Purification and characterization of acetylcholinesterase from brain tissues of Oreochromis aurea and its application in environmental pesticide monitoring

YunHua Ding*, XiaoMin Wu, JunBin Fang

Life Science Department, Huizhou University, Huizhou, Guangdong 516007, China

Purification and characterization of acetylcholinesterase from brain tissues ofOreochromis aureaand its application in environmental pesticide monitoring

YunHua Ding*, XiaoMin Wu, JunBin Fang

Life Science Department, Huizhou University, Huizhou, Guangdong 516007, China

Acetylcholinesterase (AChE) plays an important role in enzyme-based detection of pesticides in the environment. In this paper,AChE from the Triton X-100 extract of brain tissues ofOreochromis aureawas purified by (NH4)2SO4fractional precipitation,Sephadex G-100 gel filtration, and DEAE-cellulose ion exchange chromatography. Certain biochemical characterizations of the purified enzyme and inhibition of pesticides on the enzyme were also studied. The specific activity of this purified enzyme was 20.628 U/mg protein, fold of purification was 139, and recovery was 22.1%. The optimal temperature of this enzyme was between 35-40 °C, and optimal pH was between 7.5-8.0. The Michaelis constant (Km) for acetylthiocholine iodide was 0.183 mmol/L.The enzyme activity was inhibited by excess substrate, and optimal substrate concentration was 6 mmol/L. Four pesticides (dichlorvos, phoxim, triazophos, and methomyl) exhibited strong inhibitions on this enzyme with IC50less than 5 μg/mL. This study suggests thatOreochromis aurea(tilapia) could be a good enzyme source for pesticide monitoring in water environments.

Oreochromis aurea; acetylcholinesterase; purification; gel filtration; ion-exchange chromatography; pesticide sensitivity

1. Introduction

As fish are sensitive to pesticides in water environments, finding an effective and sensitive pesticide biomarker from fish has great significance in environmental pesticide monitoring. Acetylcholinesterase (AChE) is the key enzyme in biological neurotransmission, which can effectively hydrolyze neurotransmitter acetylcholine, guaranteeing the continuous nerve impulse transmission between synapses. Acetylcholinesterase is also the target enzyme of organophosphorus and carbamate pesticides. Therefore,utilizing AChE as a biomarker has been widely adopted in evaluating pesticide toxicity and environmental pollution(Liuet al., 2007).

Past researches have focused on exploring various enzyme sources to protect against pesticides. Many animal tissues, such as liver, serum, brain, and muscle, contain abundant AChE, which mingle with numerous hydrolytic enzymes (including some target enzymes with detoxification function), thus affecting the kinetic characteristics of AChE and its sensitivity to pesticides (Peiet al., 2006). Therefore, it is necessary to isolate and purify AChE from various tissues in organisms and study their enzymatic kinetic characteristics.

Currently there are very few reports on purification and characterization of AChE from freshwater fish. Liuet al.(2007) extracted AChE from carp liver and conducted pesticide sensitivity work, and Jiaet al.(2010) prepared AChE from carp brain and reported its sensitivity test. Because China has become the world’s largest producer of tilapia during the last 10 years, the object of this research was to study the methods of purifying AChE from tilapia brain tissue, the biochemical properties of AChE, and the sensitivity of AChE to four kinds of common pesticides (triazophos, phoxim, dichlorvos, and methomyl) in order to find a good enzyme source for pesticide monitoring in water environments.

2. Materials and methods

2.1. Fish

The tilapias (Oreochromis aurea) (30±2 cm) were bought from the Huizhou Fishery Institute with no exposure of pesticides, and were maintained in lab conditions for at least one week before testing.

2.2. Chemicals

The four technical grade pesticides were bought from the Huizhou pesticide market: acetylthiocholine iodide (ATch)and 5,5'_dithio-2,2'_nitrobenzoic acid (DTNB) were bought from the Sigma Company, DEAE-cellulose (DE-23) and Sephadex G-100 were purchased from the Pharmacia Company. The other chemicals were of analytical grade.

2.3. Enzyme activity determination

AChE activity was determined based on the method put forward by Ellmanet al.(1961) with a slight adjustment.ATch and DTNB were dissolved separately in pH 8.0, 0.1 mol/L sodium phosphate buffer as a 5 mg/mL solution. The enzymatic reaction systems were as follows: 50 μL enzyme solution, 50 μL DTNB, 50 μL ATch in 3 mL pH 8.0, 0.1 mol/L sodium phosphate buffer. Before ATch was added, the mixture was allowed to pre-incubate at 37 °C for 10 min.After ATch was added, the reaction system was allowed to proceed at 37 °C for 3 min. The absorbance was read on a spectrophotometer at a wavelength of 412 nm.

