Journal of Biochemical and Biophysical Methods, 20 (1990) 157-169 Elsevier
t57
JBBM 00789
Coisolation of glutathione peroxidase, catalase and superoxide dismutase from human erythrocytes Terence M. Stepanik and Donald D. Ewing Medical Biophysics Branch, Whiteshell Nuclear Research Establishment, Atomic EnerD, of Canada Ltd., Pinawa, Manitoba. Canada
(Received 26 September 1989) (Accepted 20 October 1989)
Summary Glutathione peroxidase (GSH-Px; glutathione:hydrogen peroxide oxidoreductase; EC 1.11.1.9), catalase (H 202 : H 202 oxidoreductase; EC 1.11.1.6) and superoxide dismutase (superoxide: superoxide oxidoreductase; EC 1.15.1.1) were coisolated from human erythrocyte lysate by chromatography on DEAE-cellulose. Glutathione peroxidase was separated from superoxide dismutase and catalase by thiol-disulfide exchange chromatography and then purified to approximately 9070 homoge~eity by gel permeation chromatography and dye-ligand affinity chromatography. Catalase and superoxide dismutase were separated from each other and purified further by gel permeation chromatography. Catalase was then purified to approximately 9070 homogeneity by ammonium sulfate precipitation and superoxide dismutase was purified to apparent homogeneity by hydrophobic interaction chromatography. The results for glutathione peroxidase represent an improvement of approximately 10-fold in yield and 3-fold in specific activity compared with the established method for the purification of this enzyme. The yields for superoxide dismutase and catalase were high (45 mg and 232 rag, respectively, from 820 ml of washed packed cells), and the specific activities of both enzymes were comparable to values found in the literature. Key words: Protein/enzyme purification; Glutathione peroxidase: Catalase: Superoxide dismutase
Introduction The enzymes copper, zinc superoxide dismutase (SOD, superoxide: superoxide oxidoreductase; EC 1.15.1.1), catalase (CAT, H202" H202 oxidoreductase; EC Correspondence address: T.M. Stepanik, Station 80, Whiteshell Nuclear Research Establishment, Pinawa, Manitoba, Canada ROE 1LO. Abbreviations: SOD, copper, zinc superoxide dismutase; CAT, catalase; GSH-Px, glutathione peroxidase; DE-52, DEAE-ceilulose; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis: NBT, nitro blue tetrazolium.
0165-022X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
158
1.11.1.6) and glutathione peroxidase (GSH-Px, glutathione: hydrogen-peroxide oxidoreductase; EC 1.11.1.9) play a major role in protecting aerobic cells against reactive oxygen intermediates [1-3]. SOD catalyzes the dismutation of the superoxide anion radical to 02 and H202 [4], and CAT converts H202 to 02 and H20 [5]. GSH-Px catalyzes the reduction of H202 to H2O at the expense of reduced glutathione [6-8]. In addition, a number of other hydroperoxides, including those originating from unsaturated fatty acids, thymine, and DNA, also serve as substrates for GSH-Px [9-11]. SOD and CAT may possess therapeutic value as anti-inflammatory agents and free radical scavengers during organ reperfusion [12-14]. They also appear to play a role in the modification of radiation injury [15-18]. The importance of GSH-Px to the cellular antioxidant defence mechanism has been demonstrated [19,20]. In addition, GSH-Px, SOD and CAT seem to interact in a mutually supportive fashion in providing defence against oxygen toxicity [21]. However, the determination of a therapeutic or radioprotective effect for human GSH-Px has been hindered by the lack of a simple, rapid method for the isolation of significant quantities of this enzyme. This report describes a method for the coisolation of human erythrocyte GSH-Px, SOD and CAT in high yield. The procedure uses conventional purification techniques, avoids the use of linear gradients, and is suitable for scale-up.
