THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 247, No. 11, Issue of June 10, PD. 34103414, 1972

Printed in U.S.A.

The Purification and Properties of Superoxide Dismutase

from Neurospora crassa*

(Received for publication, February 9, 197 2)

HARA P. MISRA AND IRWIN FRIDOVICH

From the Department of Biochemistry, Duke Universzty Medical Center, Durham, North Carolina 277iO

SUMMARY

Soluble extracts of Neurospora crassa contain a single, electrophoretically distinct, superoxide dismutase. This enzyme has been isolated and has been found to be a blue- green, copper- and zinc-containing enzyme, similar to that already described from bovine tissues and from garden peas. The molecular weight was ,approximately 31,000, and the enzyme appeared to be composed of 2 subunits of equal size joined only by noncovalent interractions. The Neu- rospora enzyme contains two Cu++ and two Zn++ per mol- ecule. The ultraviolet absorption spectrum indicates a lack of tryptophan. Amino acid analyses are reported as are the spectral and catalytic properties.

Superoxide dismutase seems to be present in all oxygen- metabolizing organisms and has been proposed to be an im- portant component of the defense mechanisms which allow life in the presence of oxygen (1). When isolated from bovine erythrocytes and heart muscle, this enzyme was found to have a blue-green color and to contain copper and zinc (2, 3), whereas the enzyme isolated from Escherichia coli was red-purple and contained manganese (4). How and when did this substitution of a manganese-containing enzyme by a copper- and zinc-con- taining enzyme of comparable activity occur? This question and others of evolutionary significance, dictated the desirability of examining the superoxide dismutases from a wide range of living things. The superoxide dismutase of garden peas has recently been reported (5) to be strikingly similar to that ob- tained from bovine erythrocytes. We will now describe the purification and properties of the superoxide dismutase from Neurospora crassa.

MATERIALS AND METHODS

DL-epinephrine, cytochrome c (type III), and xanthine were products of Sigma. Microgranular diethylaminoethyl cellu- lose (DE-32) was obtained from the Reeve Angel Co. Milk xanthine oxidase was purified by Mr. Ralph Wiley, from raw cream, by a procedure which did not involve exposure to pro- teolytic enzymes (6). Superoxide dismutase was assayed in

* This work was supported in full by Research Grant GM-10287 from the National Institutes of Health.

terms of its ability to inhibit the superoxide-mediated reduc- tion of ferricytochrome c by the xanthine oxidase system. This assay was performed as originally described (2) but with the modification that 5 x lOA M cyanide was added to inhibit the peroxidases which are present in crude extracts and which may otherwise interfere with this assay by catalyzing the peroxida- tion of ferrocytochrome c. Since xanthine oxidase was the last component added to the assay mixtures and since xanthine oxidase is protected against cyanide inhibition by the presence of xanthine, this level of cyanide did not interfere with the action of xanthine oxidase. This level of cyanide had no effect upon the activity of superoxide dismutase. The use of cyanide in assays of superoxide dismutase, which depended upon the reduction of nitroblue tetrazolium by O,, has been described (7). Superoxide dismutase can conveniently be assayed in terms of its ability to inhibit the autoxidation of epinephrine to adre- nochrome (8). This simple assay was used in screening column eluates. All spectrophotometric assays were performed at 25 in a Gilford model 2000 absorbance recorder. Absorption spectra were recorded with a Gary model 15 spectrophotometer. Electron paramagnetic resonance spectra were obtained with a

o...o .Q -

I I I I I I I I

0 IO PO 30 40 50 60 70 80 I

90 100 Fraction Number

FIG. 1. Elution profile. The acetone precipitate, obtained during the purification procedure, was extracted with 0.005 M potassium phosphate (pH 7.8), and this extract, after dialysis against 0.0025 M potassium phosphate at pH 7.8, was adsorbed onto a column (2.5 X 32 cm) of DE-32 equilibrated with the same buffer. A linear gradient (0.0025 + 0.050 M) in this buffer was applied in a total volume of 1000 ml, and 5 ml fractions were collected. This figure illustrates the results obtained. l -- l , absorbance at 280 nm; A- - -A, superoxide dismutase activity; 0 -----0 , conductance.

