May 7, 2013 - immobilized urease, rainbow trout and bromelain, while it is within the .... crude stem bromelain improves it sensitivity as a protease inhibi-.
© by PSP Volume 22 – No 12. 2013
Fresenius Environmental Bulletin
ASSAY FOR HEAVY METALS USING AN INHIBITIVE ASSAY BASED ON THE ACETYLCHOLINESTERASE FROM PANGASIUS HYPOPHTHALMUS (SAUVAGE, 1878) Mohd S. Aidil1, Mohd K. Sabullah2, Mohd I.E. Halmi1, Rosni Sulaiman2, Mohd S. Shukor3, Mohd Y. Shukor2, Noor A. Shaharuddin2, Mohd A. Syed2 and Amir Syahir2,* 1
School of Food Science & Nutrition, Universiti Malaysia Sabah, Malaysia Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, UPM 43400 Serdang, Selangor, Malaysia 3 Snoc International Sdn.Bhd, 1-2B Jalan Hentian 1B, Hentian Kajang, Jalan Reko, 43600 Kajang, Selangor, Malaysia 2
ABSTRACT Acetylcholinesterase (AChE) is generally used as an inhibitive assay for insecticides. A lesser-known property of AChE is its inhibition by heavy metals. In this work, we evaluate an AChE from silver catfish brain (Pangasius sp.), wasted from aquaculture industry, as an inhibitive assay for heavy metals. We discovered that the AChE was completely inhibited by Cr6+, Ag2+, Cd2+, Hg2+, Cu2+, Zn2+ and Pb2+ during an initial screening. When testing at various concentrations, the above heavy metals exhibited exponential decay type inhibition curves. The calculated IC50 for Hg2+, Cu2+, Ag2+, Cr6+, Cd2+, Pb2+, and Zn2+ were 0.071, 0.088, 0.088, 0.87, 0.913, 1.1, and 1.572 mg/L, respectively. The Limit of Detection (LOD) and Limit of Quantification (LOQ) values of the heavy metals are within the range of the Maximum Permissibility Limit (MPL) allowed by the Malaysian Department of Environment (DOE) for Class-III rivers. The IC50 values for these heavy metals are comparable, and some are lower than those of the currently known assays, such as immobilised urease, Daphnia magna, MicrotoxTM, rainbow trout, papain and bromelain assays. Field trial works on an industrial drainage site had shown that the developed assay is applicable in detecting heavy metals in the water. The assay can be carried out in less than 30 min under ambient temperature.
KEYWORDS: Pangasius sp., acetylcholinesterase (AChE), heavy metals, inhibitive assay
1. INTRODUCTION Heavy metal levels in developing countries like Malaysia with an agricultural agenda have become a problem. * Corresponding author
The health problems caused by chronic effects of heavy metals has cost millions of Ringgit to the country in healthcare and monitoring of rivers. There are 180 river basins in Malaysia, and about between 5 to 10% has been reported to be polluted with inorganics and organics [1]. Therefore, their monitoring is important. Currently, the annual levels of heavy metals in these polluted rivers are not being monitored due to high costs. One way to reduce the cost of monitoring is to use biomonitoring. The USEPA has recognized microbial- and ezyme-based methods to biomonitor toxicants [2]. Microbial-based methods, such as Microtox and Polytox, are not amenable for field trial works, as they require bulky incubators. Enzymes, such as proteases [3-5] and urease [6], could be used but they are timeconsuming as each measurement takes more than 1 h to complete. Fish have been identified to become specialized in responding to pollutants, and it has been utilized for assaying varieties of toxicants [7, 8]. Silver catfish (Pangasius sp.), locally known as ‘Patin’, is reared at a large scale for its flesh as fillet (dori) in Malaysia. Unwanted parts of the fish, such as the skin, visceral and head portion, are often thrown away [9]. Fish brain contains AChE, a source of enzymes often used for in vitro insecticide assays. Current sources of enzymes for pesticide bioassay and biosensor technology are from the fruit fly Drosophila melanogaster and the electric eel Electophorus electricus [10]. Currently, these sources are very expensive reaching several hundreds Malaysian Ringgit per mg. AChE is generally used for biomonitoring of insecticides, and is also useful to detect the presence of heavy metals [11-13]. The rapidity and simplicity of the AChE assay makes it appealing to be used in the field coupled with a portable spectrophotometer. In this work, we discovered that AChE from Pangasius sp. is sensitive to the heavy metals such as mercury, copper, silver and chromium, and we used this enzyme to detect heavy metals from several aquatic bodies in the Juru river basins.
3572
© by PSP Volume 22 – No 12. 2013
Fresenius Environmental Bulletin
gression analysis software available on the internet (www.graphpad.com).
