THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 243, No. 4, Issue of February 25, pp. 809-614, 1968

Printed in U.S.A.

Purification and Subunit Structure of Glutathione Reductase

from Bakers’ Yeast*

(Received for publication, September 27, 1967)

RICHARD D. MAVIS AND EARLE STELLWAGEN

From the Department of Biochemistry, University of Iowa, Iowa City, Iowa 52240

SUMMARY

A scheme has been devised for the purification of gluta- thione reductase from bakers’ yeast. The purified protein is homogeneous with respect to chromatographic, electro- phoretic, and sedimentation criteria. A molecular weight of 1.24 f 0.05 x lo5 was calculated for the protein from sedi- mentation equilibrium and osmotic pressure measurements. In the presence of 5 M guanidine hydrochloride, the molecular weight is reduced to 5.15 f 0.3 x lo4 calculated from sedi- mentation equilibrium experiments, assuming no preferential interaction with the solvent, or to 5.60 X lo4 calculated from osmotic pressure measurements. These values indicate that the native protein contains two polypeptide chains. The dissociated polypeptide chains appear identical with respect to net charge and molecular size. The native enzyme was found to contain 2 moles of FAD per mole of protein. Both FAD moieties can be separated from the dissociated protein by dialysis or electrophoresis, indicating that FAD is attached to the native protein by noncovalent bonds.

The flavoprotein glutathione reductase (EC 1.6.4.2, NAD- (P)Hz:glutathione oxidoreductase) catalyzes the reduction of oxidized glutathione by NADPH. Colman and Black (1) have reported that the enzyme obtained from yeast has a molecular weight of 1.18 x lo5 and contains one FAD and one reactive sulfhydryl group per mole of protein. These data suggest that the native protein contains at least two nonidentical polypep- tides. Subsequently, Massey and Williams (2) reported that yeast glutathione reductase contains two FAD moieties and two reactive sulfhydryl groups per mole, suggesting that, if the protein contains two polypeptide chains, they are identical. We have investigated the subunit. structure of the enzyme and the identity of the dissociated polypeptide chains in an attempt to clarify this issue. Because of the prohibitive cost of the

* This investigation has been aided by a grant from the Jane Coffin Childs Memorial Fund for Medical Research and by Public Health Service Research Career Program Award l-K03-GM-08737 from the National Institute of General Medical Sciences.

commercially available glutathione reductase used in the above studies, it was decided to develop a procedure for the purification of the enzyme from bakers’ yeast.

EXPERIME?r’TAL PROCEDURE

Materials-Oxidized glutathione was purchased from Calbio- them, NADPH from P-L Biochemicals, and protamine sulfate from Krishell Laboratories, Portland, Oregon. DEAE-cellulose and phosphocellulose were purchased from Schleicher and Schuell and Bio-Rad, respectively. Superbrite glass beads (type 100-5005) were obtained from Minnesota Mining and Manufacturing.

Enzymic Assay-Glutathione reductase activity was measured spectrophotometrically by following the decrease in absorbance of NADPH at 340 rnp as described by Racker (3). The assay solution contained 50 pmoles of sodium phosphate buffer, pH 7.6, 0.1 pmole of NADPH, 3.3 pmoles of oxidized glutathione, 1 I.rmole of EDTA, and 1.0 mg of bovine serum albumin in a total volume of 1.0 ml. This solution was placed in a 1.5-m] quartz cuvette with a path length of 10 mm and inserted into the thermostated sample compartment of a Gilford model 2000 recording spectrophotometer maintained at 25”. Reduction of oxidized glutathione was initiated by the addition of 5 ~1 of solution, containing sufficient enzyme to give a AA3d0 per min between 0.03 and 0.25. A unit of activity is the amount of en- zyme that catalyzes the oxidation of 1 pmole of NADPH per min. Specific activity is expressed as units of enzymic activity per mg of protein. During the purification procedure, protein concentrat.ions were determined by the method of Warburg and Christian (4).

Ultracentrifugal Measurements--All sedimentation measure- ments were made at 20” with a Spinco model E analytical ultracentrifuge. Sedimentation velocity experiments were per- formed at 59,780 rpm with the use of schlieren optics and metal- lographic photographic plates. Sedimentation coefficients were calculated as described by Schachman (5).

