ABSTRACT
This research is aimed at isolating, partially purifying and determination of arginase from the kidney of a local goat. Methods used in the isolation and partial purification of the enzyme include; Ammonium sulphate precipitation, dialysis.etc. Result from this work showed that arginase isolated from a local goat has a specific activity of 0.358 with a yield of 12.12% incubation of the enzyme with different metal ions shows that the enzyme is inhibited by Nacl and Bacl2.
CHAPTER ONE
1.0 INTRODUCTION AND LITERATURE REVIEW
1.1 INTRODUCTION
Arginase (L-arginine amidino hydrolase, EC 3.5.3.1.) is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to form L-ornithine and urea. Arginase is the terminal enzyme of the urea cycle. The function of arginase in microbes and invertebrates is mostly unknown. It is speculated that the urea cycle evolved from a biosynthetic pathway for L-arginine and appeared for the first time in amphibians as an adaptation to air-breathing in a terrestrial environment (Takiguchi et al., 1989). Many invertebrates are uricotelic organisms and eliminate excess nitrogen in the form of solid uric acid (Jenkinson et al., 1996). Nevertheless, a large number of uricotelic organisms possess arginase. It is thought that their generation of L-ornithine by arginase feeds into the production of L-proline, L-glutamate and polyamines used for collagen synthesis, energy metabolism and cell proliferation (Jenkinson et al., 1996).
In most mammals the enzyme was found to exist in two forms, has a broad tissue distribution and share ~ 60% sequence identity. The first, Arginase I, functions in the urea cycle, and is located primarily in the cytoplasm of the liver. The second isozyme, Arginase II functions primarily in L-arginine homeostasis in non-hepatic tissues (e.g., in regulating L-arginine bioavailability to nitric oxide synthase (NOS) (Jenkinson et al., 1996; Christianson et al., 1999; Morris, 2002).
Studies have shown that increased stimulation of arginase expression in animal systems leads to production of polyamines that promote tumor cell proliferation (Chang et al.,2001) and wound healing (Satriano,2003).
1.2 LITERATURE REVIEW
1.2.1 HISTORY OF ARGINASE
Arginase, the arginine hydrolytic enzyme, was first discovered by Kossel and Dakin in 1904 in the mammalian liver (Kossel and Dakin, 1904). Clementi (1916, 1918,1922) found arginase in the liver of mammals, amphibians, fishes and the turtle but not in the liver of the majority of reptiles. He found the enzyme to be present in the kidney of birds. Hunter and Dauphinee (1924) found a considerable amount of arginase in the liver o f mammals and fishes. They also established the presence of arginase in the kidney of mammals and fishes, but usually in an amount smaller than that present in the liver. In the organ of fishes other than the liver, heart and kidney, the distribution of arginase appear to be variable. In mammals, these workers did not find arginase in any organ except the liver and the kidney while in birds; they found the enzyme only in the kidney.
Edlbacher and Rothler (1925) made an extensive study of arginase content in the domestic fowl and in certain mammals. They found that was present in the liver, kidney and testis of both mammals and birds. Further, they discovered the presence of the enzyme in the placenta and thymus of some mammals. These authors were the first to find any arginase in the testis, and they also found arginase in the liver of birds which all the previous workers failed to detect. They established that in mammals, the organ richest in arginase is the liver, while in bird, it is the kidney. The relative quantity of these enzymes present per gram of body weight is far greater in mammals than in birds. They also demonstrated that the arginse value (which is the number of unit of arginase per gram of body weight) was higher in the male than in the females.
As regards the function of arginase, very little was known except that it hydrolyses arginine into ornithine and urea. Clementi (1916), who did not find any arginase in the liver of birds but found in the liver of mammals, amphibians and fishes enunciated the doctrine that arginase was present in the liver of all those animals which have what he called a “ureotelic” metabolism, that is in which urea is the final product of protein degradatition, and absent from the liver of those in which protein metabolism is “uricotelic”- ending in uric acid. The work of Edlbacher1925 as well as the result of the work of Chaudhuri 1927 did not show that arginase was totally absent from the liver of birds.
