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Objective: Isolation and purification of tyrosine hydroxylase. Isolated enzyme used for development of bioassay by using specific substrate L-tyrosine. Materials and method: Tyrosine hydroxylase found in various tissues in animals. Enzyme isolated from caudate nuclei of sheep brain using various parameters like temperature, pH, Ionic strength etc. Enzymatic activity assessment protocol was developed based on specific substrate for tyrosine hydroxylase. The enzyme purity and concentration were determined by developing gel permeation chromatographic technique Result: Maximum enzyme isolated at 370C using pH 5.8 buffer solution and 50% saturated ammonium sulphate solution. The enzyme was purified by using sephadex G 75 and assay performed by using L-tyrosine. Conclusion: The isolated enzyme was used for development of bioassay design and tested for bioactivity assays of some of drugs for cardiovascular disorders. KeywordsTyrosine Hydroxylase, Bioassay, Gel permeation chromatography, cardiovascular diseases INTRODUCTION Cardiovascular diseases are still the major factor leading to death worldwide. Atherosclerotic lesion formation which leads to not only myocardial infarction but also stroke due to weaken endothelial function and proceeding with inflammation of the vessel. This may be caused due to increased level of blood pressure, diabetes, smoking, obesity, etc. Increased level of blood pressure leads to 13% CVD, tobacco resulting in 9% as well as diabetes leading to 6% deaths. Even lack of exercise and obesity leads to 6% and 5% fatalities. Major risk factor associated with coronary artery disease, heart failure, peripheral vascular disease, impaired vision, stroke and kidney disease is increased level of blood pressure.1 CVDs are expected to impose every domain of population regardless of age, economic status, gender, and locality. Newer drugs are being discovered and designed.2 Research has identified many molecular targets playing role in controlling cardiovascular pathophysiology. They are guanylate cyclase stimulators which are soluble, tyrosine kinase inhibitors, EGF receptor blockers, Rho-kinase inhibitors, dopamine β hydroxylase, HIF hydroxylase,3-4 21-hydroxylase, 11 β hydroxylase, soluble epoxide hydroxylase,5 tyrosine hydroxylase,6 etc. Clinically, hydroxylases present a challenging but unique rewarding target for drug therapies. Hydroxylases are an important component of oxygen discerning proteins. Tyrosine hydroxylase structure shows tetramers with four identical subunits so it is called as homo tetramer. Each subunit is further divided into three domains. At the carboxyl end of the peptide chain, tetramerization is allowed by short alpha helix domain which is present there. The central 300 amino acids compose of a catalytic core, within which all the residues essential for catalysis are un rr cte d p r o f present, along with a non-covalently bound iron atom.7 Two histidine residues and one glutamate residue hold the iron in place. Thus, making it a non-iron-sulfur, non-heme iron-containing enzyme. ~150 amino acids amino terminal frame a regulatory domain, which controls the access substrates to the corresponding active site. In humans, it is thought to contain regulatory domain with four varying versions, and thus four versions of the enzyme, which rely on alternative splicing, though none of their structures have yet been properly determined. It has been put forward that this regulatory domain might be an intrinsically unstructured protein, which has no clearly defined tertiary structure, but also, there has been no evidence presented assisting this claim.8 For catalyzing the transformation of L-tyrosine amino acid to L-3, 4-dihydroxyphenylalanine i.e. L-DOPA, tyrosine 3-monooxygenase or tyrosine hydroxylase is responsible.9 It not only uses molecular oxygen, but also iron and tetrahydrobiopterin as cofactors. L-DOPA which is a precursor for dopamine, which is a precursor for important neurotransmitters, nor-epinephrine (nor-adrenaline) and epinephrine (adrenaline). In the catecholamine biosynthetic pathway, tyrosine hydroxylase is generally applicable as a rate limiting enzyme.6 MATERIALS AND METHODS LTyrosine obtained from oxford lab reagent, Bovine serum Albumin (Sigma Aldrich), Triton-X-100 (S.D. lab), Sephadex G -75 (Sigma Aldrich), Hydroxylamine hydrochloride (Sigma Aldrich) INSTRUMENT AND SOFTWARE SPECIFICATION UV-Spectrophotometer (Jasco V-630, Japan), Ultra sonicator (Spectra lab Mumbai), Gel permeation chromatography (Waters 2414), V Life MDS 4.4(V Life sciences and technology Pvt. Ltd), Cold centrifuge (Remi) EXPERIMENTAL: ENZYME ISOLATION The sheep brain was collected from slaughter house. Caudate nuclei were dissected and homogenized immediately in nine volumes of ice cold 0.25M dextrose solution at 0-4°C [10] Centrifugation of the homogenate was carried out for 15min in cold centrifuge machine at 5000 RPM and 4o C. The sediment was suspended in half the original volume of dextrose solution and