High speed sedimentation equilibrium experiments were per- formed according to the procedure of Yphantis (6). A protein solution (100 ~1) containing 0.1 mg of protein per ml was layered over 10 ~1 of fluorocarbon FC-43 (perfluorotributylamine, Min- nesota Mining and Manufacturing Company) in one sector of a filled Epon double-sector centerpiece in an assembled inter-

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810 Subunit Stru.&re of Ye ‘ast Glutathione Reductase Vol. 243, No. 4

ference cell with sapphire windows. The same volume of fluoro- carbon was placed in the other sector and layered with 100 ~1 of solvent. After equilibrium was attained, the Rayleigh inter- ferographs were photographed with the use of Kodak Spectro- scopic II-G plates. Base-line photographs were obtained after rinsing the assembled cell several times with distilled water, filling the cell with water, and then accelerating the cell to the equilibrium speed. Photographs were analyzed on a Nikon microcomparator (New York, New York) by measuring the vertical fringe displacement, D, in microns as a function of the distance from the center of rotation, r. The positions of five fringes, three black and two white, were measured at each value of r, and an average displacement of these five fringes was used to calculate molecular weight after correction for the average displacement of five fringes in the base-line photograph. Molec- ular weights were calculated from the equation

ii& = 2RT d In D

~‘(1 - tip) dr2

where d In D/d+ is the slope of a plot of In D against r2, R is the gas constant, T is the absolute temperature, p is the density of the solvent, w is the angular velocity of the rotor, and 5 is the partial specific volume of the protein. Solvent densities were measured with a 5-ml pycnometer. The partial specific volume was determined to be 0.744 by the method of Edelstein and Schachman (7).

il double-sector synthetic boundary cell was used to determine protein concentration in terms of interference fringes. Solvent was layered on top of the protein solution at a rotor speed of 9000 rpm. After layering was completed, the rotor was main- tained at that speed until individual fringes were clearly visible in the boundary. Photographs were taken with the use of spec- troscopic II-G plates. A base-line photograph was obtained after uncoupling the rotor from the drive shaft, inverting the rotor several times to mix the cell contents, and then accelerating the rotor to 9000 rpm. The number of fringes across the bound- ary was determined with the aid of a Nikon microcomparator. Corrections were made for the nonlinearity of the base-line.

Osmotic Pressure Measurementsqsmotic pressure measure- ments were performed at 25” with a Mechrolab high speed osmom- eter (Mountain View, California) and B-19 membranes obtained from Schleicher and Schuell. Protein solutions ranged in con- centration from 1 to 16 mg per ml. The osmotic pressure, ?r, in centimeter of solvent was recorded after a constant value had been established. The number average molecular weight,

nN, was calculated with the equation,

jj =RT N *lc

where R is the gas constant, T is the absolute temperature, and c is the protein concentration in mg per ml. A value for the term ?r/c at infinite protein dilution was obtained by a linear extrapolation of values measured at finite concentrations.

Zone EZectrophoresis-Starch gels were prepared by heating 50 ml of a suspension of 13% (w/v) of partially hydrolyzed starch (Connaught Medical Research Laboratories, Toronto, Canada) in buffer solution. The hot gel was poured into a Lucite mold, 3.5 x 14 cm, to a depth of 0.5 cm, covered, and allowed to cool. A portion, 0.5 x 1.0 cm, of Whatman No. 3MM paper was alternately saturated with protein solution and dried until the paper contained about 250 ng of protein; it was then inserted into a slit in the center of the gel. Gels containing 8 M urea were made with 11.5% (w/v) starch.

Disc electrophoresis in polyacrylamide gel was performed ac- cording to the procedure of Davis (8). Gels containing 8 M urea were 5% (w/v) acrylamide.

Other Measurements-Absorption spectra were obtained with a Cary model 14 recording spectrophotometer. Viscosity meas- urements were obtained with an Ostwald viscometer as described previously (9).

