Purification of NADPH-Free Glutathione Disulfide Reductase from Human Erythrocytes

PROTEIN EXPRESSION AND PURIFICATION ARTICLE NO.

13, 41–44 (1998)

PT970865

Purification of NADPH-Free Glutathione Disulfide Reductase from Human Erythrocytes ¨ gu¨s and Nazmi O ¨ zer1 I. Hamdi O Department of Biochemistry, Faculty of Medicine, Hacettepe University, Ankara 06100, Turkey

Received October 1, 1997, and in revised form November 24, 1997

Human erythrocyte glutathione disulfide reductase was purified using serially connected 2*,5*-ADP-Sepharose 4B affinity and anion-exchange columns. About 11,000-fold purification was achieved with 90% yield. The specific activity of the final preparation was 140 units per milligram of protein. The purified enzyme gave a single band on both native and SDS–PAGE with a subunit mass of 58 kDa. Its pH optimum was 7.20. The Michaelis constants determined at pH 7.4, 377C, fell within the range of previously reported values [Km(NADPH) Å 18 mM, at 30–200mM NADPH; Km(GSSG) Å 72 mM, at 40–1000 mM glutathione disulfide, both at saturating concentrations of the second substrate]. The affinity eluent NADPH and its oxidized form NADP/ were successfully removed from the enzyme on the ion-exchange column. The purification method developed is very useful when the enzyme source material is scarce (e.g., in preparations from human tissues) and may find further application in the purification of other NAD(P)H-dependent enzymes which might be inactivated by their affinity eluent(s). q 1998 Academic Press Key Words: glutathione disulfide reductase; purification; affinity chromatography; NADPH; human erythrocyte.

1. INTRODUCTION

Glutathione disulfide, GSSG,2 is reduced to glutathione, GSH, by the flavoprotein glutathione disulfide reductase ,GR (EC 1.6.4.2), at the expense of NADPH (1). The redox couple, GSH/GSSG, determines the level of metabolic activity and plays an important role in the 1 To whom correspondence should be addressed. Fax: / 90 312 311 6616. E-mail: [email protected]. 2 Abbreviations used: PBE 94, Polybuffer Exchanger 94; HxGSH, hexylglutathione; BSA, bovine serum albumin; GST 1-1, glutathione S-transferase 1-1 (isozyme); G6PD, glucose-6-phosphate dehydrogenase; 6PGD, 6-phosphogluconate dehydrogenase; GSSG, glutathione disufide.

maintenance of membrane integrity and detoxification (2). GR has been purified from erythrocytes, using different purification procedures. All reported purification procedures involve several chromatographic steps (3– 8). Some of these purification methods also involve affinity chromatography on 2*,5*-ADP-Sepharose columns, and GR is usually eluted using NADPH (8, 9). The specific activities of the enzymes obtained range from 20 to 240 units per milligram of protein (3–7). Since NADPH is an irreversible inhibitor of GR, this very high variation in specific activity of GR probably results from the presence of inactivated enzyme in purified enzyme preparations (9). Hence, a method in which the contact time of NADPH with GR is very short will be an ideal method for the purification of GR. The following is a fast and efficient method we have developed for the purification of NADP(H)-free GR. It not only gives a high enzyme yield, but also allows one to remove and separate the coenzymes NADPH and NADP/ which have very high affinity for the erythrocyte GR (10). 2. MATERIALS AND METHODS

2.1. Materials. All chromatographic materials were obtained from Pharmacia-LKB, Sweden. GSH and GSSG were from Boehringer-Mannheim, FRG. All other chemicals were the purest products available from Sigma or Aldrich, USA. Outdated erythrocytes (13–15 days old) were obtained from the Hacettepe University Blood Bank. 2.2. Activity and protein determinations. GR activity was determined in 100 mM potassium phosphate buffer (pH 7.4), containing 4 mM EDTA, 1 mM GSSG, and 0.1 mM NADPH, at 377C. The reaction was initiated by the addition of a suitable amount of GR and followed by monitoring the decrease in absorbance at 340 nm (9). A unit of enzyme is defined as the amount of the enzyme which catalyzes the oxidation of 1 mmol NADPH/min, under the specified assay conditions. 41

1046-5928/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.