1.3 OCCURENCE AND DISTRIBUTION OF ARGINASE
Arginase is an enzyme that is ubiquitous in nature and has been identified and studied for several decades in a large number of organisms (Greengard, 1970; Reyero, 1975; Grody, 1987). Prokaryotes and lower eukaryotes that contain arginase use it for some purpose other than as urea cycle component (Huggins, 1969). Arginase has been characterized from various worms, molluscs, fishes, birds, bacteria, fungi, yeast, actinomycetes, algae, plants, e.t.c. Arginase activities have also been reported in many tumors and cancer cells.
1.3.1 Bacteria
Among bacteria producing Arginase, the prominent ones include many bacilli, the mycobacteria (Zellar et al., 1954), Proteus spp. (Prozesky et al., 1973), T.aquaticus (Degryse et al., 1976), Agrobacterium-Rhizobium group, (Dessaux et al.,1976; Vissers et al., 1981) and cyanobacterium Aphanocapsa 6308 reported by (Weathers et al., 1978) and other cyanobacteria, (Gupta et al., 1981). Among the bacilli, Bacillus licheniformis, arginase has been purified by (Simon and Stalon 1976). Arginase production was studied closely in Bacillus subtilis 168 by (Baumberg and Harwood 1979). Arginase activity was reported in Streptomyces spp. (Vargha et al., 1983) and Streptomyces calvuligerus (Bascaran et al.,1989). Arginase was also reported in cyanobacterium Anabaena cycadeae(Singh and Bisen, 1994). Arginase from extreme thermophile Bacillus caldovelox has been purified and characterized(Patchett et al., 1991) andArginase production and characterization in the phototrophic bacterium Rhodobacter capsulatus E1F1 (Moreno et al., 1992).
Arginase, urease, L-ornithine 5-aminotransferase and L-ornithine cyclodeaminase activities have been detected in R. capsulatus and R. sphaeroides in the study of arginase pathway (Igeno et al., 1993). A thermostable arginase was cloned and expressed from Bacillus caldovelox (Maria et al., 1996). Arginase activity in cephamycin producers Streptomyces clavuligerus and Nocardia lactamdurans was reported(Funte et al., 1996). Bacillus brevis Nagano – gramicidin S- producing bacteria was shown to be a source of arginase (Kanda et al., 1997). The enzyme produced from bacteria is said to be highly specific for L-arginine and inducible by the addition of L-arginine to the glutamate medium. Helicobacter pylori– the gastrichuman pathogen has been reported to have rocF gene that encodes arginase (David et al., 2004). In pathogens such as Helicobacter, by consumption and out competing the host for the limited arginine available, arginase pathway is a major escape mechanism helping to evade the toxic antimicrobial effects of host nitric oxide. Arginine catabolism in the cyanobacterium Synechocystis sp.strain PCC 6803 involves urea and arginase pathway (Quintero et al., 2000).
An arginase extracted from Arthrobacter sp. KUJ 8602 acted on D-arginine, 4-guanidinobutyrate and showed little activity with Larginine and hence has been regarded as a kind of guanidinobutyrase and termed as D-arginase (Arakawa et al.,2002). In Cyanobacteria (Flores and Herrero, 2005) arginase is useful for the utilization of arginine into urea and subsequently into ammonia which is taken up by the bacteria as a nitrogen source. The spore-forming, gram positive bacterium Bacillus anthracis, the causative agent of anthrax has shown arginase activity (Raines et al., 2006). An engineered L-arginine sensor (arginine-regulated transgene, ART) of Chlamydia pneumoniae enables arginine-adjustable transcription control in mammalian cells and mice (Hartenbach et al., 2007).
1.3.2 Fungi
Arginase has been reported to be purified and characterized from Neurospora crassa (Borkovich and Weiss, 1987). Arginine specific carbamyl phosphate matabolism in mitochondria of Neurospora crassa was studied (Rowland and Janet, 1987) where it was reported that arginine efficiently feedback-inhibits intramitochondrial ornithine synthesis. Two forms of Arginase expressed by Neurospora crassa as reported(Marathe et al., 1998) is the only reported example of multiple forms of arginase in a microbial organism. The larger form was produced by mycelia growing in arginine-supplemented medium.