Materials and Methods
Preparation of cell lysate All steps were carried out at 4°C. All buffers were prepared and adjusted to their given pH values at ambient temperature. The cell lysate was prepared for chromatography by using methods similar to those described by other investigators [22,23]. Four units of outdated packed red blood cells (3-8 weeks old, Canadian Red Cross, Winnipeg, Manitoba) were washed three times with two volumes of 0.9¢~ saline. The washed packed cells were hemolyzed by addition of three volumes of distilled deionized water. After stirring for 30 min, the cell stroma was removed by centrifugation. Chromatography on DEA E-cellulose The hemolysate was dialyzed against 0.1 mM K 2HPO4 until its conductivity was one-half that of the buffer (1.5 mM potassium phosphate, (pH 6.7)) used to equilibrate the DEAE-cellulose (DE-52). The hemolysate was then recentrifuged to remove a small amount of precipitate. GSH-Px, SOD, and CAT were adsorbed onto 500 ml of DE-52 gel by a batch procedore. After 2 h of gentle stirring, the gel was washed with 1.5 volumes of 1.5 mM potassium phosphate (pH 6.7) under suction on a Buchner funnel. The gel was packed into a 5-cm diameter column and the three enzymes were released from the gel by elution with 50 mM potassium phosphate (pH 6.7) at 50 ml/h. Fractions containing the three enzymes were pooled, made up to 200 mM in NaCI, and concentrated to 20 ml by pressure ultrafiltration in an
159
Amicon cell equipped with a PM-10 membrane in preparation for thiol-disulfide exchange chromatography.
Preparation of activated thiol sepharose Glutathione-Sepharose was prepared using a procedure described by Sundberg and Porath [24]. Following epoxy activation and washing, 50 ml of activated Sepharose was coupled with one gram of oxidized glutathione at 55°C for 20 h. The gel was converted to the mixed disulfide form (activated thiol Sepharose) by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) [25]. The content of reactive thiol groups determined according to Laurell et al. [25], was found to be 12 ~amol/ml of gel.
Thiol-disulfide exchange chromatography The concentrated DE-52 pool (20 ml) containing ¢3SH-Px, SOD and CAT was first desalted on a column (2.5 x 25 cm) of Sephadex G-25, which was equilibrated and eluted with 50 mM potassium phosphate, 200 mM NaCI, 0.1 mM EDTA (pH 6.7, buffer A) at 60 ml/h. The protein-containing eluant, visible as a reddish-brown band on the column, was diverted, upon elution, directly onto a column (1.5 x 12 cm) of activated thiol Sepharose, also equilibrated and eluted with buffer A at 60 ml/h. This column was then uncoupled from the G-25 column. The thiol Sepharose gel was washed with one column volume of buffer A to elute unbound protein, including SOD and CAT. The gel was then washed with 0.5 volume of 50 mM Tris. HCI, 200 mM NaCI, 0.1 mM EDTA (pH 8.3, buffer B). This was followed by elution with 2 volumes of buffer B containing 50 mM 2-mercaptoethanol to release bound protein (including GSH-Px). The eluant, containing released protein, was diverted through a second G-25 column (1.5 x 60 cm) equilibrated with 50 mM potassium phosphate, 50 mM NaCI, 0.1 mM EDTA, 0.3 mM GSH (pH 6.0, buffer C).
Purification of GSH-Px Fractions containing protein eluted from the second G-25 column were pooled (31 ml), concentrated on PM-10 to 6 ml, clarified by centrifugation and chromatographed in buffer C on Sephacryl S-200 (2.5 x 105 cm) at 25 ml/h. Fractions containing greater than 255 of the GSH-Px activity found in the peak fraction were pooled (29.5 ml), and applied to a column (1.5 x 6 cm) of DyematrexTM green A which was then washed at 50 ml/h with 2.5 volumes of buffer C to remove unbound protein. GSH-Px was released from the gel by eluting with 2.5 volumes of buffer C (pH 7.5). The GSH-Px fractions were pooled (16.4 ml), concentrated to 5.3 ml, made 0.1~ (w/v) in sodium azide and stored at 4°C. Contaminating protein remaining bound to the gel was removed by washing with 2 volumes of 50 mM potassium phosphate, 1000 mM KCI (pH 7.5).