RESULTS

Purijication

All manipulations were performed at 4” unless indicated otherwise. All solutions contained 1 mM EDTA. Centrifuga- tion was performed at 27,300 X g for 20 min with a Sorvall RC2- B refrigerated centrifuge. The analytical details of a typical purification are shown in Table I.

Step 1. ExtractionGlutathione reductase was extracted from Fleischmann’s bakers’ yeast by homogenization with Superbrite glass beads with an Eppenbach colloid mill (Gifford-Wood Com- pany, Hudson, New York). A suspension of 2 pounds of crum- bled yeast cake and 800 ml of glass beads in 400 ml of 0.2 M so- dium phosphate buffer, pH 8.0, was homogenized for 15 min at 22”. A gap setting of 60 was used for 1 min at low speed (Volt- pat 40) to obtain a uniform solution. The gap was then closed to 30, and the suspension was circulated at top speed (Volt-pat 110). The supernatant was decanted from the glass beads and centrifuged to remove cell debris. Supernatants collected from

TABLE I Fractionation of yeast glutathione reductase

Fraction Volume Total protein Total activity Specific activity Purification

ml mg rt& u?iits/nu -fold 1. Extract................... 5,935 920,000 31,700 0.034 1 2. Protamine sulfate supernatant 4,885 275,000 17,800 0.065 2

3. Saturated (0.45-0.60) (NHd)z SO1 precipitate.. 0.20 6 610 69,000 14,000

4. Heat treatment. 735 16,200 13,500 0.83 23 5. Ethanol precipitate (33%). 5.5 162 215 1,450 8,000 6. Phosphocellulose eluate. . 75 260 45 5,000 2,200 7. DEAE-cellulose eluate. 280 22 4,500 200 5,900

Recovery -

%

100 56

44 43 25 16 14

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Issue of February 25, 1968 R. D. Mavis and E. Stellwagen 811

the homogenization of 20 pounds of yeast were pooled and processed in the following purification procedures as a single batch.

Step 2. Protamine Sulfate Treatment-A 5% (w/v) suspension of protamine sulfate in water was mixed thoroughly and adjusted to pH 6.0 with 1 K NaOH. A volume of protamine sulfate suspension containing 1 g for each 10 g of protein in the pooled supernatants from Step 1 was added slowly and with constant stirring to the pooled supernatants. The resulting suspension was st,irred for an additional 15 min and the milky precipitate was removed by centrifugation.

Step S. nmmonium Sulfate I;‘ractionationSolid ammonium sulfate was added to the supernatant from Step 2 until 0.45 saturation was attained (277 g per liter of supernatant). The precipitate was removed by centrifugation and discarded. Ad- ditional solid ammonium sulfate was added to the supernatant until 0.60 saturation was attained (99 g per liter). The pre- cipitate was collected by centrifugation and redissolved in 600 ml of 0.1 M sodium phosphate buffer, pH 6.0. The 0.60 satu- rated supernatant was discarded.

Step 4. Heat Treatment-The redissolved 0.45 to 0.60 saturated ammonium sulfate precipitate was placed in a stainless steel centrifuge vessel and heated in a water bath maintained at 55” for 30 min. The heated solution was cooled to 15” and cen- trifuged, the supernatant was collected, and the precipitate was extracted with 300 ml of 0.02 bf sodium phosphate buffer, pH 6.5. The extract was then combined with the supernatant from the heat treatment and dialyzed against 0.02 ~rl sodium phosphate buffer, pH 6.5, for 2 days.

Step 5. Ethanol Precipitation-Absolute ethanol chilled to -4” was added slowly with stirring to the dialyzed solution from Step 4 at -4” until the concentration of ethanol was 33% (v/v). This solution was stirred for an additional 30 min and then cen- trifuged, and the supernatant was discarded. The ethanol pre- cipitate was suspended in 200 ml of 0.02 M sodium phosphate buffer, pH 6.5, and dialyzed overnight against 4 liters of the same buffer. The dialyzed suspension was centrifuged, and the supernatant was collected.