re-centrifuged. Supernatants collected were combined. Enzyme was solubilized by treatment of 0.2% triton X-100 followed by centrifugation at 9000 RPM for 1.5 hours in cold centrifuge at 4oC. Supernatant collected was used for enzyme isolation at various pH conditions, temperature and ionic strength. From that parameter, one parameter was selected with greatest enzyme yield.11-13 ISOLATION OF ENZYME REGULATING PH, TEMPERATURE VALUES, IONIC STRENGTH Tyrosine hydroxylase was isolated at various pH ranges such as 5.6, 5.8, 6.0, 6.2, and 6.4 respectively. During isolation, pH of dextrose solution was adjusted to above mentioned pH respectively with 0.1N HCl and 0.1N NaOH; the further enzyme isolation at that pH was carried out. Also, at various temperature ranges and ionic strengths such as 0°C, 25°C,37°C and 10%, 20%, 30%, 40%, 50%, and 60% respectively using ammonium sulphate. After studying the entire variables, one from each variable with maximum enzymatic activity was selected by performing enzymatic assay using specific substrate. GROSS QUANTIFICATION OF TOTAL PROTEIN EXTRACTED Gross quantification of total protein extracted was done by using Biuret method for total protein estimation. For estimating protein concentration, calibration of BSA i.e. bovine serum albumin was carried out. Calibration of bovine serum albumin was used to determine unknown concentration of protein in enzyme sample. BSA concentration 0 (blank), 1mg/ml, 2mg/ml, 3mg/ml, 4mg/ml and 5mg/ml were taken in different glass tubes. un co rr cte d p roo f BSA stock solution 10mg/ml was prepared using distilled water. From this stock solution, 0 ml, 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml were added respectively. 1 ml, 0.9 ml, 0.8 ml, 0.7 ml, 0.6 ml, 0.5 ml water was added respectively to above solutions. To each test tube, 2 ml of biuret reagent was added. All the samples were incubated at 25°C for 10min and then the absorbance was recorded at 542nm using UV visible spectrophotometer (Jasco V630). ENRICHMENT OF ISOLATED PROTEIN The enzyme tyrosine hydroxylase after centrifugation was not in purified form. It was necessary to carry out purification process to eliminate other enzymes other than target enzyme. Purification of the enzyme was carried out by dialysis, column chromatography process and ammonium sulphate precipitation. The supernatant was collected and stirred using magnetic stirrer continuously at 25oC and addition of saturated ammonium sulfate was slowly carried out to bring the final concentration to 10%, 20%, 30%, 40%, 50%, and 60% respectively. Stirring was stopped once final concentration was reached and the solution was kept overnight for precipitation. The solution was centrifuged at 2000 RPM for 10 minutes on the next day so that precipitate can settle down. Pellets were collected after discarding the supernatant which contains targeted precipitated protein. By ammonium sulfate precipitation, the targeted protein gets precipitated out by altering their solubility in presence of high salt concentration. This precipitated protein was loaded in dialysis bag having 25.4 mm diameter and 42.44 mm width. The dialysis bag was kept overnight against 0.2 M sodium acetate buffer with continuous stirring by maintaining 25oC temperature and 5.8 pH conditions. The dialysis buffer was replaced after every 2 hours with freshly prepared buffer. By dialysis using a membrane which is semi permeable membrane, unwanted and small compounds from macromolecules get removed from solution by passive as well as selective diffusion. In dialysis bag, the pure targeted protein was left out which was further purified by column chromatography. ENRICHMENT OF PROTEIN BY COLUMN CHROMATOGRAPHY Sephadex G-75 column was prepared by using 0.25M dextrose solution. It was equilibrated for 24 hours at 25°C. The above collected precipitate was re-suspended in sodium acetate buffer and passed through the column. The flow rate of 0.5ml/min was maintained. The eluent fractions were collected at 10 minutes interval each. The collected fractions were used for determination of enzyme by performing assay.14 CALIBRATION OF L-TYROSINE Enzymatic activity assay protocol was developed on the basis of tyrosine hydroxylase specific substrate i.e. Ltyrosine. Enzymatic activity was determined indirectly by measuring the concentration of L-tyrosine. The Maximum wavelength of L-tyrosine having concentration 2mg/ml was determined in sucrose solution which was found to be 275nm. For determining unknown L-tyrosine concentration, calibration of L-tyrosine was plotted using 2,4,6,8,10 μg/ml concentration in 5.6 pH,5.8 pH,6.0 pH,6.2 pH,6.4 pH values. ENZYMATIC ACTIVITY ASSAY For determination of enzymatic activity and L-tyrosine content, the dialyzed enzyme and purified enzyme were taken by column chromatography using various parameters like pH of 5.6 ,5.8 ,6.0 ,6.2 ,6.4, at temperature conditions 0oC ,25oC,37oC and ionic strength of 10%, 20%, 30%, 40%, 50%, and 60% respectiv

Isolation, bioassay development and applications of tyrosine hydroxylase