Aspergillus nidulans produces arginase which enables the fungus to utilize arginine as the sole nitrogen source (Dzikowska et al., 1994). Mycelial extracts of Trichoderma sp. were reported to be a source of arginase(El-Meleigy et al., 1998). An extensive survey of higher fungi (Wagemaker et al., 2005) revealed that the presence of arginase in members of family Agaricaceae including Agaricus bisporus that led to the accumulation of urea in its fruit bodies.
1.3.3 Lichens
he purification of three forms of arginase from thallus of Evernia prunastri incubated at different time intervals(Planelles and Legaz 1987). The third form was obtained by culture grown in presence of cycloheximide. Secreted glycosylated arginase from Xanthoria parietina thallus was reported to induce loss of cytoplasmic material from Xanthoria photobionts (Molina et al., 1998). Secreted arginases from Evernia prunastri and Xanthoria parietina thalli are used(Legaz et al., 2004) to utilize arginine in a Mn2+ dependent manner and show lectin function by binding to the cell wall of both homologous and heterologous algae thus helping in further algal-lichen associations by acting as cross-recognition factors ensuring specificity in the associations.
Diaz et al., (2009) accounted the production and secretion of arginase to a medium containing arginine by Peltigera canina, a cyanolichen containing Nostoc as cyanobiont, The secreted arginase acts as a lectin by binding to the surface of Nostoc cells through a specific receptor which develops urease activity. Leptogium corniculatum, a cyanolichen containing Nostoc as photobiont, has been reported to produce and secrete arginase to culture medium containing arginine (Vivas et al., 2009).
1.3.4 Yeast
In Saccharomyces cerevisiae, arginase has been reported to form amultienzyme complex with ornithine transcarbamoylase in which arginase acts as a negative allosteric effector of ornithine transcarbamoylase (Hensley, 1988). The purification and characterization of arginase from a plasmid-containing, enzyme-overproducing, protease deficient yeast strain (S.cerevisiae)(Green et al., 1989). Arginase has been reported from Schizosaccharomyces pombe (Kang 1995). Arginase activity has been reported to be present in parts (tissues and extracts) from several plants. Reports regarding presence of arginase in Lathyrus sativus L plants (Cheema et al., 1969) and pumpkin seeds (Kollofel & Van Duke, 1975) and seedlings have been quite interesting. Evidence for arginase in apple trees was provided through studies in which 14C-labeled L-arginine was administered to trees and its subsequent metabolic products(Hill-Cottingham et al., 1973).
Sroubelakis and Kliewerin the year 1977 demonstrated the presence of arginase in grapevine- Vitis vinifera. Desai (1983) studied the properties of arginase obtained from Arachis hypogea L seedlings. Arginase obtained from cotyledons of Canavalia lineata was purified and characterized (Yu et al., 1988). Arginase has been extracted(Kang and Cho 1990) from embryonic axes of Soybean Glycine max and its properties were studied. Jack bean (Canavalia ensiformis) leaf arginase can effectively hydrolyse both L– and D–arginine (Kavanaugh et al., 1990). The activity of arginase from roots and leaves of kiwifruit vines- Actinidia deliciosa was investigated by Hale et al., (1997). Isolation and study of properties of arginase from roots of a three-year-old shade plant, ginseng (Panax ginseng C.A. Meyer) was done by Hwang et al., (2001). Regulation of loblolly pine (Pinus taeda L.) arginase in developing seedling tissue during germination and post-germinative growth was studied by Todd et al., (2001).
Arginase activity was shown to be enhanced (Millanes et al., 2005) in sugarcane plants – Saccharum officinarum cv. Mayari in response to infection by teliospores of Ustilago scitaminea. The increase in enzyme activity increases putrescine which obstructs the polarization of teliospores at the cell walls. Papadakis et al., (2005) reported that in totipotent tobacco protoplasts, extremely high levels of endogenous polyamine putrescine can be attributed to increased arginase activity.