Purification of CA T and SOD The protein fraction (32 ml) that had not bound to activated thiol Sepharose, was chromatographed at 40 ml/h on a separate Sephacryl S-200 column (3.5 x 105 cm)
160
in buffer A containing 0.3 mM GSH. CAT fractions, with absorbance ratios A 4oJA 280> 0.95, were pooled (48.5 ml) and taken for ammonium sulfate precipitation. Protein precipitating between 35-50% saturation with ammonium sulfate was collected by centrifugation, redissolved in 6 ml of buffer A, dialyzed against buffer A overnight and stored as a concentrated solution (9.6 ml) at 4°C. The SOD fractions from the S-200 chromatography were pooled (50 ml), concentrated to 8 ml, and diluted 1 : 1 (v/v) with 1400 mM potassium phosphate (pH 7.3). A precipitate was removed by passing the solution through a filter (0.8/~m, Miilex-PF). The clarified solution was applied to a phenyl Sepharose column (1.5 x 14 cm). SOD was eluted at 40 m l / h with 2 volumes of a 1 : 1 (v/v) mixture of buffer A and 1400 mM potassium phosphate (pH 7.3). The gel was then washed with 1.5 volumes of 10 mM potassium phosphate (pH 7.3) and 2 volumes of 50% (v/v) ethylene glycol/water. SOD fractions were pooled (20 ml), dialyzed or desalted on Sephadex G-25 into 15 mM potassium phosphate, 150 mM Na('! (pH 7.3), concentrated to 5.7 ml, made 0.1~ (w/v) in sodium azide and stored at 4°C.
Enzyme assays
GSH-Px activity was determined by the coupled assay procedure of Paglia and Valentine [26] as modified by Wendel [27], except that, after activation at 37°C for 10 min, the samples were cooled in air for 5 min and assayed at ambient temperature using t-butyl hydroperoxide as substrate. CAT activity was measured spectrophotometrically with H202 [28]. SOD activity was determined by measurement of the inhibition, by SOD, of the photocatalyzed reduction of nitro blue tetrazolium, (NBT) [29]. Glutathione transferase activity was measured spectrophotometrically [30] with l-chloro-2,4.dinitrobenzene.
Protein determination The protein content of the crude isolates and the final GSH-Px and SOD preparations was measured by the Coomassie protein assay procedure of Bradford [31], using reagents from Pierce (Rockford, IL). Bovine gamma-globulin was used as the standard for all samples, except the final SOD preparation, for which bovine SOD was used. The protein content of the purified CAT preparation was calculated from the absorbance at 405 nm using: l~l~! c m _ 17.1 [32].
SDS-PA G electrophoresis SDS slab gel electrophoresis was performed using a modification [33] of the Laemmli procedure [34]. Gels were stained with 0.05~ (w/v) Coomassie Brilliant Blue R250.
Analytical HPLC gel permeation chromatography Samples (50/~1) containing 75/~g of GSH-Px or 160/~g SOD were run at ambient temperature on a Biosil TSK 250 column (7.5 × 600 mm) using a Waters HPLC system composed of a M680 controller, MS10 pump, M490 detector, a Rheodyne 7125 injector and an SP4270 integrator. The column was eluted at 1.0 ml/min with 50 mM potassium phosphate, 200 mM NaCI, 0.1 mM EDTA (pH 7.0).
161
Materials
DEAE-cellulose (DE-52) was purchased from Whatman, Clifton, NJ. Sephadex G-25, Sepharose 4B, Sephacryl S-200, and phenyl Sepharose were obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. DyematrexTM green A was from Amicon, Danvers, MA. Bovine gamma-globulin used for protein determination was from Bio-Rad, Richmond, CA; bovine Cu, Zn SOD from Miles, Elkhart, IN; human erythrocyte Cu, Zn SOD from Isolab, Akron, OH, and bovine GSH-Px from Calbiochem, San Diego, CA. The molecular weight standards for HPLC were from Sigma, St. Louis, MO, while those used for SDS-PAGE were from Bio-Rad. All other chemicals were standard commercial products of high quality.
