LIST OF TABLES
Table 1………………………….. Summary of reagents used.
Table 2………………………….. Result for crude enzyme activity
Table 3………………………….. Purification Table
Table 4…………………………..Result for enzyme activity without heating after purification
Table 5…………………………..Protein content absorbance tableafter purification
Table 6 ………………………….. Enzyme activity with varying temperature after purification.
LIST OF FIGURES
Figure 1…………………………… Antioxidant enzyme schematic
TABLE OF CONTENT
TITLE PAGE………………………………………………………………….i
CERTIFICATION……………………………………………………………..ii
DEDICATION…………………………………………………………………iii
ACKNOWLEGDEMENT……………………………………………………..iv
LIST OF TABLES……………………………………………………………..v
LIST OF FIGURES…………………………………………………………….vi
TABLE OF CONTENT…………………………………………………………vii
ABSTRACT…………………………………………………………………….x
CHAPTER 1
- INTRODUCTION…………………………………………………………….1
- THE AFRICAN CATFISH……………………………………………………4
- ENZYMES AND THE GENERAL EFFECTS OF TEMPERATURE ….6
- STATEMENT OF PROBLEM ……………………………………………..7
- RESARCH OBJECTIVES ………………………………………..……..7
- SCOPE OF STUDY …………………………………..…………………8
- SIGNIFICANCE OF THE STUDY ……………………..……………….8
CHAPTER 2
LITERATURE REVIEW …………………………………………….………….10
2.1 CELLULAR REDOX ENVIRONMENT ………………………………..10
2.2 THE GLUTATHIONE PEROXIDASE (GPX) ANTIOXIDANT SYSTEM………………………………………….……………………………13
2.3 GLUTATHIONE PEROXIDASE (GPX) AND FISH ……………………14
2.4 THE EFFECT OF TEMPERATURE ON GLUTATHIONE PEROXIDASE (GPX) ACTIVITY IN FISH…………………………………17
CHAPTER 3
3.1 MATERIALS……………………………………………………………..21
3.1.1 REAGENTS/ REAGENT PREPARATIONS………………………….21
3.1.1a REAGENTS …………………………………………………………..21
3.1.1b REAGENT PREPARATION ………………………………………….22
3.1.2 APPARATUS AND EQUIPMENTS …………………………………….24
3.2 METHODS …………………………………………………………….26
3.2.1 PROTEIN DETERMINATION ………………………………………26
3.2.2 SAMPLE COLLECTION ……………………………………………..27
3.2.3 SAMPLE PREPARATION/ ENZYME EXTRACTION ……………..27
3.2.4 ENZYME ASSAY ……………………………………………………27
3.2.5 ENZYME PURIFICATION STEPS ………………………………….28
3.2.5a FIRST PURIFICATION STEPS ……………………………………..28
3.2.5b SECOND PURIFICATION STEP ……………………………………29
3.3 TEMPERATURE VARYING OF ENZYME ACTIVITIES …………32
CHAPTER 4
4.1 ENZYME ACTIVITY OF CRUDE …………………………………..34
4.2 SIZE EXCLUSION CHROMATOGRAPHY ………………………..35
4.3 ION EXCHANGE CHROMATOGRAPHY ……………………………..36
4.4 EFFECT OF TEMPRATURE ON THE PURIFIED EXTRACT ………..38
CHAPTER 5
5.1 CONCLUSION ……………………………………………………..…….41
5.2 RECOMMENDATION …………………………………………………..42
REFERENCE ………………………………………………………………..43
APPENDIX I ………………………………………………………………….51
APPENDIX II …………………………………………………………………53
APPENDIX III ……………………………………………………………….55
APPENDIX IV ………………………………………………..……………..57
APPENDIX V …………………………………………..……………………58
ABSTRACT
Glutathione Peroxidase (GPx) family of enzymes plays important roles in the protection of organisms from oxidative damage. GPx converts reduced glutathione (GSH) to oxidized glutathione (GSSG) while reducing lipid hydroperoxides to their corresponding alcohols or free hydrogen peroxide to water. This study was conducted to investigate the possible effects of temperature in vivo on Glutathione peroxidase (GPX) oxidative activity in fish tissues.An adult fully grown cat fish, (the African flathead catfish, Clariasgariepinus) about 0.7m in length and 0.15 in width, weighing 15Kg was obtained from the school pond. The fish was humanely killed and its liver was cut off from the rest of the body. Glutathione peroxidase was extracted and purified from the liver of the fish using the purification method of Ezeet al(2010).The extracted and purified GPX was then divided into 23 test tubes and labeled. Each test tube was heated at different temperatures ranging from -30°C to 50°C, after which Glutathione and Hydrogen Peroxide substrates were introduced to each test tube.Results showed that measurements were higher in test tubes were heating temperature were either exactly at or close to the acclimation temperature of the African Flathead Catfish.
