ISOLATION AND PURIFICATION OF GLUTATHIONE S-TRANSFERASE FROM RAT LIVER

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1 Journal of Al-Nahrain University Vol.12 (4), December, 29, pp Science ISOLATION AND PURIFICATION OF GLUTATHIONE S-TRANSFERASE FROM RAT LIVER Essam F. A. Al-Jumaily, Zahraa F. Ameen and Ayad H. Jassim* Biotechnology Department Genetic Engineering and Biotechnology Institute, Baghdad University, Baghdad-Iraq. *Chemistry Department Science College, Al-Nahrain University, Baghdad-Iraq. Abstract Glutathione S-transferase (EC ) was purified to homogeneity from rat liver. The enzyme was initially separated by a preparative anion exchange chromatographic step using DEAE- Cellulose, then cation exchange chromatographic CM-Cellulose was used, followed by subsequent chromatography on Sephacryl S-2, the enzyme was purified about fold with an 56.43% yield. The purified enzyme had a specific activity of 125x1-3 unit/mg protein, indicated that the enzyme is momoner of Mr (5) Daltons SDS gel electrophoresis studies. Key words: Glutathione-S-transferase; isolation, purification, molecule weight, rat liver. Introduction Glutathione S-transferases (GSTs), are a family of enzymes that play an important role in detoxification by catalyzing the conjugation of many hydrophobic and electrophilic compounds with reduced glutathione (1-3). The glutathione transferases are normally present in large quantities, representing about 1 of the extractable protein of rat liver but can be induced to greater than 2. Most researches have been carried out with enzymes from rat and human liver. But human erythrocytes and placenta, as well as sheep and mouse liver have also been sources for homogenous preparations. The enzymes have been found in all mammalian tissue tested as well as in insects, protozoa, algae, fungi, and bacteria (4). In the body biotransformation is an important biochemical protection mechanism against toxic chemical species. Cells possess an impressive array of enzymes capable of biotransforming a wide range of different chemical structures and functionalities. The enzymic detoxification of xenobiotics has been classified into three distinct phases which act in a tightly integrated manner (5). Phases I and II involve the conversion of a lipophilic, nonpolar xenobiotic into a more water-soluble and therefore less toxic metabolite, which can then be eliminated more easily from the cell (phase III). Phase I is catalyzed mainly by the cytochrome P45 system (5). This family of microsomal proteins is responsible for a range of reactions, of which oxidation appears to be the most important. Phase II enzymes catalyse the conjugation of activated xenobiotics to an endogenous water-soluble substrate, such as reduced glutathione (GSH), UDP-glucuronic acid or glycine (5). Quantitatively, conjugation to Glutathione (GSH, γ- glutamylcysteinylglycine) (6), which is catalysed by the GSTs, is the major phase II reaction in many species. GSTs can catalyse nucleophilic aromatic substitutions, Michael additions to a unsaturated ketones and epoxide ring-opening reactions, all of which result in the formation of GSH conjugates and the reduction of hydroperoxides, resulting in the formation of oxidized glutathione (GSSG) (5,7). The aim of this work to isolation and purification of glutathione S-transferase (GSTs) from rat liver to used in another project to found the interaction of GSH with the prepared complexes [Pt (IV), Au(III), pd(ii) and Cu(II)] in presence and absence of the enzyme. Materials and Methods Assay Method The enzyme activity was determined as described by (Habig.1974) (8). Determination of enzyme volume: To a one ml cuvette was added μl of.1 M potassium phosphate buffer ph 6.5, 5 μl of 2mM GSH and 5 μl of 2 mm CDNB (1-chloro-2,4-dinitro benzene). The 137