3410

Issue of June 10, 197% H. P. Misra and I. Fridovich 3411

TABLE I PuriJication

Total Total units Specific protein of enzyme activity

Fold purifi- cation

7 Cield

224,000 44* 1.0

.- %

8,150 652,000 80 1.8 100 1,800 468,000 260 5.9 72

320 245,000 766 17.4 38 36 95,400 2,650 60.2 14

Fraction Volume

6,520 1,850

120 10

Soluble extract Tsuchihashi su-

pernate. Ethanolic phase Acetone precipi.

tate Final product.

a:Homogenization of mycelia did not extract as much protein or as much superoxide dismutases as did subsequent stirring with the chloroform-ethanol mixture. This is the reason that the soluble extract, obtained by centrifugation of a homogenate of mycelia, contained less protein and enzyme than did the extract obtained by centrifugation after the homogenate had been treated with chloroform-ethanol.

* When based upon absorbance at 280 nm, this specific activity was 2.4.

I '0

I I I I I I I I I

00 2700 2900 3100 3300 350 GOUSS

FIG. 4. Electron paramagnetic resonance spectrum of t,he superoxide dismutase from Neurospora crassa. The enzyme was present at 26 mg per ml in 0.05 M potassium phosphate buffer at pH 7.8. Other conditions were: microwave frequency, 9.133 GHz; microwave power, 5 mwatts; modulation amplitude, 4 gauss; scan rate, 125 gauss per min; time constant, 1.0 s; receiver gain, 2000; and sample temperature, -100. The values of the spectral parameters are g, = 2.073 and g,, = 2.260.

.6 -

1

I I I I I

300 400 500 600 700 800 nanometers

FIG. 2. Absorption spectrum of superoxide dismutase in the visible. The enzyme was at 19.15 mg per ml in 6.65 M potassium phosphate at pH 7.8. The absorption maximum is at 660 nm, and the molar extinction coefficient at this wave length was 490.

FIG. 5. Equilibrium sedimentation of Neurospora superoxide dismutase. Protein concentration was 0.7 mg per ml dialyzed against 0.0025 M potassium phosphate, pH 7.8, and 0.1 M sodium chloride. Rotor speed was 24,000 rpm.

Varian model E-9HF equipped with a 9.5 GHz microwave bridge assembly and operated at a modulation frequency of 100 KHz. These spectra were recorded and analyzed by Dr. K. V. Rajagopalan. Molecular weight was calculated from sedimentation equilibrium data, obtained by Dr. J. Huston, with a Beckman model E ultracentrifuge. Amino acid analyses were performed by Dr. H. Steinman with a Beckman model 120 C amino acid analyzer. Metal analyses were performed by Mr. Dennis Winge using a Perkin-Elmer model 303 atomic absorption spectrophotometer. Neurospora crassa was grown at 32-34 in Fries basal medium (9) under vigorous aeration and with constant agitation for 36 hours. The mycelia were collected by filtration and, after being washed twice with cold deionized water, were stored frozen until needed. Approx-

FIG. 3. Absorption spectrum of superoxide dismutase in the ultraviolet. The enzyme was at 2.65 mg per ml in 0.05 M potas- sium phosphate at pH 7.8. The molar extinction coefficient at 258 nm was 17,400 and at 280 nm was 11,700.

nanometers FIG. 3.

Xuperoxide Disnzutase from Neurospora crassa Vol. 247, hTo. 11

FIG. 6. Polyacrylamide gel electrophoresis of Neurospora ex- tract (upper set) and of Neurospora superoxide dismutase (lower set). In each set, the outer gels were stained for protein, whereas

imately 1 kg of wet weight mycelia was obtained from 25 liters of culture medium.