2. MATERIALS AND METHODS 2.1 Chemicals
Heavy metals, such as mercury (Hg2+), molybdenum (Mo6+), cadmium (Cd2+), lead (Pb2+), copper (Cu2+), zinc (Zn2+), chromium (Cr6+) and silver (Ag2+), were prepared from atomic absorption spectrometry standard solutions of MERCK company (Merck, Darmstadt, Germany). Working solutions at concentrations of 10, 5, 2.5, 1.0 and 0.5 mg/L were prepared by diluting them in deionized water, and stored in acid-washed polypropylene containers. These solutions were prepared fresh daily. Acetylthiocholine iodide, -mercapto-ethanol and 5’-dithio-bis (2-nitrobenzoic acid) (DTNB) were purchased from Sigma-Aldrich. 2.2 Preparation of affinity purified AChE
Pangasius sutchi, with an average weight of 750 g and about 25 cm in length, were sourced from a local patin aquaculture farmer in Dengkil, Selangor, in 2012. The whole brain has been dissected and 10 g of brain was homogenized (Ultra-Turrax T25 homogenizer) in 20% (w/v) of 100 mM sodium phosphate buffer at pH 8.0. Unbroken tissues were removed by centrifugation at 15,000×g for 10 min at 4 °C, followed by ultracentrifugation of the supernatant at 100,000×g (Sorvall) for one h at 4 °C. The supernatant was subjected to affinity purification. Procainamide affinity chromatography was used to partially purify the AChE [14]. The matrix, packed in a glass column (1.6 cm x 20 cm) (Amersham) to a bed height of 10 cm, was washed with 400 ml of buffer A (20 mM sodium phosphate buffer, pH 8.0). This procedure was to clean and equilibrate the column. About 10 mg of crude extract was loaded onto the affinity matrix and washed with 500 ml of buffer A with a flow-rate of 1 ml/min. A linear gradient of 1 M NaCl in buffer A was used to elute AChE using a total volume of 100 ml. Fractions of 1 ml were then collected and assayed for activity and protein. Fractions exhibiting high AChE activity were then pooled and dialyzed in 2 L of buffer A at 4 °C overnight. The dialyzed fraction was then concentrated (Viva Spin) and stored at -20 °C until subsequent use.
2.4 Field trials
Water samples were obtained from Prai river basins within the Prai industrial estate, pristine area of Sungai Udang recreational jungle, and treated with tap water in the university for comparisons. Water samples were immediately assayed for the presence of bioavailable heavy metals using the inhibitive assay as described above with a portable spectrophotometer (Axiom, Germany). The determination of heavy metal contents in our samples was measured using atomic emission spectrometry (ICP-OES, Optima 3700DV, Perkin-Elmer, USA). All experiments were performed in triplicate. 2.5 Data and statistical analysis
The percent inhibition was calculated according to the following formula: % Inhibition = Activity of control Activity of sample × 100 Activity of control
The values shown are means ± standard deviation. Graphpad Prism version 3.0 was used to analyze all data. Comparison between groups was obtained by one-way analysis of variance (ANOVA) with post hoc analysis by Tukey’s test, or by using a student's t-test. P250 1.7-3.0 3.6 8 2.16±0.53 n.d. Pb2+ 1.572 14.6±3.2 0.27-29 0.54-5.1 0.55-2.2 n.d. 2.11 0.56 Zn2+ The time used for MicrotoxTM, Daphnia Magna, and Rainbow trout assay were 15 min, 48 h, and 96 h, respectively. a Jung et al., 1995 [6], b Hsieh et al 2004 [16], c Shukor et al., 2006 [3], d Shukor et al., 2008 [4], and n.d. is not detected. Metals
This work
tion curves. The calculated IC50 values for the heavy metals are 0.071, 0.088, 0.088, 0.87, 0.913, 1.1, and 1.572 mg/L, in the order of Hg2+ < Cu2+, Ag2+ < Cr6+ < Cd2+ < Pb2+ < Zn2+, respectively, as shown in Table 1. We concluded that the lowest concentration of a heavy metal revealing a distinguished signal before it becomes saturated is a limit of detection (LOD). LOD of a system is also typically defined as three times the standard deviation of a reference sample. The limit of quantification (LOQ) is the concentration level, which can determine the detection signal with acceptable precision (robust standard deviation in the range of 10 to 25%) and accuracy (usually 80 to 120% recovery). It is normally defined as ten times the standard deviation of a reference sample [17]. The LOD and LOQ values are given in Table 1. The targeted sensitivity of the developed assay is the Malaysian Department of Environment standard for the MPL of heavy metals, especially for water in Class-III (water supply requiring extensive treatment). The MPL level exceeding Class-III will be considered as polluted and unfit for irrigation and livestock drinking [1]. Samples that gave 10% inhibition of AChE activity in theory would contain these metal ions above the MPL level as outlined by the Malaysian Department of Environment [1]. Repeated measurements of the assay suggested that the assay was reproducible with a coefficient of variation (CV) of the replicated data ranging from 6.4 to 8.3%. Table 1 shows the comparative LC50 (lethal concentration that causes 50% toxicity), LD50 (lethal dose that
causes 50% toxicity), EC50 (effective concentration that causes 50% response), and IC50 (concentration that causes 50% inhibition) data for the metals; presented with 95% confidence intervals (CI) for different toxicity investigations. Comparison of fish AChE assay made with immobilized urease, Microtox™, daphnid (Daphnia magna), fish (rainbow trout), papain, and bromelain toxicity data are in the same table. Schenker and Gentleman [17] demonstrated that non-overlap of confidence interval usually signifies significant difference at p