The characterization of two genes LeARG1 and LeARG2 that encode arginase from Lycopersicon esculatum (tomato) and the regulation of this arginase by wounding, jasmonate and phytotoxin coronatine has been described where Arginase along with threonine deaminase (TD) has been reported to be induced by the plant hormone- Jasmonic acid in Tomato plants-Solanum lycopersicum. Often called a Jasmonic acid inducible protein (JIP), the enzyme has been shown to attack arginine in the mid-gut causing intestinal issues in Maduca sexta larvae which infest the tomato plant hence conferring plant protection against herbivores (Chen et al., 2004).
Xylem sap of the deciduous tree- Quercus ilex was reported to possess arginase by Nabais et al., (2005) which degrades arginine- a storage protein in the tree which breaks down to ornithine by the increased activity of arginase before spring time. Dabir et al., (2005) reported arginase extraction form cotyledons of Vigna catjang. Arginase has been found to be operative in cotyledons of somatic embryos of pine at late developmental stages (Rodriguez et al., 2006). Arabidopsis was reported to be a source of arginase (Palemeiri et al., 2006).
1.3.5 Insects
Two arginases A and B were reportedly produced from developing embryos of the tick Hyalomma dromedarii (Fahmy et al., 1994). Samson (2000) reported a Drosophila gene encoding a 351-amino acid-long predicted arginase (40% identity with vertebrate arginases). Tanuja et al., (2005) reported that the levels of arginase in Bombyx mori– the silk worm decreased sharply during development of the pupa but rose during adult development. Arginase from the flatworm Fasciola gigantica has been described by Mohamed et al., (2005) with two arginases- Arginase I and Arginase II.
1.4. THE SUBSTRATE; ARGININE
The amino acid arginine is an important substrate for protein synthesis and for the production of argmatine and creatine (1) it is best known however as the precursor for the imunoregulatory and vasoactive molecule nitric oxide (NO),although the conversion to NO represents only 1% of arginase plasma flux (2). Quantitatively, arginine plasma flux is determined by arginine intake, endogenous arginine synthesis and protein breakdown on one side and protein synthesis and arginine catabolism by the enzyme arginase on the other side.
Arginine is an essential amino acid which feeds into many metabolic pathways that are important for normal cell growth and differenciation, including those for proline, glutamate, gamma-aminobutyric acid (GABA), nitric oxide, and polyamines. L-arginine is cleaved into ornithine and urea by arginase, which is the final enzyme in the urea cycle.
Arginine has multiple metabolic fates in human physiology with the enormous inherent versatility of this amino acid in various pathways involved with normal growth and maintenance of the body. Besides being a precursor of nitric oxide, creatine, polyamines, agmatine and urea it is metabolically interconvertible with amino acids glutamate and proline. It has been classified as a dibasic, cationic, semi essential amino acid since it becomes necessary under periods of growth as well as recovery after injury. The body needs high level of arginine when it is under conditions of stress, illness, malnutrition or injury. There are also some genetic disorders which lower arginine levels and make the conditional supplementation of dietary arginine necessary.
The food sources high in arginine include carrot, chocolate, coconut, dairy products, gelatin, fish, poultry, meat, oats, peanuts, soyabeans, walnuts, brown rice wheat, wheat germ, sesame and sunflower seeds, popcorn and raisins. Free arginine in the body is derived from diet, endogenous synthesis and turnover of proteins. Arginine is synthesized from citrulline in many cell types in the body but a major part of endogenous synthesis is through the teamwork of epithelial cells of small intestine and proximal tubules of kidney.
During conditions of arginine deficiency caused due to catabolic stress(inflammation and infection)or due to dysfunction kidneys or small intestine, levels of arginine is not sufficient and arginine needs to be supplied through diet (Morris, 2006). Arginine has a history of safe usage as a supplement. Being a common substrate for arginase and nitric oxide synthase, its levels in the body are a result of competition and reciprocal regulation between these two key enzymes.
Medical researchers have gathered enough clinical evidence to bring L-arginine to the forefront of modern medicine as an accepted treatment for a variety of human ailments. The following are organized layout about the immense benefits of dietary arginine and its supplementation depending on its numerous roles in the body.
In aging process
Studies in aging NMRI-mouse show that L-arginine reduces kidney collagen accumulation and N-epsilon-(carboxymethyl) lysine thus inhibiting one of the primary mechanisms of aging process of crosslinking (Radner et al.,1994). Arginine increases the release of human growth hormone (HGH) (also known as the anti-aging hormone) from the pituitary gland (Gianotti et al.,2000).