RESULTS

PuriJication of Superoxide Dismutase-Two kilograms of frozen mycelia were partially thawed and then homogenized for 5 min in 4 liters of 0.005 M potassium phosphate buffer (pH 7.8) with a Sorvall Omni-Mixer which was operated at its top speed. Two liters of an ethanol-chloroform mixture (5:3) were then added to the homogenate, and the resultant thick suspension was vigorously stirred for 2 hours at room tem- perature. This mixture was clarified by centrifugation at 13,000 X g for 15 min. Solid KtHPOa (300 g per liter) was then added slowly to the clear supernatant solution while it was stirred at 23. This resulted in the salting out of a light organic phase. The phases were allowed to separate for 30 min, and the upper phase was then collected and clarified by cen- trifugation at 13,000 x g for 15 min. All subsequent steps were performed at 0 -+ 4. The organic phase was cooled to 0, and 0.65 volume of acetone, previously chilled to -2O, was added with vigorous stirring. The precipitate which formed was removed by centrifugation at 13,000 x g for 15 min and was discarded, while the supernatant solution was treated with an equal volume of chilled acetone. The pale blue pre- cipitate which then formed was collected by centrifugation at 13,000 x g for 20 min and was suspended in 120 ml of 0.005 v potassium phosphate (pH 7.8) with the aid of a Potter-Elvehjem homogenizer. Insoluble material was removed by centrifuga- tion, and the clear solution of superoxide dismutase was di- alyzed against several changes of 0.0025 M potassium phosphate buffer (pH 7.8) and was then adsorbed onto a column (2.5 x 32 cm) of DE-32 which had previously been equilibrated with this buffer. A linear gradient of potassium phosphate (0.0025 -+ 0.050 M) at pH 7.8, in a total volume of 1 liter, was then ap- plied and 5-ml fractions were collected. The results of this chromatographic procedure are shown in Fig. 1. Fractions having a specific activity in escess of 1500 units of superoxide dismutase per mg of protein were pooled and concentrated by ultrafiltration over a Diaflo UM-10 membrane. The highest specific activity observed was 3,080, and the specific activity of the pooled material was 2,650.

The results of this purification procedure are summarized in Table I. The protein concentrations of the relatively crude fractions obtained prior to column chromatography were deter- mined by the biuret method (10) whereas the protein concentra- tions of chromatographic fractions were based on absorbance in the short ultraviolet (11). In the previously reported purifica- tion of superoxide dismutase from bovine tissues (2), the protein concentrations of relatively crude fractions were based upon absorbance at 280 nm. This was also the method used in surveying the amount of superoxide dismutase present in a variety of microorganisms (1). When the specific activity of crude soluble extracts of Neurospora was determined on the basis of absorbancy at 280 nm, it was found to be 2.4. This is comparable to the specific activities found for soluble extracts of other aerobic organisms (1). On this basis, the total puri- fication achieved by the procedure outlined in Table I was

the central gel was stained for enzymatic activity. The following amounts of proteins were applied to the gels. Upper set (left to right), 100 pg, 30 pg, and 45 pg; lower set (left to right), 10 pg, 80 ng, and 15 pg.

H. P. Misra and I. Fridovich 3413 Issue of June 10, 1972

TABLE 11

Amino acid analysis

Amino acid

Lysine ...................................... 12 Histidine ................................... 11 Arginine .................................... 9 Aspartic acid. .............................. 36 Threonine .................................. Serine ...................................... Glutamic acid ............................... Proline ..................................... Glycine ..................................... Alanine ..................................... Half-cystine ................................ Valine ..................................... Methionine ................................. Isoleucine ................................... Leucine ..................................... Tyrosine .................................... Phenylalanine ...............................

26 14 20 14 39 20 3

22

13 11 2 6

I 0

Total number of residues .................... Total residues X 120 ........................

a Values are given to the nearest integer.

258 30,960

llO-fold over the first soluble extract. The specific activity of purified superoxide dismutase from N. crassa is comparable to that of the enzyme from bovine tissues (2, 3).