2 Essam F. A. Al-Jumaily reaction, which was carried out at 3 o C, was started by addition of a suitable amount of enzyme (5-5 μl); the final volume was 1ml. The reaction was monitored spectrophotometrically by the increase in the absorbance at 34 nm (ε = 9.6 mm -1 cm -1 ) Fig.(1). Correction for the spontaneous activity was made by subtracting the rate in the absence of enzyme (4). Determination of enzyme activity: The same steps above were done using 865 µl of potassium phosphate.1 M, ph 6.5 and 35 µl of enzyme. Unit of enzyme activity was defined as the amount of enzyme that catalyzes the formation of 1 μmol of S-2, 4-dinitrophenylglutathione per minute at 3 o C using 1mM concentration of GSH and CDNB (8). A specific activity was defined as enzyme activity (units) per milligram of protein (4). Protein concentration was determined as described by (1). Preparation of Cytosol Fractions: Rat liver (17 g) was cut into small pieces and homogenized in 1 mm ice-cold Tris acid ph 8. (3 ml/g liver) in a blender. The homogenate was filtrated through 2 layers of gauze to remove connective tissue. The homogenate was centrifuged at 5, rpm for one hour. The resulting supernatant was then centrifuged for 3 minute. The cytosol fraction was obtained, and stored at -2 o C (8). Purification Procedures: The purification was made through the following three steps: Ion Exchange Chromatography by DEAE- Cellulose: The ion exchanger diethylaminoethyl cellulose (DEAE-cellulose) was prepared according to the manufacturing company (Whatman) and as described by (11). Procedure (8) : The crude GSTs was dialyzed against (1 mm) Tris-HCl (ph 8.) then 34 ml of this sample was loaded onto the column (3 2.5cm) carefully until passed the exchanger. Then (15 ml) of (1 mm) Tris- HCl was added, followed by addition (15 ml) of a gradient from (1 mm) Tris-HCl buffer to (1 mm) Tris buffer containing.2 M NaCl. Fractions of (5 ml) were collected in test tubes. The absorbance in each fraction was measured spectrophotometrically at 28nm, fractions of the protein peaks were assayed for GSTs activity and Protein concentration. Fractions containing enzymatic activity were collected, and stored at 5 o C for further purification. Ion Exchange Chromatography by CM- Cellulose: The ion exchanger was prepared according to the manufacturing company (whatman) as described by (11). Procedure: The sample (2ml) was loaded onto the column (3 2.5 cm) carefully until passed the exchanger. Then (15 ml) of phosphate buffer (1 mm) (ph 6.7) was added, protein was eluted by using (26 ml) of a gradient from (1 mm) to (4 mm) phosphate buffer (ph 6.7). Fractions of (5 ml) were collected in test tubes. The absorbance in each fraction was monitored spectrophotometrically at 28 nm, fractions of the protein peaks were assayed for GSTs activity and protein concentration. The fractions containing the enzymatic activity were collected, and concentrated by ultrafiltration (12). Gel Filtration Chromatography on Sephacryl S-2 Column: Sephacryl S-2 column (63X2 cm) was prepared and packed according to the instruction of the manufacturing company (Pharmacia Fine Chemical) (13). Procedure (14,15) : A sample (1 ml) of partially purified GSTs was added to the Sephacryl S-2 column, carefully using pasture pipette. The elution of the protein was done with the application of (2 ml) phosphate buffer (.1 M) (ph 6.7). Fractions of (5 ml) were collected in test tubes. The absorbance in each fraction was monitored spectrophotometrically at 28 nm and the fractions of the protein peaks were assayed for GSTs activity and Protein concentration. Fractions containing the enzymatic activity were collected. Polyacrylamide Gel Electrophoresis under Denatured Conditions: In order to test the purity and to determined the molecular weight of the GSTs obtained 138

