Brassica Juncea, is also known as brown mustard, Chinese mustard, Indian mustard, leaf mustard, oriental mustard and vegetable mustard is a species of mustard plant. There is a sub-variety which is also known as southern giant curled mustard which includes the headless cabbage and with a mustard taste. It is also called as green mustard cabbage. Brassica Juncea is an annual developing to 0.8m through 0.3m. The flowers bloom from June to August and the seeds grow from August to September.
The species is hermaphrodite (has male and female organs) and is pollinated by means of bees. The plant is self-fertile. It is suitable for mild (sandy), medium (loamy) and heavy (clay) soils. It covered the central Asian Himalayas to china. Plant can be developed in the tropical marshes just as in a lot cooler conditions. The plant can tolerate a temperature of 6 – 37°C and a yearly precipitation of 500 – 4,000mm. Brown mustard has been cultivated for edible seed which is aorigin of oil.
This plant isused for phytoremediation to replace heavy metals for example lead, from soil. That is the important species cultivated for manufacturing of mustard oil. The living organismalso has a large scope of medicinal purposes, is utilized in the treatment of tumour in China. In Korea, the seed has been utilized for the treatment of colds and stomach disorders. The plant oil is utilized for the treatment of skin burst and ulcers. Brassica Juncea has been found to have aenormous potential to remediate cadmium, lead and zinc from contaminated conditions. This plant can be utilized as bio-remediator to decrease boron and selenium levels in contaminated soils.
Glutathione S-Transferase, generally abbreviated as GST, are a group of multifunctional proteins that work both as significant chemicals of detoxification and intra-cellular binding proteins. Glutathione S-transferase utilize glutathione in numerous responses that achieve the variation in several products such as cancer-causing agents, therapeutic substitues and products engaged with oxidative pressure. Due to the double capacity of GST, it has been the research interest of a few researchers, including pharmacologists, natural chemists, physiologists, toxicologists, and cell scholars. In their enzymatic response, they catalyze the response between the nucleophile diminished glutathione (GSH) and large number of electrophilic compounds. GST group of purifying compounds consists of numerous microsomal, mitochondrial and cytosolic proteins, which make significant molecule of the protein body. They occur in eukaryotes and prokaryotes in which they are essential in catalyzing the various responses and simultaneously accept xenobiotic and endogenous substrates. Various GST isoenzymes that are bound by cytosolic and membranes are found in eukaryotic species. Each have unique catalytic and non-catalytic binding properties. GST bind various amphipathic substitutes that they don’t use (non-substrate ligands) and had been approved to act as intracellular transport proteins for compounds that have partial solvency in water. Glutathione S-Transferase contain up to 10 percent cytosolic components in certain organs. GST catalyse the mechanism of conjugation of glutathione to electrophilic areas through sulfhydryl gathering. The motive is to enhance the solubility of compounds. Simultaneously, few compounds are detoxified, including peroxidised lipids and compounds and xenobiotics are separated. Another significant capacity did by glutathione S-Transferase is the binding of toxins, which likewise serves as a mechanism of transporting proteins. That is the reason glutathione S-transferase are referred to as ligands. Acclaimed research shows that glutathione s-transferase plays an essential role in the determination of cell sensitivity to a wide extent of harmful chemicals. The Important function carried by glutathione s transferase (GST) is to catalyse nucleophilic attack order to achieve detoxification of xenobiotics. This is performed by GSH which works under electrophilic carbon, sulphur or atoms of nitrogen. Glutathione S-transferases are best known for their capacity to conjugate xenobiotics to GSH and in this manner purify cellular products.A Glutathione S-transferase from brassica Junceadeal for the hexachlorocyclohexane pesticide was purified to homogeneity and partly characterized. Hexachlorocyclohexanes (HCHs) are substitutes of major concern in the midst of the organochlorine pesticides, which have been eagerly utilized in the past around the earth. There are eight geometric isomers of HCHs, which contrast in the centric and equatorial spot of the chlorine compounds. Hexachlorocyclohexane officially known as benzene hexachloride, is ansynthetic chemical that move in 8 chemical patterns known as isomers. This is thederivative of the manufacturing of the insecticide lindane (gamma-HCH). Gamma-HCH (also called lindane), is generated and utilized as an insecticide on fruit, vegetables, and wooded area flora, and animals, and animal bounds. It is likewise available as a prescription remedy (lotion, cream, or shampoo) to deal with control scabies (mites) and head lice in humans. Beta- HCH is one of the 5 stable isomers of technical HCH, the pesticide previously used in agriculture. Beta- HCH is used as an intermediate chemical in the manufacturing of lindane. HCH is synthetic by way of photochemical chlorination of benzene which ends up with an arrangement of particularly 5 stable HCH isomers. Beta-HCH initiate in global and aquatic food chain. Hexachlorocyclohexane (HCH) is a variety of stereoisomers used as an insecticide. Gamma-HCH is produced and used as an insecticide on fruits, vegetables, and forest crops. It is a white solid that may evaporate into the air as a colourless vapor with a slightly musty odour. All the isomers are harmful to animals to differing division and are determined in the environment. Only ?