Step 6. Phosphocellulose Chromatography-The supernatant from Step 5 was applied to a column, 4 x 33 cm, of phospho- cellulose (1 ml of resin bed per 20 units of enzymic activity) equilibrated with 0.02 M sodium phosphate buffer, pH 6.5. The column was washed with 700 ml of equilibration buffer and then with iO0 ml of 0.02 M sodium phosphate buffer, pH 7.0. En- zymic activity was eluted, batchwise, with 0.02 M sodium phos- phate, pH 7.0, containing 0.8 M sodium chloride. The active fractions were pooled and dialyzed overnight against 4 liters of 0.03 1~ Tris-HCl buffer, pH 8.0.

Step 7. DEAE-cellulose Chromatography-The dialyzed solu- tion from Step 6 was applied to a column, 2 x 25 cm, of DEAE- cellulose (1 ml of resin bed per 65 units of enzymic activity) equilibrated with 0.03 M Tris-HCl buffer, pH 8.0. The column was washed with about 300 ml of equilibration buffer and then developed with a linear gradient made from 500 ml of equilibra- tion buffer and 500 ml of the same buffer containing 0.5 M NaCl. The enzymic activity was eluted about halfway through the gradient elution system. The active fractions were pooled and dialyzed overnight against the equilibration buffer and rechro- matographed on DEAE-cellulose with the same conditions. The activity was eluted as a single boundary coincident with a

single protein boundary. The active fractions were pooled, con- centrated by ultrafiltration, and stored in 0.65 saturated ammon- iumsulfate solution at 4”.

Purijication of Commercial Enzyme

Glutathione reductase (100 mg), obtained from Boehringer und Sohne as an ammonium sulfate suspension, was dialyzed overnight against 0.02 M sodium phosphate buffer, pH 6.5. The dialyzed solution, which contained 10,000 units of enzymic activity, was applied to a column, 2 x 50 cm, of phosphocellulose equilibrated with 0.02 M sodium phosphate buffer, pH 6.5. The column was washed free of nonadsorbing material with the equilibration buffer and developed with a linear gradient made from 800 ml of equilibration buffer and 800 ml of the same buffer containing 1.0 M NaCl. Enzymic activity was eluted about halfway through the gradient elution system. The active frac- tions were pooled and contained 9,000 enzyme units in 43 mg of protein with a specific activity of 210 units per mg. The sedi- mentation coefficient, absorption spectrum, and electrophoretic mobility of this purified Boehringer preparation and the enzyme purified by the procedure described above are virtually identical. The enzyme solution was concentrated by ultrafiltration and stored in 0.65 saturated ammonium sulfate solution at 4”.

Kinetic Properties

With the use of the purified enzyme, the Michaelis constants for oxidized glutathione and NADPH were found to be 6.1 x

10-j M and 7.6 x 1O-6 M, respectively. A turnover number of 1.3 x lo4 moles of NADPH oxidized per min per mole of bound FAD was calculated. These values compare favorably with those reported by Massey and Williams (2)) who used a commer- cially prepared enzyme subjected to further purification.

Absorption Spectrum

The absorption spectrum of the purified enzyme exhibits maxima at 460, 370, and 280 rnp with shoulders at 490 and 440 mp, in agreement with published spectra (1, 2) for the commer- cial enzyme subjected to further purification. The extinction coefficient of glutathione reductase at 280 rnp was determined by measuring the refractive increment of solutions of the enzyme of known absorbance at 280 rnp relative to the solvent. The refractive increment was measured in terms of interference fringes observed across a synthetic boundary produced in the ultracentrifuge between solvent and solution. The number of fringes measured for a series of solutions of bovine serum albumin of known concentration exhibited a linear dependence on protein concentration over the range of 1 to 5 mg per ml, with an incre- ment of 3.77 interference fringes per mg per ml of protein. On the assumption that the refractive increment of glutathione reductase was the same as that for bovine serum albumin, the number of fringes observed for solutions of glutathione reductase of known absorbance at 280 rnl.r were then related to protein concentration. The extinction of a 1% solution of enzyme at 280 mp was calculated to be 15.4 f 0.3. The absorbance of the protein at 460 rnp was equivalent to that expected for 2.17 moles of bound FAD per mole of enzyme with the use of an extinction coefficient of 1.13 x lo4 M-I cm-l for enzyme-bound FAD at 460 mp (2) and a molecular weight of 1.21 X lo5 for the en- zyme.

