MOHD KHALIZAN BIN SABULLAHpsasir.upm.edu.my/26464/1/FBSB 2011 46R.pdfexamination of Mohd Khalizan bin Sabullah on his Master of Science thesis entitled “Acetylcholinesterase from

UNIVERSITI PUTRA MALAYSIA

ACETYLCHOLINESTERASE FROM Osteochilus hasselti FOR THE

DETECTION OF INSECTICIDES AND HEAVY METALS

MOHD KHALIZAN BIN SABULLAH

FBSB 2011 46

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By


Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Partial Fulfilment of the Requirements for the Degree of Master of Science

August 2011

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Dedicated to my family and friends.

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment

of the requirements for the degree of Master of Science



By


August 2011

Chairman: Assoc. Prof. Mohd. Yunus Bin Abdul Shukor, PhD

Faculty : Biotechnology and Biomolecular Sciences

The use of acetylcholinesterase to detect insecticides such as carbamate and

organophosphate is well known. However, new sources of AChE are constantly being

sought since the most crucial preparation of AChE rarely is sensitive to all

insecticides. At the same time, literature data shows that certain AChEs are sensitive

to heavy metals and by searching new sources, AChE can be applied for the detection

of heavy metals as well. In this work, an inhibitive assay for insecticide and heavy

metals was developed using partially purified fraction of AChE from Osteochilus

hasselti using Procainamid Sephacryl S-1000 with a purification fold of 8.1 and a

recovery yield of 55.3% were obtained. The partially purified fraction showed optimal

activity at pH 7.0 and optimal temperature in the range of 25oC to 30

oC.

Acetylthiocholine iodide (ATC) was chosen as specific substrate with higher maximal

activity (Vmax) of 742.43 µmole/min and the lowest Michaelis Constant (Km) at

0.0086 mM followed by Propionylthiocholine Iodide (PTC) and Butyrylthiocholine

Iodide (BTC). All of the carbamate groups such as carbaryl, bendiocarb, carbofuran,

methomyl and propoxur, and several oxonated OPs such as chlorpyrifos, diazinon,

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malathion and parathion showed significant inhibition more than 50% of AChE

activity. Partially purified AChE was not inhibited by tricholrfon, dimethoate and

acephate. ANOVA analysis showed that all of the inhibition seen was significant

compared to control (p<0.05) in the absence of insecticide. The carbamates; carbaryl,

and the oxonated OPs; diazinon showed significantly lower IC50s in O. hasselti

compared to E. electricus while parathion and diazinon showed overlapped IC50s.

Chromium, copper and mercury inhibited 80%, 61.3% and 35% of AChE activity,

respectively. The heavy metals exhibited exponential decay type inhibition curves

with calculated IC50 for copper, and chromium at 0.198 and 0.076 mg l-1

, respectively.

The effect of sample matrix in detecting insecticides using tap water, sea water and

waters from two pristine rivers samples spiked with carbamate showed no significant

differences amongst each other (p>0.05) indicating no matrix effect. Among all the

vegetable samples studied, chili shows the presence of insecticide and validated by

HPLC analysis. Sungai Juru exhibited higher inhibition proved by ICP-OES high with

amount of chromium and copper while other river samples showed low inhibition

except for Endau Rompin national water park sample. It can be summarized that O.

hasselti AChE is a new alternative source as biosensor to detect contaminant because

the sensitivity of this enzyme is almost similar compare to other commercialize

AChE. For further studies, it is recommended that full purification should be carried

out to understand the fundamental differences between this AChE and other AChEs to

further understand the mechanism of heavy metals and insecticides inhibition.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk Ijazah Master Sains

ASETILKOLINESTERES DARIPADA Osteochilus hasselti UNTUK

MENGESAN RACUN SERANGGA DAN LOGAM BERAT

OLEH


Ogos 2011

Pengerusi: Prof. Madya Mohd. Yunus Bin Abdul Shukor, PhD

Fakulti : Biotechnologi and Sains Biomolekul

Penggunaan asetilkolinesteres untuk mengesan karbamat dan organofosfat telah

diketahui ramai. Walaubagaimanapun, sumber AChE terbaru masih dikaji kerana

penyediaan penting AChE adalah amat jarang sekali dapat mengesan kesemua jenis

racun serangga. Pada masa yang sama, data literatur menunjukkan AChE tertentu

adalah sensitif dalam mengesan logam berat dan dengan mencari sumber terbaru,

AChE juga boleh diaplikasikan sebagai pengesan logam berat. Dalam kajian ini, satu