2.4. Protein content determination

Protein content was determined by the method of Bradford (1976) using bovine serum albumin as standard.

2.5. Purification of AChE

The brain tissue was homogenized with a 4 °C cold solution of 0.02 mol/L, pH 7.4 Tris-HCl buffer (W:V=1:6)containing 0.1% Triton X-100 and 0.05 mol/L NaCl; the homogenate was then centrifuged at 10,000 r/min for 30 min at 4 °C and the supernatant was marked as crude enzyme.During mild stirring, ammonium sulfate powder was added to the supernatant until 35%, 50%, and 70% saturation for fractional precipitation. The mixture was rested for 2 hrs at 4°C, then centrifuged at 6,000 r/min for 30 min at 4°C. The pellets and supernatants of each saturation were collected separately and the pellets were dissolved in pH 8.0, 0.1 mol/L sodium phosphate buffer. Enzyme activity and protein content of the pellets were determined, thus specific activity,folds of purification, and recovery were obtained. The pellet with maximum activity was run through Sephadex G-100 gel filtration. Solutions of the pellet were loaded into a Sephadex G-100 column (1.0×30 cm) and were eluted with pH 7.5, 0.05 mol/L Tris-HCl. The fractions showing AChE activity were pooled and followed by DEAE-cellulose(DE23) (1.0×20 cm). The fractions eluted with gradients of 0.2, 0.3, 0.4, 0.5, and 0.6 mol/L NaCl, pH 7.8, 0.02 mol/L sodium phosphate buffer at a flow rate of 0.3 mL/min (4 mL per tube). Those showing AChE activity were pooled and dialyzed for further study.

2.6. Pesticide sensitivity test

Three kinds of organophosphorus pesticides were used in the experiment: dichlorvos, triazophos, phoxim, and one carbamate pesticide methomyl, all of which were diluted by anhydrous alcohol to seven solutions of different concentrations. Enzyme activity was determined as 2.3, 50 μL pesticide solution was added to the reaction system substituting pH 8.0, 0.1 mol/L sodium phosphate buffer. After 10 min of inhibition at 37 °C, DTNB and ATch were added, and enzyme activities were determined based on a reaction for 3 min. The IC50(concentration that causes 50% inhibition)was calculated from the linear regression of the log concentration-inhibition percentage plot (Liuet al., 2008).

2.7. Km of AChE

The AChE activities at six concentrations (0.06-2.5 mmol/L) of substrate were determined as 2.3, and the Michaelis constant (Km) and maximal velocity (Vmax) were determined by linear regression of Lineweaver-Burk plots.

3. Results

3.1. Isolation and purification of AChE

The pellet of 50% ammonium sulfate saturation showed maximal total activity, while the pellet of 70% ammonium sulfate saturation showed the least, which indicated that the majority of enzymes were in the 50% saturation pellet during fractional precipitation (Table 1).

The crude AChE from tilapia brain tissue contained 274.127 mg protein with a specific activity of 0.148 U/mg protein. After the fractional precipitation procedure, the isolated enzyme contained 74.742 mg protein with a specific activity of 0.213 U/mg protein.

The solutions of 50% ammonium sulfate saturation pellets were loaded into a Sephadex G-100 column (1.0×30 cm)and resolved as four peaks of protein, with one peak showing AChE activity (Figure 1). After DEAE-cellulose (DE23)ion-exchange chromatography of the pooled fraction from gel filtration that showed activity, three peaks of protein were obtained with fractions that showed AChE activity quite near the third peak of protein (Figure 2). Table 2 demonstrates that from the very beginning of the DE23 ion-exchange chromatography (starting at about tube 5),certain unabsorbed AChE were eluted out, forming a small peak of AChE (Figure 2).

Table 1 Fractional precipitation of AChE at different (NH4)2SO4 saturations

Figure 1 Sephadex G-100 gel filtration

Figure 2 Ion-exchange chromatography on DEAE-cellulose DE23 column

Table 2 shows that after the complete purification procedure, the purified enzyme contained 0.436 mg protein with a specific activity of 20.628 U/mg protein, fold of purification 139.378, and recovery of activity 22.1% .

Table 2 Purification of AChE from brain tissue of Oreochromis aurea

3.2. Characterizations of AchE

3.2.1 Km of AChE

When six concentrations (0.06-2.5 mmol/L) of substrate were set, AChE activities were determined as 2.3 and represented by changes of A412 per min.; Michaelis constant (Km)and maximal velocity (Vmax) were determined by linear regression of Lineweaver-Burk plots (Figure 3). The Kmof AChE from tilapia was 0.183 mmol/L.

