residues. The proposed approach has obvious competitive advantages over other methods: the relatively low cost of the carriers (starch and gelatin) and the simple process of fabrication of the preparations with immobilized BChE combined with the simpler use because of co-immobilization of the enzyme and the DTNB indicator. Moreover, the immobilized enzyme preparations are convenient to use because they are dosage forms intended for a single measurement, and they retain their activity over long storage times (more than one year). The preparations based on starch gel are more sensitive to OPCs and, thus, are better candidates for assay. After the incubation of the preparation with the immobilized enzyme in the solution of the analyte, the malathion and pirimiphos-methyl detection limits were below the MACs of these pesticides.

6. Patents

Patent RF 2546245. Enzyme preparation based on immobilized butyrylcholinesterase and method of its preparation. Esimbekova E.N., Lonshakova-Mukina V.I., Kratasyuk V.A.

Patent RF 2704264. Express method for determination of butyrylcholinesterase inhibitors in water and aqueous extracts. Esimbekova E.N., Lonshakova-Mukina V.I., Kratasyuk V.A.