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Assessment of Acetylcholinesterase from Channa micropeltes as a Source of Enzyme for Insecticides Detection.

Byline: Mohd Sha Aidi Sharif , M. Izuan Effendi Halmi, Amir Syahir, Wan Lutfi Wan Johari and M. Yunus Shukor _: Abstract

Abstract

In this work we assess the potential of acetylcholinesterase (AChE) from Channa micropeltes (Toman) as a sensitive test for the presence of insecticides. The partial purification and characterization of a soluble AChE from C. micropeltes brain tissues using affinity chromatography gel (procainamideSephacryl S-1000) showed that the partially purified AChE was most active on acetylthiocholine (ATC) but had low activities on propionylthiocholine (PTC) and butyrylthiocholine (BTC), indicating that the partially purified fraction was predominantly AChE. Soluble AChE was partially purified 9.27-fold with a 91.12% yield. The partially purified AChE displayed the highest activity on ATC at pH 7 and at 30oC using 0.1 M Tris buffer. The enzyme exhibited Michaelis-Menten kinetic constants, Km, for ATC, BTC and PTC at 36, 77 and 250 M, respectively, and the maximum velocities, Vmax, were 18.75, 0.12 and 0.05 mol/min/mg protein, respectively. Moreover, the AChE from C. micropeltes presented comparable sensitivity to carbamates and organophosphates insecticides than that from Electrophorus electricus by comparing half maximal inhibitory concentration values, therefore the enzyme is a valuable source for insecticides detection in Malaysian waters at lower cost. Copyright 2014 Friends Science Publishers

Keywords: AChE; Channa micropeltes; Affinity Chromatography; BiomarkerIntroduction

The extensive use of organophosphate (OP) and carbamate pesticides is a concern due to the neurotoxicity properties of the compounds (Gill et al., 2011). In Malaysia, the presence of pesticides were reported in the Selangor River, which is one of the major rivers used as a source of drinking water, due to intense agricultural activities (Leong et al., 2007). Another study conducted along Malaysian shore also indicate the detection of pesticides in fishes although the levels were below the maximum residue limits most of the time (Santhi et al., 2012).Aquatic organisms have been widely used as biomarkers to detect various pesticides and toxicants, which can inhibit activities of cholinesterases (de la Torre et al.,2002; Shaonan et al., 2004; Tham et al., 2009; Assis et al.,2010). An example of the effect of pesticides on fish has been tested on a snakehead fish, Channa striata (Van Cong et al., 2006). Similar types of fish that are natives to Malaysian waters, such as C. striatus, C. micropeltes and C. lucius (Mohsin and Ambak, 1983), are therefore potentially useful as biomarker agents for pesticides or insecticides. Moreover, mussels (Peric and Petrovic, 2011) and snails (Chitmanat et al., 2008) have been employed to detect the presence of pollutants by linking it to the inhibition of cholinesterase activities.Two classes of cholinesterase, i.e. acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), can be specifically distinguished based on the substrate (Radic et al., 1991). AChE is found in nervous system and its functions are not yet fully studied (Carlock et al., 1999). The hydrolysis rate of acetylcholine by AChE is faster than BChE. Only BChE can hydrolyze both butyrylcholine and acetylcholine while AChE is inactive on butyrylcholine. Nevertheless, AChE is easily inhibited by OP and carbamate insecticides, providing a convenient and rapid means of monitoring the presence of pollutants in the environment.A previous in vivo work on a snakehead fish has shown that the brain cholinesterase activity from the organism is very sensitive to insecticides (Van Cong et al.,2006) and hence can be a replacement for the expensive Electrophorus electricus, which is commonly used as biosensor for the detection of insecticide (Tham et al.,2009). The main aim of this work is to assess the sensitivity of AChE from C. micropeltes on carbamates and OPs assay

in vitro. The sensitivity of AChE from C. micropeltes in comparison of AChE from Electrophorus electricus will be then evaluated. This work proves that AChE from C. micropeltes is the potential source for a cheap and local indicator of OP and carbamate insecticides in the tropics.

Materials and Methods

Chemicals

Carbofuran, methomyl, carbaryl, parathion, malathion, diazinon, bendiocarb, chlorpyrifos, acephate, dimethote and trichlorfon were purchased from Dr. Ehrenstorfer (Augsburg, Germany). Bromine, acetylthiocholine iodide (ATC), propionylthiocholine chloride (PTC), AY- mercaptoethanol, procainamide hydrochloride, 1,4- butanediol diglycidyl ether and sodium borohydride were purchased from Sigma-Aldrich. 5'-dithio-bis (2- nitrobenzoic acid) (DTNB) and butyrylthiocholine iodide (BTC) were purchased from Fluka Chemie GmbH. Commercial AChE preparation from eel (E. electricus, 349 units/mg solid) was purchased from Sigma (St. Louis, USA). Biorad Protein Assay was purchased from Bio-Rad Laboratories Inc. Vivaspin4 was from Vivascience. All other chemicals used in this study were of analytical or special grade.