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812 Subunit Stru&ure of Yeast Glutathione Reductase Vol. 243, No. 4

Sedimentation Measurements

The purified enzyme sedimented as a single symmetrical boundary both in neutral buffer and in 5 M guanidine hydro- chloride, as shown in Fig. 1. The sedimentation coefficient of the native protein exhibited a slight dependence on protein con- centration as shown in Fig. 2, giving a value of 6.88 S at infinite

dilution. In the presence of 5 M guanidine hydrochloride, the sedimentation coefficient was reduced from 6.9 S to 1.7 S at a concentraGon of 3 mg per ml. A similar reduction in the sedi- mentation coefficient of the enzyme was observed in 8 M urea

and in 1 y0 sodium dodecyl sulfate. The equilibrium gradients for glutathione reductase in the

presence and absence of guanidine hydrochloride are shown in Fig. 3. A partial specific volume of 0.744 f 0.025 was calculated from the gradients observed in HZ0 and 96% DzO with the

NATIVE DISSOCIATED

S 20,w=6.9 S S 20,w= I.7 s

FIG. 1. Sedimentation velocity patterns of glutathione reduc- tase. The direction of sedimentation is from Zeft to right. Native, 10 mg per ml of protein in 0.05 M sodium phosphate buffer, pH 6.8. The photograph was taken 32 min after attaining a speed of 59,780 rpm, with the use of a phase plate angle of 50”. Dis- sociated, 3 mg per ml of protein in 5 M guanidine hydrochloride and 0.01 M mercaptoethanol. The photograph was taken 16 min after attaining a speed of 59,780 rpm, with the use of a phase plate angle of 55”. In this experiment the protein boundary was preformed in a double-sector synthetic boundary cell. The pro- tein sedimented as a single boundary for at least 64 min.

6.90 , I I I I I 1

PROTEIN CONCENTRATION, mg /ml

FIG. 2. Dependence of sedimentation coefficient of native glu- tathione reductase on protein concentration. The solvent was 0.05 M sodium phosphate, pH 6.8.

492

Y2, cm2 FIG. 3. Equilibrium sedimentation of glutathione reductase in

various solvent systems. The ordinate scale is the natural loga- rithm of the fringe displacement, and the abscissa gives the square of the distance from the axis of rotation. 0, in 0.05 M sodium phosphate, pH 6.8, in HsO at 16,200 rpm for 15 hours; 0, in 0.05 M sodium phosphate, pH 6.8, in 96% D*O at 16,200 rpm for 15 hours; A, in 5 M guanidine hydrochloride and 0.01 M mercapto- ethanol at 42,040 rpm for 20 hours. All experiments were per- formed at 20” with a protein concentration of 0.1 mg per ml.

0.6 I I I

0.2-c "

I I I I 0 6 16

CONCENTRATION, f?-Ig / tTll

FIG. 4. Osmotic pressure measurements of glutathione reduc- tase. 0, in 0.10 M sodium phosphate, pH 6.8; 0, in 5 M guanidine hydrochloride and 0.01 M mercaptoethanol.

method of Edelstein and Schachman (7). The average molecular

weight of native glutathione reductase is 1.22 f 0.06 x lo5 calculated from five sedimentation equilibrium measurements. The average molecular weight of the protein is reduced to 5.15 f 0.30 x 104 in 5 M guanidine hydrochloride based on five sedimen- tation equilibrium measurements. No corrections were made for any preferential binding of either water or guanidine hydro- chloride.

Osmotic Pressure Measurements

The dependence of the osmotic pressure of glutathione re- ductase on protein concentration in the presence and absence of 5 M guanidine hydrochloride is shown in Fig. 4. Molecular

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Issue of February 25, 1968 R. D. Matis and E. Xtellwagen 813

STARCH POLYACRYLAM I DE

GEL

ORIGIN+

t

A B

FIG. 5. Electrophoretic patterns of . . _ .