Results and Discussion The results for the purification procedure are given in Table 1. It was not possible to determine accurate values for GSH-Px activity in intact human erythro-
TABLE 1 PURIFICATION OF GLUTATHIONE PEROXIDASE, CATALASE AND SUPEROXIDE DISMUTASE a Step
Volume (ml)
Activity (units)
Protein (mg)
Specific activity (u/rag)
Yield (9~)
Giutathione peroxidase DE-52 TDEC b S-200 MDAC e Concentration
136 31 29.5 16.4 5.3
1200 c 620 470 360 280
2580 120 13.3 6.1 4.5
0.5 5.2 35.3 59.0 62.2
100 52 39 30 23
136 32 48.5
21500 • 17 300 18000
2 580 1830 350
8.3 9.5 51.6
100 80 84
13700
230
59.1
64
410000 r 330000 230000 170000
2580 ! 830 300 ND s
159 181 757 ND
100 80 56 41
45
3 500
39
Catalase DE-52 TDEC S-200 Ammonium sulfate precipitation
9.6
Superoxide dismutase DE-52 TDEC S.200 Phenyl sepharose Buffer exchange. concentration
136 32 50 20 5.7
160000
a Based on 820 ml of washed packed erythrocytes; b Thiol-disulfide exchange chromatography: c One unit equals 0.868.A[NADPH]'min-I'[GSH]o t [27]; d Matrex dye affinity chromatography; e Rate constant for a first order reaction k (s - t ) [28]: f One unit causes a 50~ inhibition of the photoreduction of NBT [29]; g ND. not determined.
162
cytes or in the diluted hemolysate under the experimental conditions (1 mM GSH, ambient temperature) used in the enzyme assay procedure. In addition, the SOD value determined for the diluted hemolysate after chloroform-ethanol treatment to remove hemoglobin was consistently less than that for the DE-52 pool which was not subjected to chloroform-ethanol treatment. Thus, for uniformity, the percentage yield for all three enzymes has been calculated by comparison with the enzyme activity found in the DE-52 pool prior to concentration. GSH-Px, CAT and SOD were separated from hemoglobin in the hemolysate by anion-exchange chromatography on DE-52. After concentrati61~ of the DE-52 pool, a low molecular weight component(s) capable of reducing the mixed disulfide in activated thiol Sepharose was removed by desalting on Sephade× G-25~ GSH-Px was retained by the activated thiol Sepharose gel, SOD and CAT were not. After washing the column to remove unbound protein, the pH was raised to 8.3 to permit efficient release of GSH-Px from the gel using 50 mM 2-mercaptoethanol. The eluant containing GSH-Px was then diverted through a seco~id Sephadex G-25 column to remove the 2-nitro-5-thiobenzoate anion, which coelutes with GSH-Px from the thiol Sepharose column [25], and to promote rapid buffer exchange into buffer C, which is used in the final two steps of the purification procedure. Thiol-disulfide exchange chromatography has been used to purify GSH-Px from rat liver [35]. A recovery of 37~ was realized by applying the sample at pH 7.0 and eluting GSH-Px at the same pH with 0.3 mM cysteine. For native human erythrocyte GSH-Px, elution with 50 mM 2-mercaptoethanol at pH 8.3 results in a recovery of 52~. Similar recoveries were found starting with 6 units of red cells, indicating that the capacity of the column had not been exceeded. Following chromatography on activated thioi Sepharose, GSH-Px was chromatographed on Sephaeryl S-200 (Fig. 1). Fractions containing at least 25~ of the activity of the peak fraction were pooled and chromatographed on Matrex-gel green A (Fig. 2). Matrex gel red A could substitute for green A, but the other Matrex gels (blue A, blue B,and orange A) and bromosulfophthalein-glutathione agarose failed to bind GSH-Px at pH 6.0. After removal of unbound protein by washing with buffer C at pH 6.0, GSH-Px was released from the gel by using buffer C at pH 7.5. The enzyme could also be released at pH 6.0 by increasing the NaCI concentration of buffer C to 250 mM (results not shown). These results are consistent with a binding mechanism involving predominantly ionic interactions. Such interactions have been identified as being important for the binding of proteins by dye-ligand adsorbents [36l. A purification procedure for human erythrocyte GSH-Px has been reported by Awasthi et al. [37]. These authors stated that GSH-Px does not bind to ion-exchange resins like DEAE-cellulose (DE-52) in the presence of hemoglobin and consequently used two ammonium sulfate precipitations and cation-exchange chromatography to remove hemoglobin. We have found that GSH-Px binds to DE-52 in the presence of hemoglobin providing the ionic strength of the hemolysate is sufficiently low (less than or equal to that of a 5 mM potassium phosphate buffer, pH 6.7). In Awasthi et al.'s procedure, anion-exchange chromatography, gel permeation chromatography and a second anion-exchange chromatography were used to complete the isolation