-HCH has any appreciable insecticidal action. The only known use of HCH is as an insecticide, formerly widely utilized specially in the limit of cotton microbes. In certain nations, it might be used for control of leaf hoppers, stem borers in lowland rice, as seed treatment for decrease of wireworm harm in winter and spring planted grains and for control of irritations of oats, sugar beets and oilseed rape. Technical HCH is a mixture of alpha, beta, gamma and delta isomers and these isomers contrast qualitatively and quantitatively in biological activity. The alpha and gamma isomers are central nervous stimulants and beta and delta are depressants. In a mixture of these isomers, one component may check the impact of another component and the last impact may rely upon the composition. HCH is essentially synthetized by a reaction between chlorine gas and benzene in industrial production. The technical HCH is regularly a mix of a few stereoisomers, which generally contains four isomers, to be specific, ?-HCH, ?-HCH, ?-HCH, and ?-HCH, representing 67%70%, 5%6%, 13%15%, and 6% of the specialized HCH, individually; and ?-HCH, additionally named lindane, is the best part of specialized HCH in destroying pests. Technical grade HCH and ?-HCH are considered as likely human cancer-causing agents while beta-HCH is viewed inthe potential human cancer-causing agent.
REVIEW OF LITERATURE
Glutathione S-transferase (GSTs, EC 18.104.22.168), encoded in themassive group of genetic code, plays an essential function in the vital protocol of degradation in plant cells . GST is a broad and comprehensive enzyme that removes large number of electrophilic xenobiotics from the biological systems by binding to tripeptide glutathione and engaged in the detoxification framework by different mechanisms . GST compound removes some toxic substances in the flow by covalent or non-covalent bonding and are phase 2 enzymes of xenobiotics detoxification [11,13]. The isoenzymes of the GST are found in all life forms as diverse as organisms, fungi, insects, fish, warm blooded animals and plants [1,12]. The most widely recognized tissues of the GST are the liver, particularly the organs, for example, kidney, small digestive system, lung and breast [16,32]. The GST compound establishes 5% of the absolute protein material of the cytosols of these organs. Due to of hugepresence of the GST in these organs, it is straightforwardly in contact with xenobiotics taken all things considered. Most GSTs described to date are dimers made out of 22-30 kDasubsets, and every subunit have a glutathione (GSH) restricting site (G-site) and an adjoining electrophilic substrate restricting site (H-site) [17,41]. These enzymes are for the most part found in the cytosol of organisms and may be isolated into a few forms, for example, phi, tau, theta, zeta, lambda and dehydroascorbate reductase, among which phi and tau classes are specific in plants . One of the essential enzymes that enhance the importance of biotransformation in pesticide toxicology is the GST. Therefore, the study of oxidative pressure chemicals is significant regarding biological, toxicological and evalutional perspectives . GST enzyme activity has been identified in many eukaryotic organisms including plants, insects and animals. Isozymes have been purified from human. Each of the purified isozymes has a distinctive example of reactivity relying upon the carbon skeleton or the reactive class of the substrate. However, in a given tissue those enzymes collectively have an enormous scope of catalytic capability. In wide, the transferases are dimers and havemolecular weights in the scope of 45000 and 50000. In plants, GST functions to sequester the conjugates into vacuoles rather focusing on them for discharge excretion observed in animals. Numerous types of GST are available in many organisms and each shows affinity for a wide variety of substrates . In plants, a significant role of GST is the degradation of herbicides . The ability of GST to inactivate herbicides gives plants a barrier against chemical exposure. Therefore, characterization of GSTs present in plant species can give basic data to the design and application of herbicides. Plant GSTs likewise react to pathogen attack, heavy metal toxicity and oxidative pressure resulting from normal metabolic procedures. In addition, their job in xenobiotic detoxification, GSTs also act as intracellular transporter proteins . GSTs mediate transportation of endogenous compounds among different cell compartments. GSTs are engaged with such a transportation  and subsequently have a significantly job in the existence cycle and advancement of plants. However, there are a few isotypes of GST present in a living being or plant . These GSTs are probably going to change in their explicitness for various substrates and have a significant role in life cycle and development of plants. In this way, it is essential to obtain GSTs as an initial step to investigate a relationship in their structure and function. They are likewise known to kill endogenous secondary metabolite framed during oxidative pressure . These compounds happen in numerous forms and catalyse a large number of responses including electrophilic functional groups . The GST has a primary job during the process of inactivating a wide scope of exogenous/endogenous poisonous molecules and to transform them into water soluble compounds. GSTs increased fundamental importance in parasites as the main part of detoxification system due to the absence of cytochrome P450 (CYP450) activity .