3 Absorbance at 34 nm Journal of Al-Nahrain University Vol.12 (4), December, 29, pp Science from the gel filtration step, protein polyacrylamide gel electrophoresis was performed for the purified enzyme (9,16,17). To determine the molecular weight of Glutathione S-transferase enzyme, The distance of the bromophenol blue dye transfer from the top of the gel to the center of the dye band was measured. Also the distance from the top of the gel to the center of separated standard protein bands was measured. The relative mobility (R m ) was calculated for each protein according to the following equation: Relative mobility (R m ) = distance of protein / distance of bromophenol blue dye The relation between R m and log molecular weight of the standard protein was drown, then the molecular weight of the enzyme was determined by using the R m value of the enzyme from the standard curve. Results and Discussion Determination of Enzyme Volume for Enzyme Assay: GSTs activity was determined for the supernatant of the crude sample, it has been noticed that 35 µl is the best volume of crude enzyme which gave the highest absorption at 34 nm as shown in Fig.(1), and this volume was used to measure the enzyme activity in the next steps of purification Volume of Enzyme (µl) Fig.(1): Standard curve of enzyme activity at different volumes after 3 seconds. Crude Extract: After determining GSTs activity and protein concentration for the supernatant of the crude sample, it had been found that the crude sample had demonstrated a specific activity of U/mg protein with fold (1) and 1% yield, as shown in Table (1). Purification by Ion Exchange Chromatography: A- DEAE-Cellulose: The first step in Purification of GSTs was done by using ion exchange chromatography using DEAE-Cellulose column (18), after being dialyzed. Fig.(2) shows the protein peaks obtained from ion exchang chromatography using DEAE-Cellulose and it reflects the presence of three proteins by reading the absorbance at (28 nm). Estimation of GSTs activity in all eluted protein peaks showed that the first peak contain GSTs activity. While the second and third peaks, which appeared after addition of.2 M NaCl in the same buffer, contain no GSTs activity. Thus DEAE-Cellulose column retained about 8% of the applied protein whereas enzyme activity, as assayed by 1-chloro-2, 4-dinitrobenzene, was not adsorbed (9). The fractions of the first peak were then all collected and after determining GSTs activity (and protein concentration, it was found that the partially purified enzyme had demonstrated a specific 139

4 Absorbance at 28 nm (unit /ml) Absorbance at 28 nm (Unit / ml) Essam F. A. Al-Jumaily activity of U/mg protein with purification folds and 96.45% over all yields, as shown in Table (1). 3 Absorbance Wash Elution Fraction NO. Fig.(2): Purification of crude GSTs on DEAE-Cellulose column (3 2.5cm). the column was eluted by using 1mM tris buffer (ph 8.), then eluted by using gradient of 1mM tris buffer (ph 8.) containing.2 M NaCl, at flow rate of (5 ml/hour). The second step in the purification of GSTs was done by ion exchange chromatography using CM-Cellulose column. The results shown in Fig.(3), indicate that after washing with (15 ml) of phosphate buffer (1 mm) (ph 6.7), two protein peaks were observed by reading the absorbance at (28 nm). Plotting (26 ml) gradient of phosphate buffer (4 mm) (ph 6.7), two further protein peaks were obtained. Estimation of the GSTs activity in all protein peaks showed that the first couple washing peaks contains no GSTs Absorbance Wash Elution Fraction NO. Fig.(3): Purification of GSTs on CM-Cellulose column (3 2.5cm). The column was eluted by using 1mM phosphate buffer (ph 6.7), then eluted by using gradient of 4mM phosphate buffer (ph 6.7), at flow rate of (5ml/hour). 14

5 Absorbance at 28 nm (unit / ml) Journal of Al-Nahrain University Vol.12 (4), December, 29, pp Science activity, while GSTs activity for the second couple was found to be concentrated in the second peak fractions. Therefore the fractions of the second peak of couple two were collected and after determining GSTs activity and protein concentration, it had been found that the partially purified enzyme had demonstrated a specific activity of ( U/mg protein) with (24.85) purification folds and yield of GSTs of (64.59%)., as shown in Table (1). Partially purified GSTs represented by the collected fractions from step B above were subjected to further purification using gel filtration column (19). By reading the absorbance at (28 nm), two major protein peaks were observed as shown in Fig.(4). Estimation of the GSTs activity in the two peaks showed that the first one contains no GSTs activity, and it was found to be concentrated in the second one. The fractions of the second peak were then collected and after determining GSTs activity and protein concentration it had been found that the purified enzyme had demonstrated a specific activity of U/mg protein with purification folds and 56.43% over all yields, as shown in Table (1)..25 Absorbance Fraction No. Fig.(4) : Purification of GSTs on Sephacryl S-2 column (63 2cm). Eluent:.1M phosphate buffer (ph 6.7), at a flow rate of (3ml/hour). 141