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t57
JBBM 00789
Coisolation of glutathione peroxidase, catalase and superoxide dismutase from human erythrocytes Terence M. Stepanik and Donald D. Ewing Medical Biophysics Branch, Whiteshell Nuclear Research Establishment, Atomic EnerD, of Canada Ltd., Pinawa, Manitoba. Canada
(Received 26 September 1989) (Accepted 20 October 1989)
Summary Glutathione peroxidase (GSH-Px; glutathione:hydrogen peroxide oxidoreductase; EC 1.11.1.9), catalase (H 202 : H 202 oxidoreductase; EC 1.11.1.6) and superoxide dismutase (superoxide: superoxide oxidoreductase; EC 1.15.1.1) were coisolated from human erythrocyte lysate by chromatography on DEAE-cellulose. Glutathione peroxidase was separated from superoxide dismutase and catalase by thiol-disulfide exchange chromatography and then purified to approximately 9070 homoge~eity by gel permeation chromatography and dye-ligand affinity chromatography. Catalase and superoxide dismutase were separated from each other and purified further by gel permeation chromatography. Catalase was then purified to approximately 9070 homogeneity by ammonium sulfate precipitation and superoxide dismutase was purified to apparent homogeneity by hydrophobic interaction chromatography. The results for glutathione peroxidase represent an improvement of approximately 10-fold in yield and 3-fold in specific activity compared with the established method for the purification of this enzyme. The yields for superoxide dismutase and catalase were high (45 mg and 232 rag, respectively, from 820 ml of washed packed cells), and the specific activities of both enzymes were comparable to values found in the literature. Key words: Protein/enzyme purification; Glutathione peroxidase: Catalase: Superoxide dismutase
Introduction The enzymes copper, zinc superoxide dismutase (SOD, superoxide: superoxide oxidoreductase; EC 1.15.1.1), catalase (CAT, H202" H202 oxidoreductase; EC Correspondence address: T.M. Stepanik, Station 80, Whiteshell Nuclear Research Establishment, Pinawa, Manitoba, Canada ROE 1LO. Abbreviations: SOD, copper, zinc superoxide dismutase; CAT, catalase; GSH-Px, glutathione peroxidase; DE-52, DEAE-ceilulose; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis: NBT, nitro blue tetrazolium.
0165-022X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
158
1.11.1.6) and glutathione peroxidase (GSH-Px, glutathione: hydrogen-peroxide oxidoreductase; EC 1.11.1.9) play a major role in protecting aerobic cells against reactive oxygen intermediates [1-3]. SOD catalyzes the dismutation of the superoxide anion radical to 02 and H202 [4], and CAT converts H202 to 02 and H20 [5]. GSH-Px catalyzes the reduction of H202 to H2O at the expense of reduced glutathione [6-8]. In addition, a number of other hydroperoxides, including those originating from unsaturated fatty acids, thymine, and DNA, also serve as substrates for GSH-Px [9-11]. SOD and CAT may possess therapeutic value as anti-inflammatory agents and free radical scavengers during organ reperfusion [12-14]. They also appear to play a role in the modification of radiation injury [15-18]. The importance of GSH-Px to the cellular antioxidant defence mechanism has been demonstrated [19,20]. In addition, GSH-Px, SOD and CAT seem to interact in a mutually supportive fashion in providing defence against oxygen toxicity [21]. However, the determination of a therapeutic or radioprotective effect for human GSH-Px has been hindered by the lack of a simple, rapid method for the isolation of significant quantities of this enzyme. This report describes a method for the coisolation of human erythrocyte GSH-Px, SOD and CAT in high yield. The procedure uses conventional purification techniques, avoids the use of linear gradients, and is suitable for scale-up.