Hexachlorocyclohexane (HCH) is a six-chlorine substituted cyclohexane; it collectively represents the eight isomers of 1,2,3,4,5,6-hexachlorocyclohexane. Hexachlorocyclohexane is an organochlorine insecticide which has been broadly utilized in horticulture previously. It is as yet utilized for public health and medicinal purposes. Hexachlorocyclohexane is the term used for each of the 8-isomers of HCH . Among the isomers, just gamma HCH, normally known as lindane, has insecticidal properties. During the production of gamma HCH up to 85% of the item comprised of different isomers, mostly alpha, beta and delta HCH . HCH is commercially manufactured by the photochemical chlorination of benzene in the existence of UV fluorescence. The specialized evaluation of HCH originated in this way contains mainly ?ve variously stable isomers: alpha (60 to 70%), beta (5 to 12%), gamma (10 to 12%), delta (6 to 10%), and ? (3 to 4%) . These isomers vary in the spatial directions of the chlorine molecules around the cyclohexane ring. Although only the gamma isomer is insecticidal , specialized HCH was used broadly as a cheap yet successful insecticide in developing countries. It is possible to isolate and purify the gamma-HCH isomer from specialized HCH, and the puri?ed gamma-isomer, generally called as lindane, was likewise normally used as an insecticide, especially in developed countries.
The manufacturing and the use of HCH/lindane has led to two major residue issues. One includes the point source contamination of very high concentrations because of the open-air stockpiling of waste isomers during lindane production [40,42] the production of 1 ton of lindane produces 8 to 12 tons of “HCH sludge,” comprising fundamentally of the alpha, beta, and delta-HCH isomers. The beta isomer is reliably found in higher concentration in human fat and blood though alpha and gamma HCH are most predominant isomers in soil. Every one of the four isomers of HCH are dangerous and considered worldwide pollutants [42,43]. In spite of the fact that the use of lindane and BHC (a variety of HCH isomers) has been prohibited or seriously fixed in various nations, including India, the ban has not stopped HCH deposits from entering the environment. Subsequently, HCH residues are being detected in different environment niches, in India as well as everywhere throughout the world [7,34,35,39]. While gamma HCH is industrious in the environment yet degradable, the alpha and beta isomers are the more tricky of the diverse HCH isomers in view of their low solvency in water and subsequently their high potential for bioaccumulation. There is likewise proof that the alpha and gamma isomers of HCH are converted into the beta isomer in living beings . An overview for HCH residues in different examples directed over numerous years demonstrated that most are contaminated with alpha and beta HCH [18,19,25]. The persistence of HCH isomers in aerobic environments is essentially because of the absence of microorganisms that can degrade them [34,35,36,37]. HCH isomers are known to degrade gradually under aerobic conditions, however there are not many reports of HCH degradation under aerobic conditions [25,27,36].
Diminished use of specialized HCH in to two decades has prompted decreased environmental weights of alpha-HCH [3,41]. Late estimations in the Arctic show that alpha-HCH is currently revolatilizing from the western Arctic Ocean and its territorial oceans [40,41]. This inversion is support by chiral examination of the alpha-HCH in air and surface water . In the specialized blend alpha-HCH exists as a racemate of two enantiomeric structures which are non-superimposable perfect representations. Convergence of HCHs in air and water are exhibited promotion used to decide the condition of air-water gas trade .
The Aim of this study is:
Partial Purification and Characterization of Glutathione S-Transferase.
Determination of unit activity.
Estimation of protein content by Lowrys method.