asai perencatan untuk racun serangga dan logam berat telah pun dibangunkan

menggunakan fraksi penulenan separa AChE dari Osteochilus hasselti menggunakan

Prosainamida Sephacryl S-1000 dengan penulenan sebanyak 8.1 kali ganda dan hasil

perolehan sebanyak 55.3% telah pun diperoleh. Fraksi separa tulen menunjukkan

aktiviti AChE adalah optimum pada pH 7 dan suhu optima pada jarak 25oC hingga

30oC. Asetiltiokolin Iodida (ATC) telah dipilih sebagai substrat spesifik dengan

kelajuan maksima tertinggi (Vmax) iaitu 742.43 µmole/min dan pekali Michaelis (Km)

terendah iaitu 0.0086mM diikuti Propioniltiokolin Iodida (PTC) dan Butiriltiokolin

Iodida (BTC). Kesemua kumpulan karbamat seperti karbaril, bendiokarb, karbofuran,

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methomil dan propoxur, dan beberapa organofosfat oxon seperti klorpirifos, diazinon,

malathion and parathion menunjukkan perencatan ketara melebihi 50% pada aktiviti

AChE. AChE separa tulen tidak direncatkan oleh triklorfon, dimethoat dan asefat.

Analisis ANOVA menunjukkan kesemua perencatan adalah ketara berbanding dengan

kawalan (p<0.05) tanpa kehadiran racun serangga. Karbamat; carbaryl dan OP oxon;

diazinon menunjukkan IC50 rendah yang ketara dalam O. hasselti berbanding dengan

E. electricus manakala parathion dan diazinon menunjukkan pertindihan IC50.

Kromium, kuprum dan merkuri telah merencat 80%, 61.3% dan 35% aktiviti AChE,

masing-masing. Apabila diuji pada kepekatan yang berbeza, logam berat

menunjukkan lekuk kemerosotan eksponan dengan pengiraan IC50 untuk kuprum dan

kromium pada 0.198 dan 0.076 mg l-1

, masing-masing. Kesan sampel matrik untuk

mengesan racun serangga menggunakan air paip, laut dan sungai tidak tercemar yang

telah ditambah bersama karbamat menunjukan masing-masing tiada perbezaan ketara

(p>0.05) yang menunjukan tiada kesan matrik. Daripada kajian sampel sayur-sayuran,

cili menunjukan kehadiran racun serangga dan dibuktikan melalui analisis HPLC.

Sungai Juru menunjukkan perencatan tinggi dimana menunjukkan kepekatan yang

tinggi bagi kromium dan kuprum dibuktikan melalui ICP-OES sementara sampel

sungai lain menunjukkan perencatan yang rendah kecuali sampel dari Taman Negara

Endau Rompin. Ini boleh disimpulkan bahawa AChE O. hasselti merupakan alternatif

terbaru pengesan biologi untuk pencemaran kerana kesensitifan enzim ini hampir

sama berbanding AChE komersil yang lain. Bagi kajian akan datang, adalah

disyorkan untuk menjalankan penulenan penuh untuk memahami perbezaan prinsip

antara AChE ini dan AChE yang lain bagi kefahaman lebih lanjut tentang mekanisma

perencatan logam berat dan racun serangga.

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ACKNOWLEDGEMENT

I would like to send my grateful to my supervisor, Dr. Mohd. Yunus bin

Abdul Shukor for his invaluable attention, guidance and constructive suggestions

regarding to this project. I also take this opportunity to express my appreciation to

Prof. Dr Mohd Arif Syed for his advice and encouragement, and all lecturers who are

gave a lot of information also for helping me in this project. Special thanks to all the

PhD student, Master student, academics staff and laboratory assistance at the

Department of Biochemistry for their cooperation.

To all my members in enzymology, bioremediation and toxicology lab, I like

to give my humble gratitude for their suggestion and support for this project. Not

forget to Prof. Dr. Nor Aripin Shamaan for his advises, comment, sharing experiences

and knowledge to me.