Materials and Methods
Preparation of cell lysate All steps were carried out at 4°C. All buffers were prepared and adjusted to their given pH values at ambient temperature. The cell lysate was prepared for chromatography by using methods similar to those described by other investigators [22,23]. Four units of outdated packed red blood cells (3-8 weeks old, Canadian Red Cross, Winnipeg, Manitoba) were washed three times with two volumes of 0.9¢~ saline. The washed packed cells were hemolyzed by addition of three volumes of distilled deionized water. After stirring for 30 min, the cell stroma was removed by centrifugation. Chromatography on DEA E-cellulose The hemolysate was dialyzed against 0.1 mM K 2HPO4 until its conductivity was one-half that of the buffer (1.5 mM potassium phosphate, (pH 6.7)) used to equilibrate the DEAE-cellulose (DE-52). The hemolysate was then recentrifuged to remove a small amount of precipitate. GSH-Px, SOD, and CAT were adsorbed onto 500 ml of DE-52 gel by a batch procedore. After 2 h of gentle stirring, the gel was washed with 1.5 volumes of 1.5 mM potassium phosphate (pH 6.7) under suction on a Buchner funnel. The gel was packed into a 5-cm diameter column and the three enzymes were released from the gel by elution with 50 mM potassium phosphate (pH 6.7) at 50 ml/h. Fractions containing the three enzymes were pooled, made up to 200 mM in NaCI, and concentrated to 20 ml by pressure ultrafiltration in an
159
Amicon cell equipped with a PM-10 membrane in preparation for thiol-disulfide exchange chromatography.
Preparation of activated thiol sepharose Glutathione-Sepharose was prepared using a procedure described by Sundberg and Porath [24]. Following epoxy activation and washing, 50 ml of activated Sepharose was coupled with one gram of oxidized glutathione at 55°C for 20 h. The gel was converted to the mixed disulfide form (activated thiol Sepharose) by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) [25]. The content of reactive thiol groups determined according to Laurell et al. [25], was found to be 12 ~amol/ml of gel.
Thiol-disulfide exchange chromatography The concentrated DE-52 pool (20 ml) containing ¢3SH-Px, SOD and CAT was first desalted on a column (2.5 x 25 cm) of Sephadex G-25, which was equilibrated and eluted with 50 mM potassium phosphate, 200 mM NaCI, 0.1 mM EDTA (pH 6.7, buffer A) at 60 ml/h. The protein-containing eluant, visible as a reddish-brown band on the column, was diverted, upon elution, directly onto a column (1.5 x 12 cm) of activated thiol Sepharose, also equilibrated and eluted with buffer A at 60 ml/h. This column was then uncoupled from the G-25 column. The thiol Sepharose gel was washed with one column volume of buffer A to elute unbound protein, including SOD and CAT. The gel was then washed with 0.5 volume of 50 mM Tris. HCI, 200 mM NaCI, 0.1 mM EDTA (pH 8.3, buffer B). This was followed by elution with 2 volumes of buffer B containing 50 mM 2-mercaptoethanol to release bound protein (including GSH-Px). The eluant, containing released protein, was diverted through a second G-25 column (1.5 x 60 cm) equilibrated with 50 mM potassium phosphate, 50 mM NaCI, 0.1 mM EDTA, 0.3 mM GSH (pH 6.0, buffer C).
Purification of GSH-Px Fractions containing protein eluted from the second G-25 column were pooled (31 ml), concentrated on PM-10 to 6 ml, clarified by centrifugation and chromatographed in buffer C on Sephacryl S-200 (2.5 x 105 cm) at 25 ml/h. Fractions containing greater than 255 of the GSH-Px activity found in the peak fraction were pooled (29.5 ml), and applied to a column (1.5 x 6 cm) of DyematrexTM green A which was then washed at 50 ml/h with 2.5 volumes of buffer C to remove unbound protein. GSH-Px was released from the gel by eluting with 2.5 volumes of buffer C (pH 7.5). The GSH-Px fractions were pooled (16.4 ml), concentrated to 5.3 ml, made 0.1~ (w/v) in sodium azide and stored at 4°C. Contaminating protein remaining bound to the gel was removed by washing with 2 volumes of 50 mM potassium phosphate, 1000 mM KCI (pH 7.5).
