A COMPARATIVE STUDY
TO DETERMINE IF
TURNIPS CAN BE USED AS
AN ALTERNATIVE SOURCE
OF PEROXIDASES TO
HORSERADISH
BY HENNA YAHYA
Coventry University
Priory St,
Coventry
CV1 5FB
1
,Henna Yahya 4837224
Contents
1. Introduction .......................................................................................................................................... 4
2. Methodology......................................................................................................................................... 8
2.1 Materials ............................................................................................................................... 8
2.2 Preparation of sodium phosphate buffer at pH 7 .................................................................. 8
2.3 Bradford Method .................................................................................................................. 8
2.3.1 Protein estimation using Bradford Method ....................................................................... 8
2.3.2 Determination of protein concentration of horseradish and turnip extracts ........ 8
2.4 Enzyme assay using ABTS as substrate ............................................................................... 9
2.5 Determination of horseradish peroxidase activity ................................................................ 9
2.6 Peroxidase extraction from vegetables ............................................................................... 10
2.7 Ammonium Sulphate Precipitation .................................................................................... 10
2.7.1 Preparation of ammonium sulphate pilot study ................................................. 10
2.7.2 Ammonium sulphate pilot study ....................................................................... 10
2.7.3 Ammonium sulphate precipitation of vegetable extract .................................... 11
2.8 Gel Filtration of turnip and horseradish ............................................................................. 12
2.9 Calculations for purification table ...................................................................................... 12
2.10 SDS-PAGE ....................................................................................................................... 13
2.11 Effect of temperature on peroxidases ............................................................................... 13
3. Results ................................................................................................................................................ 14
3.1 Determination of protein concentration .............................................................................. 14
3.2 Commercial horseradish peroxidase activity ...................................................................... 14
3.3 Turnip and horseradish peroxidase activity in crude extract .............................................. 15
3.4 Ammonium sulphate precipitation ..................................................................................... 16
3.4.1 Ammonium sulphate pilot study ....................................................................... 16
3.4.2 Ammonium sulphate precipitation of horseradish and turnip ........................... 19
3.5 Gel Filtration of turnip and horseradish ............................................................................. 19
3.6 Purification table ................................................................................................................ 20
3.7 SDS-PAGE ......................................................................................................................... 22
3.8 Effect of temperature on activity of peroxidase ................................................................. 24
4. Discussion .......................................................................................................................................... 26
4.1 Bradford Method ................................................................................................................ 26
4.2 Enzyme assay using ABTS as substrate ............................................................................. 26
4.3 Extraction of peroxidases from vegetable sources ............................................................. 27
4.4 Ammonium sulphate pilot study ........................................................................................ 27
4.5 Ammonium sulphate precipitation of vegetable extracts ................................................... 28
4.6 Gel filtration of vegetable extracts ..................................................................................... 28
4.7 Purification table ................................................................................................................ 29
4.8 SDS-PAGE ......................................................................................................................... 30
4.9 Effect of temperature on peroxidase activity ...................................................................... 31
4.10 Limitations and Improvements ......................................................................................... 31
4.11 Conclusion........................................................................................................................ 32
References .............................................................................................................................................. 34
FINAL WORD COUNT: 5434
2
,Henna Yahya 4837224
ACKNOWLEDGEMENTS
In the name of Allah, the most gracious, the most merciful
I would like to thank my project supervisor Dr Katrine Wallis for being a supportive
supervisor and always promptly responding to my numerous emails. I would also like
to thank the lab technicians Angela and Patrick for telling me where all the equipment
was in the lab
I especially want to thank Hafsah, Zoe and Ash for their continuous support and for
putting up with my constant moaning about horseradish and turnips. Without these
three I wouldn't have got through the data collection period.
3
, Henna Yahya 4837224
ABSTRACT
Horseradish is a recognised source of peroxidase and has many commercial uses and
research is being done on other sources of peroxidases. Peroxidase was extracted and
assessed from turnip and horseradish. Both were purified using ammonium sulphate
precipitation and gel filtration. Enzyme activity was measured using an artificial
substrate known as 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS).
Yield of horseradish peroxidase came out to be six times higher than turnip
peroxidase. Horseradish peroxidase was purified by 18.66 folds whereas turnip
peroxidase was purified by 5.40 folds. SDS-PAGE revealed the molecular mass of
turnip peroxidase to be 37 kDa. Thermal stability was carried out on turnip and
horseradish peroxidase in order to assess the activity of these peroxidases at different
temperatures.
1. INTRODUCTION
Peroxidases are heme-containing enzymes which are found in various plants and
microorganisms. Peroxidases catalyse many reactions in the presence of peroxides
such as hydrogen peroxide:
Some of these reactions include: oxidation of phenols and amines, detoxification of
soil and neutralisation of contaminated water (Hamid and Rehman 2009). Peroxidases
can be used in diagnostic kits for determining the quantity of uric acid, glucose or
cholesterol and can be simply used for detection of toxins, pathogens and potential
cancer causing materials in the bladder and prostate (Hamid and Rehman 2009). Some
4
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