Lastly, my uncountable appreciation to my beloved parent and brother, Mohd Khalid

bin Sabullah for their help, support, sharing their experience to gave a lot of

information, encourage, advices and the importance is believing in me be a good and

special man. For those who are the name that I have not mentioned, your support will

be remembered and may The God bless you all.

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I certify that an Examination Committee met on 3 August 2011 to conduct the final

examination of Mohd Khalizan bin Sabullah on his Master of Science thesis entitled

“Acetylcholinesterase from Osteochilus hasselti for detection of insecticides and

heavy metals” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act

1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The

committee recommends that the candidate be awarded the relevant degree. Members

of the Examination Committee are as follows:

Dr. Nor’Aini Abdul Rahman, PhD

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia,

(Chairman)

Dr. Suraini Abd. Aziz, PhD

Professor


Universiti Putra Malaysia,

(Internal examiner)

Dr. Umi Kalsom Md Shah, PhD

Associate Professor


Universiti Putra Malaysia

(Internal examiner)

Dr. Bechan Sharma, PhD

Professor

Department of Biochemistry,

University of Allahabad,

Allahabad,

India.

(External Examiner)

_________________________________

NORITAH OMAR

Assoc. Professor/Deputy Dean

School of Graduate Studies


Date :

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This thesis was submitted to the Senate of Universiti Putra Malaysia has been

accepted as fulfilment of the requirements for the degree of Master of Science. The

members of the Supervisory Committee were as follows:

Mohd Yunus Abdul Shukor, PhD

Associate Professor



(Chairman)

Mohd Arif Syed, PhD

Professor



(Member)

________________________________

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies


Date :

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DECLARATION

I declare that the thesis is my original work except for quotation and citations which

have been duly acknowledged. I also declare that it has not been previously, and is not

concurrently, submitted for any other degree at Universiti Putra Malaysia or at other

institutions.

_______________________________


Date: 3 August 2011

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LIST OF TABLES

Table

Page

1 The of polluted river in Malaysia

21

2 The MRL of insecticides in agriproduce

23

3 List of chemicals 29

4 List of equipment 31

5 The purification of various procedure of AChE from Osteochilus

hasselti

51

6 Comparison between work by Tham et al., (2009) with this study 51

7 The comparison of Vmax and Km for ATC, BTC and PTC

56

8 Comparisons of the sensitivity of O. Hasselti AChE to various

insecticides with AChEs from E. electricus

66

9 Comparison of the assay to MicrotoxTM

, Daphnia magna, fish,

papain and bromelain assays

71

10 Summry of insecticides studies with limit of detection (LOD) and

limit of quantitation (LOQ) value

74

11 Percentage activity of AChE remain after incubated with different

of water samples

81

12a Sungai Juru water sample (SJ1) has been tested with Inductive

coupled plasma (ICP-OES) and indicated numerous of metal ions

85

12b Sungai Melaka water sample (SM2) has been tested with Inductive

coupled plasma (ICP-OES) and indicated numerous of metal ions

85

12c The water sample from protected area Sungai Pinji, Perak (SP2)

has been tested with Inductive coupled plasma (ICP-OES) and

indicated numerous of metal ions

86

12d The water sample from protected area Endau-Rompin National

Park (ER3) has been tested with Inductive coupled plasma (ICP-

OES) and indicated numerous of metal ions

87

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TABLE OF FIGURES

Figure

Page

1 Acetylcholinesterase

4

2 Break down or hydrolysis reaction of AChE

5

3 The hydrolysis mechanism of acetylcholine towards enzyme

AChE

9

4 Distribution of toxicant after entry into biological system via

inhalation, ingestion and skin penetration

11

5 General structure of Carbamate

13

6 Thiophosphoryl group of OP insecticides undergoes oxidative

desulfuration to phosphoryl group (oxon)

15

7 Mechanism inhibition of Acetylcholinesterase by

Organophosphate.