In cardiovascular system
Most of the significant effects of arginine in therapy of the cardiovascular system arise from its being a metabolic precuesor of nitric oxide an endogenous neurotransmitter that helps to prevent vasoconstriction and which initiates vasodilation by relaxing the smooth muscle cells of the blood vessels.in patients of moderate congestive heart failure, arginine significantly increases stroke volume and cardiac output (without effect on heartbeat)as well as increase in vasodilation (leading to increase blood circulation)in congestive heart failure patients (Koifman et al.,1995). It improves blood circulation, improves exercise capability and facitates vasodilation in angina patients (Ceremuzynski et al., 1997) in studies conducted on young men with coronary artery disease, oral L-arginine has been shown to improve endothelium-dependent dilation and reduces monocyte adhesion to endothelial cells thus helping to prevent atherosclerosis and reducing the severity of existing atherosclerosis (Adams et al.,1997).
Cigarette smoking has been observed to be associated with increased human monocyte adhesion to endothelial cells (an underlying event in the course of atherosclerosis whose inhibition has been reported by use of arginine (Adams et al., 1997). L-arginine treatment directly influences the kinetics of nitric oxide and superoxide production thus improving blood circulation (Huk et al., 1997) thereby also helping to prevent free radicals-induced damage to the lining of blood vessels.Reports recorgnise(Wallace et al.,1999) that L-arginine infusion dilates coronary vasculature in patient undergoing coronary bypass surgery.
Khosh & Khosh (2001) reported that Arginine lowers blood pressure in some hypertensive patients (by facilitating the body’s production of nitric oxide (NO) and by inhibiting the angiotensin converting enzyme (ACE). The combination of arginine with cholesterol lowering stain namely Atorvastatin has beenshown to hinder the spread of atherosclerotic plaques as compared with monotherapiesthereby opening a new area of therapeutic strategy (Rasmusen et al.,2005) . Miller (2006) shoed from an experimental study that a moderate dose of sustained-release L-arginine can improve endothelial function and blood pressure.Tripathi and Misra (2009) have reported that free radical scavenging role of L-arginine is of immense therapeutic value to patients of myocardial infarctions and acute angina since it inhibits pro-oxidative enzymes and act as an anti-oxidant by scavenging harmful free radicals that cause substantial damage to cardiomyocytes.
In digestive system
While it’s deficiency can cause constipation , the supplementation of L-arginine may decrease the incidence of gallstones. Arginine reduces intestinal permeability (due to arginine’s role in the production of nitric oxide) (Miller,1997) and alleviates many cases of ulcerative colitis by promoting the healing of the ulcers that occur in the colon of ulcerative colitis patients (Sagela,2000) and helps to prevent post surgical damage after intestinal manipulation (Thomas et al.,2001).
In excretory system
Oral arginine reduces the pain and discomfort with intestinal cystitis (Smith et al.,1997) and significantly improves the function of the kidney and helps to prevent age-related degradation of the kidney (Reckelhoff et al.,1997).
In immune system
Arrest of liver cancer (Weisburger,1969) and of the mammary tumor growth by L-arginine was reported by Cha-Chung in 1980 where it was observed that arginine boosted the ability of the immune system to fight breast cancer. Later it was observed that supplemental arginine increases thymic cellularity in normal and murine sarcoma virus inoculated mice and increases the resistance to murine sarcoma virus tumor (Rettura et al.,1979). Arginine retards the growth of tumors and cancer by enhancing immune function. The process underlying this function is the ability of arginine to enhance the size and health off thymus gland- the “director” of immune system which produces enzyme thymosin which in turn stimulates the spleen and lymph nodes to manufacture high levels of active and effective T-lymphocytes (T-cells) (Morigushi et al.,1987).
Kirk et al.,(1993) accounted that arginine stimulates wound healing and immune function in elderly human being as well as decreases inflammation in general (Effron et al.,1998). Arginine helps to prevent bacterial and viral diseases in person with suppressed immune system (Field et al.,2000). Patterns of arginine and nitric oxide in patients with sickle cell with vaso-0cclusive crises and acute chest syndrome suggested that arginine alleviates sickle-cell disease (Moris et al., 2000). Arginine enhances the ability of the immune system to recover from elective surgery and accidental injury (Wilmore, 2001).