C D

glutathione reductase. All experiments were performed at room temperature. A, in 0.02 M sodium phosphate, pH 6.5, at 20 ma for 4 hours; B, in 8 M

urea and 0.02 M sodium phosphate, pH 6.5, at 15 ma for 24 hours; C, in 0.375 M Tris-HCl, pH 9.5, at 25 ma for 2 hours; D, in 8 Y urea-O.375 M Tris-HCl, pH 9.5, at 22 ma for 13 hours.

GEL

c ORIGIN

weights of 1.26 X 105 and 5.60 x lo4 were calculated from the extrapolated values of w/c at infinite dilution for the native pro- tein and t’he protein in 5 M guanidine hydrochloride, respec- tively.

Viscosity L!feasurements

The reduced viscosity of the native protein in 0.05 M sodium phosphate buffer, pH 6.8, is 3.09 ml per g at a protein concen- tration of 5.1 mg per ml.

Electrophoresis

The purified enzyme migrated toward the anode as a single yellow band when subjected to starch gel electrophoresis at pH 6.5, as shown in Fig. 5A. In the presence of 8 M urea, the protein migrated as a single, colorless, rather diffuse band, as shown in Fig. 5B. A yellow band was observed to migrate rapidly through the starch gel and into the anode chamber during the first few hours of electrophoresis in 8 M urea. Disc electrophoresis in polyacrylamide gel at pH 9.5 yielded a single protein band in the presence and absence of 8 M urea, as shown in Fig. 5, C and D.

DISCUSSIOS

The yeast glutathione reductase purified by this procedure appears to be monodisperse when examined by chromatographic, electrophoretic, and sedimentation procedures. The specific activity is comparable to that of other preparations if the same extinction coefficient is used for measurement of protein con- centration. The K, values for both substrates agree well with previously published values. The sedimentation coefficient at infinite dilution, 6.88 S, is slightly higher than the value of 6.45 S reported by Colman and Black (1). The dependence of the sedimentation coefficient on protein concentration is also lower than reported (1). These observations suggest that the protein prepared by the procedure described here has a more compact conformation than the commercially prepared enzyme. The

low value of 3.1 ml per g for the reduced viscosity indicates that the native conformation is quite symmetrical.

The extinction coefficient at 280 rnp, E&, was calculated to be 15.4 from refractive index measurements. This value is intermediate between the values of 10.5 (1) and 14.5 (2) ob- tained from dry weight measurements and the value of 18.6 (2) determined by the biuret method. Since only limited quantities of the enzyme were available, small errors in determining the dry weight of the enzyme could account for the lack of agreement in the extinctions calculated by this procedure. The biuret method, although circumventing this problem, assumes that all proteins give the same calorimetric response, an assumption that may not be warranted for glutathione reductase. Whereas the same criticism may be applied to the refractive index method, which employs bovine serum albumin as a standard, it is well known that the refractive increment of all proteins examined does not vary by more than ~1~2.5s (10).

Molecular weights of 1.22 f 0.06 x lo5 and 1.26 X 105 for the native protein were calculated from sedimentation equilib- rium and osmotic pressure measurements, respectively, in good agreement with the value of 1.18 x lo5 reported by Colman and Black (1). A molecular weight of 1.16 x lo5 was calculated for the native protein from the Scheraga-Mandelkern equation (ll), with the use of the measured value for s& ,ul of 6.88 S, ti of 0.744, reduced viscosity of 3.1 ml per g and the assumption of a value for /? of 2.12 X 106.

The addition of a variety of protein denaturants, 1% sodium dodecyl sulfate, 5 M guanidine hydrochloride, or 8 M urea, lowered the sedimentation coefficient of the protein from 6.9 S to 1.7 S, which suggested that the native protein had been dissociated into its constituent polypeptide chains. The same reduction in sedimentation coefficient was observed with these reagents in the presence and absence of 0.01 M mercaptoethanol, which in- dicated that interchain disulfide bonds were not present. In the presence of 5 M guanidine hydrochloride, a molecular weight of 5.15 f 0.3 X 104 was calculat.ed from sedimentation equilib- rium measurements. This value is 42$& of the molecular weight of the native protein, which suggested that glutathione reductase contains two polypeptide chains. However, some uncertainty exists in the calculation of molecular weights in 5 M guanidine hydrochloride because of possible preferential interaction of some component of the solvent with the dissociated chains.