Specimen

C. micropeltes was used as the fresh water test organisms in this study. The fishes (weight: 900-1200 g; average length:36 cm) were obtained from Snoc International Sdn Bhd, Selangor, Malaysia. The selected fish was decapitated and the whole brain was promptly removed. The process of obtaining crude extract of the brain cells were laid out by Tham et al. (2009). The supernatant of the crude extract wasfurther purified using the following procedures.

Preparation of Affinity Chromatography Columns

Epoxy (bisoxirane) activation: Affinity procainamide chromatography was prepared according to the modified method of Ralston et al. (1983). Briefly, 100 mL of Sephacryl S-1000 (settled gel, Sigma, St. Louis, USA) was washed with 1 L of deionized water in a sintered glass tunnel, dried, and then transferred to a 500-mL beaker. 75 mL of 0.6 M sodium hydroxide containing 150 mg sodium borohydride was subsequently added and continuously stirred before the addition of approximately 75 mL of 1,4- butanediol diglycidylether. The mixture was left stirred at room temperature overnight. The activated gel was then thoroughly washed with water to remove excess reagent until there was no oily film (representing epoxy compound) on the surface of the gel. Acetone was used to completely remove bisoxirane groups. The gel was resuspended in water for ligand coupling. Ligand coupling of procainamidesephacryl S-1000 gel: The epoxy-activated Sephacryl S-1000 was washed with deionized water on a sintered glass filter. The gel slurry was transferred onto a coupling solution of 12 mM of borate buffer (pH 11.0) containing 0.2 M of procainamide. The pH was then adjusted to 12 by the addition of sodium hydroxide. The mixture was incubated at 25C for 96 h on a shaking incubator. The gel was washed in sequence with 10 volumes each of 0.1 M sodium acetate (pH 4.5), 12 mM sodium borate (pH 10) and deionized water. The gel was then suspended in 100 ml of 1.0 M ethanolamine (pH 9.0) to block excess active groups and the mixture was stirred for 6 hours. The gel was finally washed thoroughly with 1 L of1.0 M NaCl followed by 5 L of deionized water.

Partial Purification of AChE using AffinityProcainamide Chromatography

The procedure for partial purification of AChE using affinity procainamide chromatography has been previously described in details (Tham et al., 2009). For quantitative measurement of proteins, Bradford protein assay (1976) was employed using bovine serum albumin as a standard.

Activity and Effect of Substrates

The measurement of AChE activities and the effect of substrates have been previously described (Ellman et al.,1961; Tham et al., 2009). Briefly, AChE activity was measured from the development of yellow color over time through reaction of thiocholine and 5, 5'-dithio-bis-2- nitrobenzoate (DTNB). AChE catalyzes the hydrolysis of acetylthiocholine iodide (ATC) producing thiocholine and acetate. The activity is defined as the hydrolysis of ATC (mol) per minute.In order to analyze the efficiency of the enzyme catalysis, the substrate specificity was determined through enzyme kinetics utilizing Michaelis-Menten equation. Three different substrates i.e. ATC, butyrylthiocholine iodide (BTC) and propionylthiocholine iodide (PTC) were tested. Experiment was performed in triplicates. The apparent Km value (Km,app), was determined by non-linear regression analysis using Graphpad PRISM 4.OPs were activated prior to assaying according to the modified method of Villatte et al. (1998). The pesticide (25L) was incubated in 5 L of 0.01 M pure bromine solution at room temperature for 20 min. 20 L of 5% ethanol was added to stop the activation process. Preliminary experiments showed that bromine and ethanol at givenconcentration did not inhibit AChE activities. The halfmaximal inhibitory concentration (IC50) was determined using at least five different concentrations of carbamate and OPs. The assay mixture contained 0.1 M potassium phosphate buffer (pH 8.0), 0.067 mM DTNB, pesticides and enzymes, and was incubated in the dark (10 min) following methods by Tham et al. (2009). A 20 L of 0.5 mM ATCwas subsequently added into the mixture and was left for 10 min. The absorbance of the reaction mixture was read at 405 nm. The experiment was conducted at room temperature (Tham et al., 2009).

Effect of pH and Temperature

The effect of pH and temperature for purified AChE was determined by measuring the enzyme activities at the different pH using an overlapping buffer system consisting of acetate buffer (pH 4 to 6), potassium phosphate buffer (pH 6 to 8) and Tris-Cl (pH 7 to 9) and temperatures (15 to80C) for 10 min and enzyme activity was assayed withATC as previously described.