In metabolism
Arginine deficiency can cause alkalosis in the body however, its presence scavenge the super-oxide production and therefore exert anti-oxidant effect in the body. Studies carried out on animals proved that L-arginine lowers serum LDL level (Ryzenhov et al., 1984) and normalizes endothelial function (Rossitch et al., 1991) in cerebral vessels from hypercholesterolemic rabbits by reducing their total cholesterol levels.In diabetes, arginine has been shown to reverse damages of diabetes (Giugliana et al., 1997) and in some cases prevent diabetes (Mohan and Cas, 1998). By stimulating the release of human growth hormones (HGH) from the pituitary gland,arginine alleviates obesity and d facilitates weight loss (Gianotti et al., 2000).
Piatti et al., (2001) stated that arginine reduces insulin resistance and improves blood sugar disposer in diabetes type 2 patients. Due to its roles in production of nitric oxide which inturn improves blood circulation via vasodilation, arginine has been shown to increase oxygen uptakes in the lung in persons with hypoxia and altitude sickness (Beall et al., 2001).
In muscoskeletal system
By producing nitric oxide, arginine causes the relaxation of smooth muscle. It facilitate muscle growth (by inhibiting muscle loss) and is required for the transport of the nitrogen used in muscle metabolism (Barbul, 1986). By stimulating the release of human growth hormones (HGH) which is an important mediator of bone formation and bone turnover and stimulation of nitric oxide synthesid which is a potent inhibitor of osteoclasts that cause the reabsorption of bone, arginine may prevent and alleviate osteoporosis (Visser & Hoekman, 1994)
Stevens et al., (2000) reported that arginine enhanced high intensity dynamics human muscle performance.The roles of arginine in sepsis has been studied by Luiking et al., (2005) who reported that arginine supplementation could reduce the effects of infection and sepsis through the NO pathway.
In nervous system
As stated by Cestaro (1994), arginine is essential for the regeneration of damaged axons of neurons by acting as an agent for degrading proteins that have been damaged through axons injury. By stimulating the production of nitric oxide (NO) –a neurotransmitter responsible for the potential (storage) of long term memory, it serves as a memory enhancer (Pautler, 1994). In athletes, arginine supplementation improves pituitary responsiveness and modulates hormonal control (di Luigi et al., 1999). Due to its ability to repair damaged axons by increasing polyamine level, arginine may be useful for the treatment of Alzheimer,s disease (Tarkowski et al., 2000).
In sexual system
Now known as the ‘Natural Viagra’ the role of arginine has been accounted since long in improving sperm count and sperm motility (Keller et al., 1975) Due to its involvement in the manufacture of endogenous spermidine resulting in improvement of sperm count and sperm motility, arginine alleviates male infertility. It enhances sexual performance in woman too (Block, 1998).
Chen et al., (1999) reported that arginine lessens male impotence by alleviating erectile dysfunction. In animal models, arginine resulted in endogenous nitric oxide-mediated relaxation and nitrinergic innervations which suggests that it may improve prostate function (Aikawa et al., 2001).
In skin
Arginine is essential for and accelerates the healing of wounds by stimulating the release of human growth hormones (HGH), stimulating the production of collagen and by stimulating the proliferation of fibroblasts (Barbul et al., 1978). Arginine stimulate wound healing and immune function in elderly human beings by proliferation of fibroblasts (skin cells) (kirk et al., 1993 ). Apart from accelerating the healing of burns (Yu et al., 1996) when applied topically it keeps the skin young by increasing the level of vascular endothelial growth factor in the skin (Block,1998) besides improving scleroderma (Freedman et al., 1999).It has shown to dramatically speed up the healing of wounds in patients with high risk of infection after cardiac surgery (Tepaske et al., 2001).