CHAPTER ONE
INTRODUCTION
- INTRODUCTION
1.1.1 FISH: SOURCE OF FOOD FOR MAN.
Food is one of the basic needs of man (Morey, 1940; Pierce, 2010). Since agriculture produces the food that provides the calories and micronutrients essential for a healthy and productive life, it is interlinked in many important ways to human nutrition and health (Michael, 2011). These nutrients include carbohydrates, proteins, fats and oil, minerals, vitamins and water. Of these nutrients, it is the proteins that supply the body with amino acids necessary for growth and repair of damaged tissues. The sources of protein include plants and animals. Animal sources are preferred because of the presence of essential amino acids and higher digestibility. However, the major disadvantage is higher cost. The animal sources include fish, poultry, dairy, pork, snail, and rabbit.
Fish accounts for one fifth of world total supply of animal protein (FAO, 1991; Olagunjuet al., 2007). It is an important source of protein to a large number of Nigerians. It provides 40% of the dietary intake of animal protein of the average Nigerian (FDF, 1997; Sogbesanet al., 2006). According to Adekoya and Miller (2004), fish and fish products constitute more than 60% of the total protein intake in adults especially in rural areas.
Amiengheme (2005) enumerated the importance of fish in Human Nutrition as follows:
- Fish food has a nutrient profile superior to all terrestrial meats (beef, pork and chicken) being an excellent source of high quality animal protein and highly digestible energy;
- Fish is a good source of sulphur and essential amino acids such as lysine, leucine, valine and arginine. It is therefore suitable for supplementing diets of high carbohydrate contents;
- It is an extremely rich source of polysaturated (Omega III) fatty acids, which are important in lowering blood cholesterol level and high blood pressure, reducing the risk of sudden death from heart attacks and reduces rheumatoid arthritis, lowering the risk of age- related muscular degeneration and vision impairment; decreasing the risk of bowel cancer and reducing insulin resistance in skeletal muscles.
- Fish is also a good source of thiamine fat soluble vitamins (A, D and E), water soluble vitamins (B complex) and minerals (calcium, phosphorus, iron, iodine and selenium);
In Nigeria, fisheries occupy a unique position in the agricultural sector of the economy (Kudi et al., 2008). Its contribution to Gross Domestic Product (GDP) rose from 76.76 billion in 2001 to N162.61 billion in 2005 (CBN Report, 2005). Nigerians are large consumers of fish, with an annual average demand estimate at 1.4 million metric tonnes, (Kudiet al., 2008). Domestic fish production of about 0.5 million metric tonnes is supplied by artisan fishermen (85%), and fish farmers (15%) (Adekoya and Miller, 2004; Emokaro, 2010; BusinessDay, 2011). However a demand and supply gap of at least 0.7 million metric tonnes exists nationally with import making up the short fall at a cost of 400 billion United States dollars per year. According to FAO (2007), this figure (0.7 million metric tonnes) makes Nigeria the largest importer of fish in the developing world.
To take advantage of the large market created by this deficit, the immediate past and current Governments has promoted a backward integration plan for increased fish farming and production in Nigeria, Nigerians are also complementing Government efforts by increasing their participation in aquaculture, with many fish farmers focusing on African flathead catfish, Clariasgariepinus as they have been shown to have a potential market value of two to three times that of other cultivable species like Tilapia and Heterobranchus (FAO, 2000; Fafioye and Oluajo, 2005; Emokaroet al., 2010; Businessday, 2011).