16

8 Mechanism inhibition of acetylcholinesterase by carbamate

16

9 Summary of mechanism of acetylcholinesterase activity toward

ACh, carbamate and organophosphate

17

10 Osteochilus Hasselti, ikan terbul

29

11 Map of Malaysia showing location of sampling sites

46

12 Elution profile of partially purified AChE from C. batrachus on

custom synthesized Procainamide–Sephacryl S-1000

chromatography column with assayed of AChE total activity

(U/min) and protein content determination

50

13 Denaturing polyacrylamide gel electrophoresis (SDS-PAGE)

protein profile from brain tissue of Osteochilus hasselti in a 10%

polyacrylamide gel

52

14 The pH profile on the activity of partial purified AChE from

Osteochilus hasselti

53

15 The temperature profile on the activity of partial purified AChE

from Osteochilus hasselti

54

16 Three synthetic substrates with different concentrations

incubated with partial purified AChE to prove it specificity

55

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17 Effect of various insecticides on the enzymatic activity and

partially purified AChE from Osteochilus hasselti

56

18 Effect of various oxonated OP on the enzymatic activity


59

19 Effect of various metal ions on the enzymatic activity and


60

20a The activity of Osteochilus hasselti AChE has been screened at

different concentration of bendiocarp

63

20b The activity of Osteochilus hasselti AChE has been screened at

different concentration of carbaryl

63

20c The activity of Osteochilus hasselti AChE has been screened at

different concentration of carbofuran

64

20d Theactivity of Osteochilus hasselti AChE has been screened at

different concentration of methomyl

64

20e The activity of Osteochilus hasselti AChE has been screened at

different concentration of propoxur

65


different concentration of chlorpyrifos

68


different concentration of diazinon

68

21c The activity of Osteochilus hasselti AChE has been screened at

different concentration of malathion

69

21d The activity of Osteochilus hasselti AChE has been screened at

different concentration of parathion

69


different concentration of chromium

73


different concentration of copper

74

23a Partial purified AChE from Osteochilus hasselti has been

incubated in different water sources which has been spiked with

different concentration of carbofuran. DW, WF, R and S show

deionized water, water fall from Gunung Ledang Recreational

Jungle, river from Kuala Tanah National Park and sea from

76

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South China Sea at Chempedak Bay

23b Partial purified AChE from Osteochilus hasselti has been


different concentration of malathion. DW, WF, R and S show




76

23c Partial purified AChE from Osteochilus hasselti has been


different concentration of chromium. DW, WF, R and S show




77

24 Partial purified AChE from Osteochilus hasselti has been

incubated in different vegetable source from UPM

78

25 The ability of partial purified Osteochilus hasselti AChE has

been tested to detect anticholinesterase compound by incubated

with different selected rivers sample. SJ, SM, SK, SP and ER

are stand for Sungai Juru, Sungai Melaka, Sungai Kuyuh,

Sungai Pinji and Endau Rompin. The numbers show different

sampling location

80

26a Tap water was tested with High Performance Liquid

Chromatography (HPLC) as the control for the water samples

83

26b The water sample from Sungai Pinji (SP2) was tested with High

Performance Liquid Chromatography to detect the existence of

malathion at the wavelength of 210nm which the arrow pointed

to malathion was detected at retention time of 5.489 minute

indicated lowest peak

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LIST OF ABBREVIATIONS

Å Angstrom

≈ Almost equal to

°C degree Celsius

% percent

3D Three Dimensional

AChE Acetylcholinesterase

ACh Acetylcholine

ATC Acetylthiocholine Iodide

BuChE Butyrylcholinesterase

BTC Butyrylthiocholine Iodide

ChE Cholinesterase

ChO Choline Oxidase

CE Carbofuran equivalence

DDVP Dichlorvos

DTNB 5,5’-dithiobis(2-nitrobenzoic acid)

DTT dichlorodiphenyltrichloroethane

g gravity (Relative Centrifugal force)

HPLC-MS High Performance Liquid Chromatography Mass

Spectrophotometer

HCL Hydrochloric acid

IC50 fifty percent inhibition concentration

GC-MS Gas Chromatography Mass Spectrophotometer

kDa kilodalton

Km (app) Apparent Michaelis-Menten Constant

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M molar

mg miligram

mM milimolar

mg/l milligram per liter

µl microliter

nm nanometer

NaOH Sodium hydroxide

OP Organophosphate

OD optical density

pH -log concentration of H+ ion (Puissance hydrogene)