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Specific activity was calculated on the basis of protein concentration determined by the method of Bradford, with bovine serum albumin as standard (11). 2.3. Determination of Michaelis constants and pH optimum. Kinetic parameters were determined in 100 mM potassium phosphate (pH 7.4) buffer at 377C, containing 4 mM EDTA, at 40–1000 mM GSSG and 30– 200 mM NADPH. The reaction was initiated by the addition of 3.3 nM GR. The dependence of enzymatic rate on pH was determined using potassium phosphate and Tris–HCl buffers. At each pH, rates observed were extrapolated to zero buffer concentration. The pH optimum was obtained from a plot of the extrapolated rates versus pH (12). 2.4. Polyacrylamide gel electrophoresis (PAGE). Discontinuous PAGE (7%; 0.75 1 100 mm) was carried out under nondenaturing, nonreducing conditions, essentially as described by Laemmli for denaturing gels (13), omitting sodium dodecyl sulfate. SDS–PAGE was carried out on 12.5% gels, exactly as in (13). After application of the samples, electrophoretic separation was carried out at room temperature. Staining and destaining of the gels were done according to (13), omitting the acid fixation step. 2.5. Enzyme purification. Except where specified, all purification procedures were carried out at 0–47C. Erythrocytes (120 ml), obtained by centrifugation of 380 ml of blood at 700g for 20 min, were washed three times using physiological saline supplemented with 1 mM EDTA. Washed erythrocytes were hemolyzed in 1 vol of water containing 0.006 vol of cold toluene and centrifuged at 13,200g for 20 min. The supernatant was filtered through Celite, 12.5 g Celite per liter of supernatant, to ensure the complete removal of the membrane fragments. The filtrate was diluted by the addition of an equal volume of water and any precipitate formed was removed by brief centrifugation at 13,200g. The supernatant (åhemolysate, 323 ml) was dialyzed overnight against 10 mM potassium phosphate (KP) buffer, pH 7.4, containing 1 mM EDTA and 1 mM e-aminocaproic acid (Buffer A). The clear dialysate (388 ml) was then applied to serially coupled HxGSH-Sepharose 4B (1.6 1 8 cm) and 2*,5*-ADPSepharose 4B (2 1 2 cm) columns, preequilibrated with Buffer A. The columns were washed with 1 liter of Buffer A. During both the application of the sample and the washing step, the flow rate was kept at 55 ml per hour. After the Buffer A wash, the two columns were disconnected. [From the first column (HxGSHSepharose 4B), human erythrocyte glutathione Stransferase was purified using the published procedure for human jejunal glutathione S-transferases (14)]. The 2*,5*-ADP-Sepharose 4B column was washed with 400 mM KP (pH 7.4)–1 mM EDTA buffer until the absorbance at 280 nm was õ0.010. About 10 bed

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FIG. 1. Gradient elution of GR and coenzymes from the PBE 94 column. Buffer: 10/250 mM potassium phosphate, pH 7.4. Flow rate is 27 ml/h. Fraction volume is 2.5 ml. Notations: l, activity/10; —, A260 ; s, A280 ; — , A340 ; ---, A462 ; —, buffer gradient. 10/250 mM.

volumes of buffer was used. The column was further washed with Buffer A until the absorbance at 280 nm returned to õ0.010. A postcolumn of PBE 94 (2 1 2.5 cm) preequilibrated with 10 mM KP, pH 7.4 (Buffer B), was connected to the affinity column. GR was eluted from the affinity column using 0.5 mM NADPH in Buffer B (30 ml). The effluent was directly passed on to the PBE 94 column. Following the NADPH elution step, the two columns were disconnected and the elution of the PBE 94 column was continued with a 100ml gradient of 20 to 250 mM KP, pH 7.4. In the washing steps and elution from the affinity and PBE 94 columns, the flow rate was 27 ml per hour. GR eluted from the column at 35 mM KP. Enzymatic activity and absorbances at 235, 260, 280, 340, and 462 nm were measured in all fractions. Fractions containing GR activity (tubes 3–6 Å 10 ml) were combined, divided into 0.5-ml aliquots, and stored at 0807C. 3. RESULTS AND DISCUSSION

Human erythrocyte glutathione disulfide reductase was purified 11,000-fold from human erythrocytes, with a yield of 90%. The specific activity of the purified enzyme was 140 units per milligram of protein (details of the purification are given in the legend to Fig. 1 and

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PURIFICATION OF NADPH-FREE GLUTATHIONE DISULFIDE REDUCTASE TABLE 1

Purification of Human Erythrocyte Glutathione Reductase Purification (steps)

Volume (ml)

Activity (U/ml)

Total activity (units)

Hemolyzate (Celite eluate) Dialyzate HxGSH-Sepharose 4B, 2*,5*-ADP-Sepharose 4B, PBE 94 eluate

323 388

0.26 0.24

84 93

10

7.58

76

Table 1). The preparation gave a single band on both native (7%, not shown) and SDS–PAGE (12.5% gel; subunit Mr 58 kDa, Fig. 2). The pH optimum was found

FIG. 2. SDS–PAGE (12.5%) of the human jejunal glutathione reductase. The arrowheads correspond to the points of migration of the standards (starting from the cathode: bovine serum albumin, 66,000; ovalbumin, 45,000; rat liver GST 1-1, 25,000). Amounts of the protein applied per well were GR, 3.5 mg; BSA, 4 mg; ovalbumin, 5 mg; and GST 1-1, 2.6 mg.