A survey by Addo (2005) revealed that Nigerian children below the age of 18 years, who make up about 47% of our total population are still victims of stunting, wasting and under-weight, so with the increased establishment of more aquaculture in Nigeria, it is possible to reverse this trend of malnourishment among Nigerians in this age bracket.
- THE AFRICAN CATFISH
The African catfish – Clariasgariepinus, an omnivorous freshwater fish, is a popular delicacy in Nigeria. It is a prominent culture species because of its fast growth rate and resistance to diseases and stress factors like over-stocking and poor water quality (Olojoet al., 2005). It is distributed mainly in fresh waters of Africa hence the name African catfish, although it is also seen in Asia. It is named ‘catfish’ because they possess prominent barbels which resemble cat’s whiskers. It has a slender body, flat bony head and broad terminal mouth with four barbels. The pectoral fins have spines. Its dendritic organ is an accessory breathing organ and it is a modification of the gill arches (Ahmed et al., 2008). The adult of about 1.5m weighs up to 29kg (Teugel, 1986).
Habits: They are nocturnal omnivorous animals feeding on living as well as dead organic matter. They are capable of swallowing large prey because of large mouth (Teugel, 1986).They can crawl on the ground to escape drying pools but can also survive in shallow mud for long periods of time between rainy and dry seasons. They can produce croaking sound. They spawn mostly at night in the shallow areas of rivers, lakes and streams. Development of the larva is rapid as they are able to hatch 24 hours and swim within 48 – 72 hours after fertilization.
1.3 ENZYMES AND THE GENERAL EFFECTS OF TEMPERATURE
The living cell is the site of tremendous biochemical activity. Catalysis, through enzymes, makes possible biochemical reactions, which are necessary for all life processes. Enzymes are responsible for bringing about almost all of the chemical reactions in living organisms. Without enzymes, these reactions take place at a rate far too slow for the pace of metabolism.
Like most chemical reactions, the rate of an enzyme-catalyzed reaction is affected by temperature. Variations in reaction temperature as small as 1 or 2 degrees may introduce changes of 10 to 20% in the results. In the case of enzymatic reactions, this is complicated by the fact that many enzymes are adversely affected by high temperatures.
The enzyme Glutathione Peroxidase which catalyses the reduction of hydrogen peroxides is the main focus of this study. Understanding the effects of temperature on the activity of this enzyme in the catfish, could help in determining the best storage temperature for this fish such that harvested catfish can be kept fresh for a longwhile for consumption after
1.4. STATEMENT OF PROBLEM
Despite the clear increase in interest for aquaculture and fish consumption in Nigeria, a major problem of storage exists. This is clearly observed in the lack of fresh iced or well dried catfish options in the market. It is also manifest in the ‘point and kill’ marketing phenomenon, which has invariably restricted catfish farming to retail/subsistence levels intended for consumption by members of the immediate locale were the farm is sited, and discourages large scale catfish farming intended for consumers across state and even national borders.
1.5. RESARCH OBJECTIVES
The aim of this study is to isolate the enzyme Glutathione Peroxidase (GPx) from the liver of the African catfish (Clariasgariepinus) and carryout investigations in vitro to determine
- The optimum temperature range wherein the enzyme Glutathione peroxidase best catalyses the reduction of hydroperoxides and other free radicals, thereby protecting the tissues of the Flat Head Catfish from oxidative stress and damage.
- The temperature range wherein rate of the catalytic reduction activity of the enzyme Glutathione peroxidase is lowest.
1.6. SCOPE OF STUDY
This study will be carried on only the African Catfish (Clariasgariepinus), as survey has shown that it is the by far the catfish variety most consumed by inahbitants of Eastern Nigeria.
1.7. SIGNIFICANCE OF THE STUDY
The importances of this study are as follows:
- Results obtained at the end will accurately advise the best storage temperature for the Flat Head Catfish that will ensure optimum protection of its muscles and other edible tissues from oxidative stress and damage,
- Thereby increasing its shelf life,
- Encourage farmers to engage in larger cultivation of this popular species of fish for wider consumption,
- This in turn reduces production and processing costs; and wastage and loss due to deterioration and decay,
- Ensure that fish which is an important but currently expensive source of protein is made cheaper and consumed more frequently by citizens of all income and age brackets;
- And finally help reduce the malnutrition problem earlier mentioned in the last.
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