Purification of CA T and SOD The protein fraction (32 ml) that had not bound to activated thiol Sepharose, was chromatographed at 40 ml/h on a separate Sephacryl S-200 column (3.5 x 105 cm)
160
in buffer A containing 0.3 mM GSH. CAT fractions, with absorbance ratios A 4oJA 280> 0.95, were pooled (48.5 ml) and taken for ammonium sulfate precipitation. Protein precipitating between 35-50% saturation with ammonium sulfate was collected by centrifugation, redissolved in 6 ml of buffer A, dialyzed against buffer A overnight and stored as a concentrated solution (9.6 ml) at 4°C. The SOD fractions from the S-200 chromatography were pooled (50 ml), concentrated to 8 ml, and diluted 1 : 1 (v/v) with 1400 mM potassium phosphate (pH 7.3). A precipitate was removed by passing the solution through a filter (0.8/~m, Miilex-PF). The clarified solution was applied to a phenyl Sepharose column (1.5 x 14 cm). SOD was eluted at 40 m l / h with 2 volumes of a 1 : 1 (v/v) mixture of buffer A and 1400 mM potassium phosphate (pH 7.3). The gel was then washed with 1.5 volumes of 10 mM potassium phosphate (pH 7.3) and 2 volumes of 50% (v/v) ethylene glycol/water. SOD fractions were pooled (20 ml), dialyzed or desalted on Sephadex G-25 into 15 mM potassium phosphate, 150 mM Na('! (pH 7.3), concentrated to 5.7 ml, made 0.1~ (w/v) in sodium azide and stored at 4°C.
Enzyme assays
GSH-Px activity was determined by the coupled assay procedure of Paglia and Valentine [26] as modified by Wendel [27], except that, after activation at 37°C for 10 min, the samples were cooled in air for 5 min and assayed at ambient temperature using t-butyl hydroperoxide as substrate. CAT activity was measured spectrophotometrically with H202 [28]. SOD activity was determined by measurement of the inhibition, by SOD, of the photocatalyzed reduction of nitro blue tetrazolium, (NBT) [29]. Glutathione transferase activity was measured spectrophotometrically [30] with l-chloro-2,4.dinitrobenzene.
Protein determination The protein content of the crude isolates and the final GSH-Px and SOD preparations was measured by the Coomassie protein assay procedure of Bradford [31], using reagents from Pierce (Rockford, IL). Bovine gamma-globulin was used as the standard for all samples, except the final SOD preparation, for which bovine SOD was used. The protein content of the purified CAT preparation was calculated from the absorbance at 405 nm using: l~l~! c m _ 17.1 [32].
SDS-PA G electrophoresis SDS slab gel electrophoresis was performed using a modification [33] of the Laemmli procedure [34]. Gels were stained with 0.05~ (w/v) Coomassie Brilliant Blue R250.
Analytical HPLC gel permeation chromatography Samples (50/~1) containing 75/~g of GSH-Px or 160/~g SOD were run at ambient temperature on a Biosil TSK 250 column (7.5 × 600 mm) using a Waters HPLC system composed of a M680 controller, MS10 pump, M490 detector, a Rheodyne 7125 injector and an SP4270 integrator. The column was eluted at 1.0 ml/min with 50 mM potassium phosphate, 200 mM NaCI, 0.1 mM EDTA (pH 7.0).
161
Materials
DEAE-cellulose (DE-52) was purchased from Whatman, Clifton, NJ. Sephadex G-25, Sepharose 4B, Sephacryl S-200, and phenyl Sepharose were obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. DyematrexTM green A was from Amicon, Danvers, MA. Bovine gamma-globulin used for protein determination was from Bio-Rad, Richmond, CA; bovine Cu, Zn SOD from Miles, Elkhart, IN; human erythrocyte Cu, Zn SOD from Isolab, Akron, OH, and bovine GSH-Px from Calbiochem, San Diego, CA. The molecular weight standards for HPLC were from Sigma, St. Louis, MO, while those used for SDS-PAGE were from Bio-Rad. All other chemicals were standard commercial products of high quality.