Results

Partial Purification

Fig. 1 shows the elution profile of enzyme activity and protein content from the procainamide-based affinity chromatography. AChE activity was detected in the washed fraction and this was due to column overloading. The bound protein was eluted from the column at a flow rate of 0.2 mL/min with high ionic strength phosphate buffer (1.0 M NaCl in 20 mM sodium phosphate buffer pH 7.4). Tubes exhibiting high AChE activities were pooled. Changing of the ionic strength of the solution using 1 M NaCl lowered the energy of the bond between AChE and the procainamide ligand, leading to the desorption of AChE from the matrix. It is probable that the explanation for protein peaks that were not coinciding with enzyme activity peaks was due to non-specific hydrophobic binding of protein via other interactions (Scopes, 1994). The purification yielded 55 mg protein, with specific AChE activity of 793,674mol/min/mg. 91.12% of enzyme activity was recovered and a 9.27-fold purification was obtained (Table 1).

Kinetic Studies

Table 2 shows the Michaelis-Menten constant (Km,app) and maximal velocity (Vmax,app) of the enzyme in hydrolyzing three different substrates; ATC, BTC and PTC. Both BTC and PTC recorded higher Km values than that of ATC, indicating that the affinity of the partially purified enzyme towards BTC and PTC were lower compared to ATC. The enzyme also displayed higher rates of hydrolysis when incubated with ATC but showed far less activity in the presence of BTC and PTC, based on the Vmax values. Based on the table of Km,app and Vmax,app, ATC gave the highest Vmax,app and the lowest Km,app. The catalytic efficiency calculated from the Vmax,app/Km,app shows that ATC exhibited the highest efficiency followed by BTC and PTC. This supported the evidence that the enzyme that was partially purified was indeed AChE.The Effect of pH on AChE Activity

The assays were carried out using an overlapping buffer system consisting of acetic acid buffer (pH 3.0 to 5.5), sodium phosphate buffer (pH 5.0 to 8.0) and Tris-Cl buffer (pH 7.0 to 10.0). The partially purified AChE displayed highest activity towards ATC at pH 7, using 0.1 M Tris buffer,###with###a###specific###enzyme###activity###of###34.12mole/min/mg protein (Fig. 2).

Table 1: Purification table of partially purified AChE from Channa micropeltes

Procedure###Volume (ml)###Total activity (U) Total protein (mg) Specific activity (U/mg)a###Purification fold (X)###Yield (%)

Homogenate###12.5###48,205,875###563###85,582###1.00###100.00

Ultracentrifugation at 100,000A-g for 1 h###7.5###35,033,818###293###119,708###1.40###72.68

Affinity-Procainamide Sephacryl S1000###2###43,924,625###55###793,674###9.27

The Effect of Temperature on AChE ActivityFig. 3 shows a bell-shaped graph from the temperature profile, where AChE achieved its maximum activity at30C. At lower temperature, the enzyme is retarded but not denatured, resulting in low activity.The Effect of Insecticides on AChE Activity

Screening of insecticides showed that all of the carbamatesand the OPs malathion, parathion and diazinon gave stronginhibition while other OPs such as trichlorfon, acephate anddimethoate showed less than 50% inhibition to AChE activity (Fig. 4). IC50s for various insecticides chosen for further studies are shown in Table 3 in comparison with another source of AChE. Other comparisons on IC50s of fish cholinesterases on pesticides can be found elsewhere (Assis et al., 2011). From the experimental data, if there is no overlap between two associated confidence intervals, it usually indicates significant difference at the pless than 0.05 level. An overlapped confidence interval indicates that moreexperimentation are required to evaluate non-significance(Schenker and Gentleman, 2001). Based on this premise, the AChE from C. micropeltes showed comparable sensitivity to carbamates and organophosphates than that from E. electricus. AChE from C. micropeltes was more sensitive to the carbamates carbaryl, methomyl and bendiocarb than that from E. electricus. The latter was more sensitive to the carbamate carbofuran while propoxur showed similar sensitivity towards both sources with an overlapped confidence interval. As for organophosphate insecticides, C. micropeltes was more sensitive to parathion and diazinon than E. electricus while the latter was more sensitive to malathion while chlorpyrifos showed similar sensitivity towards both sources with an overlapped confidence interval (Table 3).

Discussion

C. micropeltes is highly prized for its medicinal properties in healing wounds of internal organs and is widely found in Malaysian fresh water aquatic bodies (Muhamad and Mohamad, 2012). The results from this study will be useful for comparison purposes on other local fish species and as a precursor for the development of an assay for insecticide pollutants. The purification fold and yield obtained are within the reported range for other AChEs using the custom made procainamide based affinity gel (Talesa et al., 1992; Forget and BocquenACopyright, 1999; Gao and Zhu, 2001).