Determination of specific activity of Glutathione S-Transferase.
Purification of GST by Ammonium sulphate precipitation and Dialysis.
Molecular weight identification by SDS-PAGE.
MATERIALS AND METHODS
COLLECTION OF SEEDS
Brassica Juncea (Mustard) seeds were collected from Punjab Agricultural University, Ludhiana.
Fig 1: Brassica Juncea (Mustard) Seeds.
GERMINATION OF SEEDLINGS
Brassica Juncea seeds were washed with distilled water for 5 min, sterilized with sodium hypochlorite for 2 min and then rinsed with distilled water for 5 min. The seeds were allowed to germinate in petri plates containing Whatman filter paper moistened with distilled water and stored in laboratory at 25 degree C for 120 hours.
Fig 2: Germination of seeds Day 3
Fig 3: Germination of seeds Day 5
PREPARATION OF CRUDE EXTRACT
The germinating seeds at different time intervals (24, 48, 72, 96 and 120 h), cotyledons were isolated from seedlings. Seed/ Cotyledons (3g) were ground to a fine powder and then homogenized in 25ml of 250mM Tris-HCl (pH 7.8), 0.5mM DTT and 1mM EDTA for 10 min. The homogenized extract was centrifuged at 10,000 rpm for 3 min. After centrifugation, the pellet was discarded and only supernatant (crude extract) was used for further purification.
Fig 4: Isolation of cotyledons from seedlings.
Fig 5: Preparation of enzyme extract.
Fig 6: Separation of layers after centrifugation.
Fig 7: Supernatant was transferred to a fresh tube.
The supernatant (70ul) was used as assay and treated with 2 ml of 0.05M phosphate buffer (pH 7.5). 1 ml of 0.01M glutathione, 1 ml of 0.01M CDNB. The unit activity of GST is detected by measuring the absorbance at 340 nm for 3 min with 30 seconds interval.
DETERMINATION OF PROTEIN CONTENT
The protein concentration was carried out spectrophotometrically at 660 nm by using Lowry protein assay. Bovine serum albumin was used as a protein standard. Test tubes (T1-T9) were taken as standard and T10-T11 as sample test tubes and T12 as blank. 0.1 ml of crude extract was added in 5 ml of alkaline copper solution. Then 0.5 ml of Folins reagent solution was rapidly added, mixed well and placed for 30 min at room temperature. Absorbance was recorded at 660 nm wavelength by using distilled water as blank on spectrophotometer.
Fig 8: T1-T9 are standards (Bovine Serum Albumin as standard protein).
Fig 9: T10-T11 are test samples and T12 as Blank.
AMMONIUM SULPHATE PRECIPITATION
GST enzyme was precipitated by ammonium sulphate precipitation for partial purification of enzyme. Various centralizations of 30%, 40%, half, 60%, 70% lastly 80% were prepared. The suspension was mixed on magnetic stirrer for two hours at room temperature. After sufficient shaking, the solution was centrifuged at 6000 rpm for 20 min and resultant supernatant and pellet were isolated. Pellet was dissolved in 1 ml of 0.1 M phosphate buffer (pH 7) and enzyme assay of both the pellet and supernatant was performed utilizing spectrophotometer .
Maximum amount of enzyme acquired in 80% concentration of ammonium sulphate precipitation was dialyzed for desalting utilizing dialysis bag likewise called as dialysis layer. Prior to dialysis, the dialysis layer was boiled in 0.1M sodium carbonate till boiling point of solution and afterward held the arrangement containing dialysis film for overnight. From that point onward, precipitated sample was poured in dialysis pack and it was dialyzed against 200 ml, 0.1M phosphate buffer (pH 7) on magnetic stirrer for 2 hours .
MOLECULAR WEIGHT IDENTIFICATION BY SDS-PAGE
SDS-PAGE was performed by the method of Laemmli  using a Mini-Protean A apparatus. Protein was detected by assaying unstained gel slices from the same gel. Each gel slice was tested for enzyme activity by incubating with 1-chloro2,4-dinitrobenzene. A gel slice with the corresponding enzyme activity was then added to a small volume of SDS-PAGE sample buffer, boiled for 5 min, and then subjected to SDS-PAGE .
RESULT AND DISCUSSION
12 seeds were allowed to germinate in a petri plates containing Whatman filter paper moistened with distilled water out of which only 10 seeds were germinate. So, the percentage of seed germination was 83.33%.