PTC Propionylthiocholine Iodide

PMSF phenylmethylsulfonylfluoride

PAGE polyacrylamide gel electrophoresis

PrChE Propionylcholinesterase

SEM Standard Error Mean

SDS sodium dodecyl sulphate

TEMED N,N,N’,N’-tetramethyl-ethylenediamine

Vmax (app) Apparent Maximum velocity

v/v volume/ volume

w/v weight/ volume

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TABLE OF CONTENTS

Page

ABSTRACT iii

ABSTRAK v

ACKNOWLEDGEMENT viii

APPROVAL ix

DECLARATION xi

LIST OF TABLES xii

LIST OF FIGURES xiii

ABBREVIATIONS xvi

CHAPTERS

1 INTRODUCTION 1

2 LITERATURE REVIEW 2.1 Acetylcholinesterase (AChE) 3

2.2 Acetylcholine and function of AChE 4

2.3 AChE structure 5

2.4 Active site of AChE 6

1.4.1 Anionic site 7

1.4.2 Esteric site 7

2.5 The hydrolysis mechanism 8

2.6 Cholinesterase (ChE) molecular weight 9

2.7 Water and xenobiotics pollution. 10

2.7.1 Xenobiotic and distribution in living system 10

2.8 Pesticides 11

2.8.1 Insecticides 12

2.8.1.1 Carbamate 13

2.8.1.2 Characteristic and categories of carbamates 14

2.8.1.3 Organophosphate 15

2.9 Heavy Metals and its pollution 17

2.10 Heavy metals pollution in Malaysia

21 2.10.1 Pesticides, heavy metals pollution and

contamination 22 in Malaysia

2.10.2 Modern detection methods for

insecticides and 23 heavy metals

2.11 Osteochilus hasselti

26

3 MATERIALS AND METHODS

3.1 Materials 28

3.1.1 Specimen 28

3.1.2 Chemical 29

3.1.3 Equipment 31

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3.2 Methods 32

3.2.1 Enzyme assay 32

3.2.2 Affinity chromatography 34

3.2.2.1 Procainamide–Sephacryl S-1000 affinity gel 34

3.2.2.2 Epoxy (Bisoxirane) activation 34

3.2.3 Preparation of crude homogenate 35

3.2.4 Partial purification using affinity chromatography 36

3.3 Electrophoresis 37

3.3.1 Denaturing PAGE (SDS-PAGE) 37

3.3.2 Non-denaturing PAGE (Native PAGE) 38

3.3.3 Staining process 39

3.4 Protein content determination 40

3.5 Calculation of purification fold and yield 40

3.6 Determination of enzyme parameter 41

3.6.1 pH profile 41

3.6.2 Temperature profile 41

3.6.3 Substrate specificity 42

3.7 Inhibition studies 43

3.7.1 Pesticides and heavy metals screening 43

3.7.2 Activation process for organophosphate 44

3.7.3 IC50 value determination 44

3.8 Trial works using real samples 44

3.8.1 Collection and treatment of water samples 44

3.8.2 Extraction of insecticides from vegetable samples 45

3.8.3 Xenobiotics determination in water and vegetables 46

sample.

3.9 Statistical analysis 47

4 RESULTS AND DISCUSSION

4.1 Purification of Acetylcholinesterase (AChE) 49

4.1.1 Partial purification of AChE from Osteochillus hasselti 49

4.2 AChE characterization 52

4.2.1 Effect of pH on AChE activity 53

4.2.2 Effect of temperature on AChE activity 54

4.2.3 Substrate specificity 55

4.3 Inhibition studies 57

4.3.1 The effect of AChE on selected insecticides 58

4.3.2 Secondary screening of organophosphate 58

4.4 IC50 value determination 61

4.4.1 IC50 value of selected carbamates 61

4.4.2 IC50 value of selected oxonated organophosphate 65

4.4.3 IC50 value of selected heavy metals 70

4.5 The use of AChE in the detection of xenobiotics compound 75

4.5.1 Spiked water samples 75

4.5.2 Assay of insecticides in vegetables sample 77

4.6 Assay of the toxicity of various water samples 78

5 CONCLUSION 87

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REFERENCES 88

APPENDICES 102

APPENDIX I 102

APPENDIX II

103

BIODATA OF STUDENT 105

Documents

MOHD KHALIZAN BIN SABULLAHpsasir.upm.edu.my/26464/1/FBSB 2011 46R.pdfexamination of Mohd Khalizan bin Sabullah on his Master of Science thesis entitled “Acetylcholinesterase from