Rather than attempting to evaluate preferential solvent inter- actions, the molecular weight in 5 M guanidine hydrochloride was determined by osmotic pressure, a method that is independent of preferential solvent binding. Extrapolation of osmotic pres- sure data to infinite dilution gave a molecular weight of 5.60 X 104, a value that is 46’% of the molecular weight of the native protein. With the use of a molar extinction of 1.13 X lo4 at 460 rnp for enzyme-bound FAD (2), the minimum molecular weight per FAD calculated from the absorption spectrum and $,, was 5.65 X 104, in agreement with similar values calculated for glutathione reductase from pea seedlings (12) and yeast (2). These results, taken together, indicate that native glutathione reductase contains two polypeptide chains. The In D versus ~2 plot (Fig. 3) for the dissociated protein in 5 M guanidine hydro- chloride is linear, which indicates that the two polypeptide chains are identical, or nearly so, in molecular size. The dis- sociated protein migrated as a single band when subjected to zone electrophoresis in 8 M urea, which indicates that the two polypeptide chains are identical nrith respect to net charge at pH 6.5 and 9.5.

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814 Subunit Structure of Yeast Glutathione Reductase Vol. 243, No. 4

Since glutathione reductase contains two apparently equivalent polypeptide chains and two FAD moieties, it is likely that each chain in the native protein has one bound FAD. The FAD must be attached to the polypeptide chains by noncovalent bonds, since FAD was separated from the dissociated and pre- sumably unfolded protein by dialysis and by electrophoresis. Colman and Black (1) have suggested that the FAD is bound covalently, possibly to a cysteinyl residue, because the FAD could be dissociated from the polypeptide chain in 6 M urea only in the presence of NADPH. These authors observed that the addition of 6 M urea to glutathione reductase produces multiple sedimenting species when examined by sedimentation velocity, in contrast to the single boundary observed here in 8 M urea. Clearly, 6 M urea is not adequate to achieve complete disruption of the native conformation but does produce a series of partially unfolded structures, which, apparently, largely retain the ability to bind FAD. The addition of NADPH to the protein in 6 M

urea may release the FAD by displacing the equilibrium between the folded and the unfolded forms toward the latter.

Acknowledgment-We are indebted to Mr. Frank Castellino for performing the osmotic pressure measurements.

REFERENCES 1. COLMAN, R. F., AND BLACK, S., J. Biol. Chem., 240,1796 (1965). 2. MASSEY, V., AND WILLIAMS, C. H., JR., J. Biol. Chem., 240,

4470 (1965). 3. RACKER, E., J. Biol. Chem., 217, 855 (1955). 4. WARBURG, O., AND CHRISTIAN, W., Biochem. Z., 310,334 (1942). 5. SCHACHMAN, H. K., in S. P. COLOWICK AND N. 0. KAPLAN

(Editors), Methods in enzumolos~, VoZ. IV, Academic Press, tiewYo&, 1957, p. 32. - --.

6. YPHANTIS. D. A.. Biochemistrv. 3. 297 (1964). 7. EDELSTEI&, S. J.; AND SCHAC&A&, H. K., J’. Biol. Chem., 242,

306 (1967). 8. DAVIS. B. J.. Ann. N. Y. Acad. Sci.. 121,40 L (1964). 9. STELL~AGE~, E., J. Biol. Chem., 2&Z, 602 (1967).

10. DOTY, P., AND GEIDUSCHEK, E. P., in H. NEURATH AND K. BAILEY (Editors), The proteins, VoZ. 14, Academic Press, New York, 1953, p. 493.

11. SCHERAGA. H. A.. AND MANDELKERN. L.. J. Amer. Chem. Sot.. 76, 179 (1953). ’

I I

12. MAPSON, L. W., AND ISHERWOOD, F. A., Biochem. J., 86, 173 (1963).

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Richard D. Mavis and Earle StellwagenYeast

Purification and Subunit Structure of Glutathione Reductase from Bakers'

1968, 243:809-814.J. Biol. Chem. 

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