Table 2: Comparison of Km app and Vmax app of AChE from Channa micropeltes for ATC, BTC and PTC

Parameter###unit###ATC###BTC

Vmax app###mol/min/mg###18.75

Km app###M###36###77

Vmax app/Km app###L/min/mg###0.52

Table 3: Comparisons of the sensitivity of C. micropeltes AChE to various insecticides with AChEs from E. electricus

Insecticides###C. micropeltes IC50###E.

###(Confidence Interval) mg/L (Confidence

Carbofuran###0.0081 (0.0074-0.0089)###0.006

Carbaryl###0.07922 (0.0697-0.0917)###0.133

Methomyl###0.0192 (0.0178-0.0208)###0.026

Propoxur###0.0679 (0.0616-0.0757)###0.060

Bendiocarb###0.0379 (0.0341-0.0427)###0.015

Parathion-oxon###0.0316 (0.0279-0.0363)###0.068

Malathion-oxon###0.0242 (0.0192-0.0327)###0.014

Diazinon-oxon###0.0599 (0.0554-0.0652)###0.177

Chlorpyrifos-###0.0522 (0.0418-0.0693)###0.060

The enzyme has high affinity for ATC followed by PTC and BTC as apparent from the low Michaelis-Menten constant (Km,app) values indicating that the sample was a true AChE. The Km,app value obtained for ATC is within the range obtained from other freshwater fish such as Pseudorasbora parva, Carassius auratus auratus, Oncorrhychus mykiss (Shaonan et al., 2004) and Cnesterdon decemmaculatus (de la Torre et al., 2002). Catalytic efficiency calculated from the ratio indicated significant differences between ATC and the other substrates tested. The catalytic efficiency value was within the range reported for ATC from Cyprinus carpio and Cnesterodon decammaculatus (de la Torre et al., 2002).The optimum pH value obtained in this study coincided with the near neutral pH values reported for AChE from mammalian, insects and fish tissues (Villatte et al., 1998; Forget and BocquenACopyright, 1999; Anitha et al., 2004; Sahin et al., 2005). The pH profile clearly shows that at the extreme ends of the pH range, the enzyme activity declines significantly. This is due to the change of charge distribution at the substrate-binding site thereby directly influencing the enzyme-substrate complex interaction. The active site of AChE contains side chains with charge groups, which stabilize enzyme-substrate complex via hydrogen and ionic bonds. The ionic states of these charged groups are sensitive to pH. Thus, a change in pH values would dramatically change the ionic states of the active sites and molecular areas, leading to a loss in the enzyme activity (Dziri et al.,1997).With an optimal temperature recorded at 30C, this temperature skews from the general optimal rates of mammalian cholinesterase which is reported to be around37C to 40C. It was reported that the optimal temperature for insects or any other organisms might be lower (Fairbrother et al., 1991). Since fish are poikilotherms, it ispossible that warmer temperatures will denature andinactivate AChE. Beyond the optimum temperature, the energy of the vibration within the molecule is great enough to disrupt the non-covalent bonds that maintain the three- dimensional structure of the enzyme, resulting in conformational changes. At 70C and above, the enzyme activity is negligible. It is important to control the temperature during the catalysis due to the effects that it will bring to the kinetic parameters of the enzymatic reaction (Copeland, 2000). The insecticides screening results showed that the AChE from this organism could be further developed into a sensitive inhibitive assay for insecticides. The bromine oxidation technique in this work was adequate to fully oxidize the organophosphates. However, oxonation using bromine is limited to OP compounds that require oxidative desulfuration for activiation. OP compounds that are oxygen analog in the active form are activated by other procedures (Villatte et al., 1998).Pollutions such as azo dyes (Pathak and Madamwar,2010; Ruiz-Arias et al., 2010), detergents (Malaviya and Sharma, 2011; Carvalho-Neta et al., 2012), hydrocarbons (Chavan and Mukherji, 2010; Kang et al., 2010; Hung et al.,2011; Tripathi et al., 2011), heavy metals (Sani et al., 2010; Kavita et al., 2011) and insecticides (Deshpande et al.,2011; Rama Krishna and Philip, 2011) are serious health threats and the development of an assay for insecticides ishoped to increase the biomonitoring efficiency of toxic xenobiotics. A previous work has shown that C. punctatus could be used as a biomarker for monitoring the presence of insecticide (Agrahari et al., 2006) and there is a possibilitythat AChE from this organism could be used as a newsource of AChE for the detection of insecticides.

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Author:Sharif, Mohd Sha Aidi; Halmi, M. Izuan Effendi; Syahir, Amir; Johari, Wan Lutfi Wan; Shukor, M. Yunu
Publication:International Journal of Agriculture and Biology
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Date:Apr 30, 2014
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