6 Purification Step Vol. (ml) Table (1) Purification steps of Glutathione S-transferase. (U/ml) X1-3 Protein Conc. (mg/ml) Specific (U/mg)x1-3 Total (U)X1-3 Essam F. A. Al-Jumaily Purific ation Fold Yield (%) Crude Extract Ion exchange Chromatography (DEAE- Cellulose) Ion exchange Chromatography (CM-Cellulose) Gel Filtration (Sephacryl S-2) The purity of the purified GSTs was determined by polyacrylamide gel electrophoresis under denatured conditions. The purified enzyme preparation showed only single protein band on the gel after staining with commassie brilliant blue R-25, indicating the probability of reaching the apparent homogeneity of purification process Fig.(5).The location of the protein band on the gel could be as a result of the low molecular weight of the protein, since the electrophoresis mobility of protein in the gel essentially depends on the molecular weight of the protein in addition to its net charge (2), this low molecular weight was determined by comparison with standard protein of known molecular weight. Fig.(5): The Electrophoresis of patter GSTs enzyme taking from mouse liver in acrylamide gel in presence of SDS and mercaptoethanol and determining the molecular weight of purified enzyme. The standard curve Fig.(6) of standard proteins in acrylamide gel in presence of SDS, was used to calculate the molecular weight of the enzyme subunit by electrophoresis method. The molecular weight obtained was 5 Dalton. 142

7 Logarithm of Molecular weight Journal of Al-Nahrain University Vol.12 (4), December, 29, pp Science 5 Phosphorylase-b (94) 4.8 Albomin (67) 4.6 GSTs Ovalbomin (432) Carbonic anhydrase (3) 4.4 Trypsin-inhibitor (2) 4.2 α-lactoalbumin (144) References [1] J.H. Keen, W.H. Habig, and W.B.Jakoby, The Journal of Biological Chemistry, 251(2), Issue of October 25, 6183, (1976). [2].M.Warholm, H. Jensson, M.K.Tahir, and B. Mannervik, Biochemistry, 25, 4119 (1986). [3] J. Csiszar, M. Szabo, E.llles and K. Kurucz, Acta. Biologica Szegediensis, 46(3-4), 79, (22). [4] S.P.Colowick and N.O.Kaplan, "Methods in Enzymology", 113, (1985). [5] D. Sheehan, G. Meade, V.M.Foley and C.A.Dowd, Biochem. J., 36, 1, (21). [6] C.LU. Shelly, The FASEB Journal, 13, 1196, (1999). [7] J. A. Redick, W. B. Jakoby and J. Baron, The journal of Biological Chemistry, 257(24), Issue of December 25, 152, (1982). [8] W. H. Habig, M. J. Pabst and W. B. Jakoby, The journal of Biological Chemistry, 249(22, 25), 713, (1974). [9] Y.R.Al-Hussuna, M.Sc. Thesis, Al- Nahrain University, Iraq (25). [1] M. M. Bradford, Anal. Biochem., 72, 248, (1976). Relative Mobility (Rm) Fig.(6) : Molecular weight determination for purified GSTs extracted from rat liver, using acrylamide gel in presence of SDS. [11] J. R. Whitaker and R. A. Bernhard. "Experiments for: An Introduction to Enzymology". The Whiber Press, (1972). [12] M. J. Pabst, W. H. Habig and W. B. Jakoby, The Journal of Biological chemistry, 249(22), Issue of November 25, 714, (1974). [13] K.H.Tan, D.J.Meyer, J.Belin and B.Ketterer, Biochem.J., 22, 243, (1984). [14] R. P. Saneto, Y. C. Awasthi and S. K. Srivastava, Biochem.J., 25, 213, (1982). [15] F. H. M. Al-Shikirhy, M. Sc. Thesis, Baghdad University, Iraq (24). [16] P. C. Simons and D. L. Vander Jagt, Analytical Biochemistry, 82, 334, (1977). [17] Dayagi and Y. Degini, "The Chemistry of Carbon-Nitrogen Double Bond", S. Patai, John Wiley and Sons interscience, New York, 64 (197). [18] A. A. Al-Sheikhly. Ph. D. Thesis, Saddam University, (1999). [19] N. H. Al-Mudallal, E. F. A. Al-Jumaily, A. W. R. Hamad, S. J. Hamza, Al- Mustansiriya J.Sci., 14(1), (23). 143

8 Essam F. A. Al-Jumaily [2] P. J. Blackshear, "Systems for polyacrylamide gel electrophoresis. In: Methods in Enzymology'. (ed. Jakoby W.B.) 14, 237, Academic Press, New York (1984). (E.C ) S (SDS) DEAE-Cellulose CM-Cellulose S