Results and Discussion The results for the purification procedure are given in Table 1. It was not possible to determine accurate values for GSH-Px activity in intact human erythro-
TABLE 1 PURIFICATION OF GLUTATHIONE PEROXIDASE, CATALASE AND SUPEROXIDE DISMUTASE a Step
Volume (ml)
Activity (units)
Protein (mg)
Specific activity (u/rag)
Yield (9~)
Giutathione peroxidase DE-52 TDEC b S-200 MDAC e Concentration
136 31 29.5 16.4 5.3
1200 c 620 470 360 280
2580 120 13.3 6.1 4.5
0.5 5.2 35.3 59.0 62.2
100 52 39 30 23
136 32 48.5
21500 • 17 300 18000
2 580 1830 350
8.3 9.5 51.6
100 80 84
13700
230
59.1
64
410000 r 330000 230000 170000
2580 ! 830 300 ND s
159 181 757 ND
100 80 56 41
45
3 500
39
Catalase DE-52 TDEC S-200 Ammonium sulfate precipitation
9.6
Superoxide dismutase DE-52 TDEC S.200 Phenyl sepharose Buffer exchange. concentration
136 32 50 20 5.7
160000
a Based on 820 ml of washed packed erythrocytes; b Thiol-disulfide exchange chromatography: c One unit equals 0.868.A[NADPH]'min-I'[GSH]o t [27]; d Matrex dye affinity chromatography; e Rate constant for a first order reaction k (s - t ) [28]: f One unit causes a 50~ inhibition of the photoreduction of NBT [29]; g ND. not determined.
162
cytes or in the diluted hemolysate under the experimental conditions (1 mM GSH, ambient temperature) used in the enzyme assay procedure. In addition, the SOD value determined for the diluted hemolysate after chloroform-ethanol treatment to remove hemoglobin was consistently less than that for the DE-52 pool which was not subjected to chloroform-ethanol treatment. Thus, for uniformity, the percentage yield for all three enzymes has been calculated by comparison with the enzyme activity found in the DE-52 pool prior to concentration. GSH-Px, CAT and SOD were separated from hemoglobin in the hemolysate by anion-exchange chromatography on DE-52. After concentrati61~ of the DE-52 pool, a low molecular weight component(s) capable of reducing the mixed disulfide in activated thiol Sepharose was removed by desalting on Sephade× G-25~ GSH-Px was retained by the activated thiol Sepharose gel, SOD and CAT were not. After washing the column to remove unbound protein, the pH was raised to 8.3 to permit efficient release of GSH-Px from the gel using 50 mM 2-mercaptoethanol. The eluant containing GSH-Px was then diverted through a seco~id Sephadex G-25 column to remove the 2-nitro-5-thiobenzoate anion, which coelutes with GSH-Px from the thiol Sepharose column [25], and to promote rapid buffer exchange into buffer C, which is used in the final two steps of the purification procedure. Thiol-disulfide exchange chromatography has been used to purify GSH-Px from rat liver [35]. A recovery of 37~ was realized by applying the sample at pH 7.0 and eluting GSH-Px at the same pH with 0.3 mM cysteine. For native human erythrocyte GSH-Px, elution with 50 mM 2-mercaptoethanol at pH 8.3 results in a recovery of 52~. Similar recoveries were found starting with 6 units of red cells, indicating that the capacity of the column had not been exceeded. Following chromatography on activated thioi Sepharose, GSH-Px was chromatographed on Sephaeryl S-200 (Fig. 1). Fractions containing at least 25~ of the activity of the peak fraction were pooled and chromatographed on Matrex-gel green A (Fig. 2). Matrex gel red A could substitute for green A, but the other Matrex gels (blue A, blue B,and orange A) and bromosulfophthalein-glutathione agarose failed to bind GSH-Px at pH 6.0. After removal of unbound protein by washing with buffer C at pH 6.0, GSH-Px was released from the gel by using buffer C at pH 7.5. The enzyme could also be released at pH 6.0 by increasing the NaCI concentration of buffer C to 250 mM (results not shown). These results are consistent with a binding mechanism involving predominantly ionic interactions. Such interactions have been identified as being important for the binding of proteins by dye-ligand adsorbents [36l. A purification procedure for human erythrocyte GSH-Px has been reported by Awasthi et al. [37]. These authors stated that GSH-Px does not bind to ion-exchange resins like DEAE-cellulose (DE-52) in the presence of hemoglobin and consequently used two ammonium sulfate precipitations and cation-exchange chromatography to remove hemoglobin. We have found that GSH-Px binds to DE-52 in the presence of hemoglobin providing the ionic strength of the hemolysate is sufficiently low (less than or equal to that of a 5 mM potassium phosphate buffer, pH 6.7). In Awasthi et al.'s procedure, anion-exchange chromatography, gel permeation chromatography and a second anion-exchange chromatography were used to complete the isolation
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