In treating heatstroke
A study conducted in mice showed that heatstroke induced death in mice could be controlled by administration of arginine at appropriate concentration and time by increasing in Th-2-cytokines and levels of splenic and hepatic arginine (Chatterjee et al., 2006)
As accounted above ,being vital for an enormous numbers of processes in the body, the determination of arginine is of prime importance in physiological fluids. The levels of arginine have been found to vary in condition of metabolic disorders and disease. Thus, arginine level in the body constitutes an important marker for diagnosis and treatment of various diseases. L-arginine levels in patients undergoing surgeries such as thoracoabdominal surgery (Nijveldt et al.,2000) and heart surgeries are particularly reduced and in such cases careful monitoring of arginine is necessary for speedy recovery.. Being a precursor of the vasodilator NO, quantification of arginine levels is imperative in NO related processes such as atherosclerosis and strokes.
L- Arginine levels in blood have proved to be a maker of nitric oxide – mediated brain damage in acute stroke (Armengou et al., 2003). In tumor cells undergoing therapy with arginine degrading enzymes ( argininase, arginine deiminase and arginine decarboxylase), arginine quantification is particularly vital to assess the efficacy of the anti-cancer agent as well as the stage/phase of tumour removal . Regarding foods, arginine is one of the most abundant amino acids present in grape juice and its monitoring in grape juice is essential for winemakers to realize hazard analysis critical control point (HACCP) since they are the raw material for making wines and must.Elevated arginine concentrations in grape juice and must can lead to an increased potential for Ethyl Carbamate (named as a potent carcinogen by FDA) concentrations in wine (Austine and Butzke, 2000).
Arginine determination has been mainly carried out in fruit juices such as pineapple juice (Elkins et al., 1997),green tea (Lvova et al., 2003 ) and grape juice (Spayd and Anderson-Bagge, 1997; Hua et al., 2008) by means of spectrophotometric (in pineapple & grape juice) and by biosensor ( in green tea) methods. Ough et al., (1988) reported that arginine concentration higher than 1000 mg/L could produce 15µg/L of ethyl carbamate which is currently the voluntary limit of EC in wines in the US while Canada has legalised this limit to 30µg/L (Orduna et al., 2001). Arginine present in wines can be converted to ammonia and citrulline by heterofermentative malolactic bacteria during malolactic fermentation in grape must and wine. Ammonia produced increases the pH and hence the risk of growth by spoilage bacteria and citrulline is a precursor to the formation of potential carcinogenic ethyl carbamate (urethane) (Orduna et al., 2001 ).
Also alcoholic fermentation by yeast produces urea from arginine which can spontaneously react with ethanol to form ethyl carbamate (Uthurry et al.,2006). Ethyl carbamate content in wines with malolactic fermentation as determined at different points in vinification process in a recent study conducted by Masque et al.,(2010) was reported to be lower than the existing legal limit after malolactic fermentation but observed to increase with a storage time of 8 months. Fruits such as watermelon have been found to be good and novel sources of arginine and their regular consumption has been shown to increase plasma levels of arginine (Mandel et al., 2005; Collins et al., 2007).
A significant finding has been the potential that dietary sources of arginine (such as watermelon juice) may have found use in treating severe forms of malaria due to providing the body with the requisite precursor ( arginine) for nitric oxide (NO) which helps cure fever and others forms of complications in the disease (Lopansri et al., 2003; Hobbs et al ., 2003). The above facts make the quantification of arginine in biological and food samples absolutely necessary and indispensable.
1.5 SCIENTIFIC CLASSIFICATION OF LOCAL GOAT
Kingdom Animalia
Phylum Chordata
Class Mammalia
Order Ariodactyla
Family Bovidae
Sub family caprinae
Genus Capra
Specie C.aegagrus
Sub species C.A hircus (Linnaeus 1758).
The local goat (capra aegagrus hircus) is a subspecies of goat domesticated from the wild goat of southwest Asia and Eastern Europe. The goat is a member of the family Bovidae and a closely related to the sheep as both are in the goat-antelope subfamily caprinae. Goats are one of d oldest domesticated species.
1.6. AIM AND OBJECTIVES
The specific aim and objective of this research was:
i. To isolate Arginase from the kidney of a local goat by partial purification of the enzyme.
ii. To determine effect of metal ions on the enzyme (arginase).