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Protein (mg/ml)

Total protein (mg)

Specific activity (U/mg protein)

Yield (%)

Purification (fold)

20.5 16.6

6621 6449

0.0127 0.0144

100 111

1 1.13

0.054

0.54

140

90

11,000

to be 7.20. The Michaelis–Menten constants were determined from rates observed at 377C, in 100 mM KP (pH 7.4)–4 mM EDTA, using 40–1000 mM GSSG and 30–200 mM NADPH. Plots of 1/Vm versus 1/GSSG (and 1/NADPH) (15) yielded Km(NADPH) Å 18 mM and Km(GSSG) Å 72 mM, in agreement with previously reported values (3–7). Glutathione disulfide reductase binds to the 2*,5*ADP-Sepharose 4B column with high affinity. Increasing the phosphate buffer concentration to 400 mM caused the elution of almost all proteins (e.g., G6PD, 6PGD) which were nonspecifically or specifically bound to the affinity column (16). The elution of GR was effected by subsequently decreasing the phosphate buffer concentration to 10 mM (Buffer B, pH 7.4) and supplementing Buffer B with 0.5 mM NADPH. NADPH inactivation of GR is directly related to the contact time of the enzyme with NADPH. To obtain a high yield of enzyme activity, an ion-exchange column was serially coupled to the affinity column so that the enzyme eluted from the affinity column could immediately enter the ion-exchange column and the coenzymes which have higher affinity for the ion-exchange column could dissociate from the enzyme. With the KP (pH 7.4) buffer gradient used, the elution order was: first, GR and GR-bound FAD (at 35 mM); second, NADP/ (at 75 mM); and third, NADPH and free-FAD (at 230 mM) (Fig. 1). Elution positions and concentrations were determined using the characteristic absorption spectra of protein, NADP/, NADPH, and FAD. The amount of the NADP/ in fraction 10 was determined using the extinction coefficient of NADP/ at 260 nm and it was estimated that less than 5% of NADPH in Buffer B was converted into NADP/ during chromatography. Using the specific absorption characteristics, at different wavelengths, of different coenzymes, the contents of the different fractions were to be: fractions 4–6, GR and GR-bound FAD; fraction 10, NADP/; fractions 17–20, FADH2 ; and fractions 33–35, FAD. The very high absorbance observed at 260 and 340 nm after fraction 25 was due to NADPH. Fractions 4–6 were the only protein-containing fractions, as determined by the method of Bradford (11).

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The purification protocol described above may easily be used for the purification of GR from other sources, as well as for the purification of other NAD(P)H-dependent enzymes which might be inactivated by their eluants. This method appears especially advantageous, where the enzyme source material is scarce (e.g., in preparations from human tissues).

8.

9.

10.

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by the use of affinity chromatography on 2*,5*-ADP-Sepharose 4B and crystallization of the enzyme. Anal. Biochem. 98, 335– 340. Thieme, R., Pai, E. F., Schirmer, R. H., and Schulz, G. E. (1981) ˚ resThree-dimensional structure of glutathione reductase at 2 A olution. J. Mol. Biol. 152, 763–782. ¨ gu¨s, I. H., and O ¨ zer, N. (1991) Human jejunal glutathione reO ductase: Purification and evaluation of the NADPH- and glutathione-induced changes in redox state. Biochem. Med. Met. Biol. 45, 65–73. Pai, E. F., Karplus, P. A., and Schulz, G. E. (1988) Crystallographic analysis of the binding of NADPH, NADPH fragments, and NADPH analogues to glutathione reductase. Biochemistry 27, 4465–4474. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Gast, R., Valk, B. E., Mu¨ller, F., Mayhew, S. G., and Veeger, C. (1976) Studies on the binding of FMN by apoflavodoxin from peptostreptococcus elsden II: pH and NaCl concentration dependence. Biochem. Biophys. Acta 446, 463–471. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680– 685. ¨ zer, N., Erdemli, O ¨ ., Sayek, I., and O ¨ zer, I. (1990) Resolution O and kinetic characterization of glutathione S-transferases from human jejunal mucosa. Biochem. Med. Met. Biol. 44, 142–150. Segel, I. H. (1975) ‘‘Enzyme Kinetics,’’ pp. 505–845, Wiley Interscience, New York. Carlberg, I., and Mannervik, B. (1985) Glutathione reductase. In ‘‘Methods in Enzymology’’ (Colowick, S. P., and Kaplan, N. O., Eds.), Vol. 113, pp. 484–490, Academic Press, San Diego.

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