3.2.2 Effects of substrate on activity and inhibition of excess substrate

A series of substrate solutions (0.2-12 mmol/L) of ATch were used to determine the activities of AChE. The results showed that the velocities (reaction rates) significantly increased as the concentration of ATch increased. However,the increase of velocity slowed as the concentration of ATch further increased, and at some point (concentration of 6 mmol/L) the velocity gradually decreased, showing a phenomenon of substrate inhibition (Figure 4).

3.2.3 Optimal pH and temperature of AChE

As AChE activities were determined at different temperatures (20-45 °C) and different pH values, it became clear that the optimal temperature of AChE was between 35-40 °C and the optimal pH was between 7.5-8.0, which were very similar to other reports (Zhuet al.,2006).

3.3. Inhibition of AChE by pesticide and sensitivity test

Figure 5 shows that three kinds of organophosphorus pes-ticides (dichlorvos, triazophos, phoxim, and one carbamate pesticide methomyl) all have strong inhibition on AChE. According to ecological toxicology, IC50(concentration that causes 50% inhibition) is often used to evaluate the sensitivity of AChE to pesticide. The smaller the IC50, the stronger the inhibition of the pesticide on AChE. Table 3 displays that all of the IC50values of the four pesticides on AChE were less than 5 μg/mL, and the IC50values of the three organophosphorus pesticides were less than that of the carbamate pesticide methomyl. Dichlorvos showed the strongest inhibition with an IC50of 0.0398 μg/mL, while methomyl showed a relatively weaker inhibition with an IC50of 4.4668 μg/mL.

Figure 3 Michaelis constant (Km) of AChE for acetylthiocholine iodide

Figure 4 Effect of acetylthiocholine iodide on AChE activity

Figure 5 Inhibition of four pesticides on AChE activity

Table 3 IC50 of four insecticides on AChE from brain tissue of Oreochromis aurea

4. Discussion

Excess substrate inhibition is an important characteristic of AChE that differs from other cholinesterases (Shiet al.,1981). The AChE from tilapia brain tissue showing substrate inhibition as the concentration of ATch exceeded 6 mmol/L suggests that this purified enzyme in the experiment was a true AChE. The optimal substrate concentration of 6 mmol/L was higher than that ofNebia albiflora(0.5 mmol/L) (Shiet al., 1981),Scomberomorus niphonius(2.5 mmol/L) (Zhuetal., 2006), andPseudorasbora parva(0.6 mmol/L) (Liet al.,1997). The interspecies variation may be due to innate variation or different conditions of determination of activity.

As a characteristic constant of enzyme, the Michaelis constant (Km) can approximately reflect affinity between an enzyme and substrate. In this study, as long as concentrations of the substrate were in a certain range (less than 6 mmol/L), the relationship between velocities and concentrations of the substrate can be represented by the Michaelis-Menten equation. Kmof AChE from tilapia brain tissue was 0.183 mmol/L, while Kmof AChE fromNebia albifloramuscle was 0.10 mmol/L (Shiet al., 1981),Scomberomorus niphoniusbrain tissue was 0.311 mmol/L(Zhuet al., 2006), andPseudosciaena croceamuscle was 0.125 mmol/L (Dong, 1995). It seemed that there was no significant difference in Kmof the AChE despite the environment variation (sea water/fresh water) and tissue difference (muscle/brain).

In this experiment the IC50values of four pesticides on AChE were less than 5 μg/mL, showing their strong inhibition. This result was very similar to that of carp (Liuet al.,2007), but different from that of housefly (Weiet al., 2009)andChironomus kiiensis(Liuet al., 2008). The variation may be due to different binding forces of AChE from various species with the same pesticide. After a complete purification procedure as detailed in this paper, 0.5 mg purified AChE can be obtained from 10-g brain tissues of tilapia.Thus, compared with the scarce and expensive torpedoes(Torpediniformes) as well as electric eels (Electrophorus electricus), brain tissue of tilapia could be a good enzyme source for pesticide monitoring in water environment.

Acknowledgment:

This project is supported by Huizhou Science and Technology Planning Project Foundation (Grant No. 2008P60).

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*Correspondence to: Associate Professor, YunHua Ding, Huizhou University, No.46, Yanda Road, Huizhou, Guangdong 516007, China. Tel: +86-752-2529077; Email: dingyunhua@hzu.edu.cn

27 January 2011 Accepted: 10 May 2011