Absorption Spectra-The purified superoxide dismutase was blue-green and exhibited an absorption maximum at 660 nm, whose Em was 490. This absorption in the visible region of the spectrum is shown in Fig. 2. The spectrum of the enzyme in the ultraviolet region was similar to the absorption spectrum of phenylalanine and is shown in Fig. 3. This spectrum, which lacks the 280 nm maximum usually associated with proteins, indicates that the Neurospora superoxide dismutase, like the corresponding bovine enzyme (2, 3), is devoid of tryptophan. The electron paramagnetic resonance spectrum of Neurospora superoxide dismutase was characteristic of Cu*+ and is shown in Fig. 4. Double integration of this signal indicated 2.04 moles of Cu++ per 31,100 g of enzyme. The parameters of the electron paramagnetic resonance signal were g, = 2.073 and gll = 2.260.

Molecular Weight-The purified enzyme was brought to sedimentation equilibrium at 24,000 rpm while dissolved in 0.0025 M potassium phosphate, 0.10 M NaCl at pH 7.8 and 17.4, in an An-D rotor. Fig. 5 presents In fringe displacement as a function of the square of the distance from the center of rotation. The data, when so plotted, do fit a straight line, which indicates homogeneity with respect to sedimentation properties. From the slope of the line in Fig. 5 and assuming a partial specific volume of 0.73, the molecular weight was calculated by the method of Yphantis (12) to be 31,100.

were stained for protein and the central gel was stained for en- zymatic activity. The amount of protein that was applied onto the gels was as follows. Upper se6 (left to right), 25 pg, 100 ng, and 20 pg; lower set (left to right), 10 fig, 80 ng, and 15 Pg. Upper set

Fro. 7. Effect of freezing and thawing a concentrated solution was frozen and thawed at 26 ng per ml; lower set was frozen and of Neurospora superoxide dismutase. Within each set, outer gels thawed at 2.6 mg per ml.

3414 Superoxide Dismutase from Neurospora crassa Vol. 247, X0. 11

Polyacrylamide Gel Electrophresh-The crude soluble ex- tract of Neurospora was analyzed by gel electrophoresis (13), as was the purified superoxide dismutase. Protein was vis- ualized by staining with Amido black, whereas superoxide dismutase activity was localized by its ability to prevent the reduction of nitroblue tetrazolium by photochemically gen- erated superoxide radicals (7). Fig. 6 illustrates the results of these manipulations. The crude extracts of Neurospora exhibited at least 18 protein zones but only one band of super- oxide dismutase activity. The purified enzyme gave only one discernible band of protein which coincided with the zone of enzymatic activity.

Gel electrophoresis of purified superoxide dismutase, before and after freezing, demonstrated that freezing concentrated solutions (26 mg per ml) of the Neurospora enzyme resulted in the generation of multiple active components. This effect is illustrated in Fig. 7. Freezing of dilute solutions (2.6 mg per ml) of this enzyme, under otherwise identical conditions, did not result in generation of multiple components.

Subunit Structure-Gel electrophoresis in the presence of sodium dodecyl sulfate, with and without @-mercaptoethanol, was used to explore the quaternary structure of the enzyme (14). The gels were calibrated with the following molecular weight standards: transferrin, 77,000; human serum albumin, 67,500; catalase, 60,000; ovalbumin, 43,000; pepsin, 35,000; carbonic anhydrase, 29,000; trypsin, 23,000; bovine superoxide dismutase subunits, 16,500. In the absence of /3-mercapto- ethanol the enzyme gave a molecular weight of 16,800 and in its presence of 18,000. These results imply that the Neurospora superoxide dismutase is composed of 2 subunits of equal size which are associated by noncovalent interractions.

Amino Acid Analysis-Triplicate 0.2-mg-samples of the en- zyme were sealed in vacw, in Pyrex tubes containing 1.0 ml of 6 N HCl, 0.1% phenol, and were then incubated at 110 for 24, 48, and 72 hours. These tubes were then opened, the contents evaporated to dryness in vacua, and the residues redissolved in 1.0 ml of 0.01 N HCl, 0.1% phenol. These samples were then analyzed on a Beckman model 120 C amino acid analyzer. The results of these analyses, corrected for time-dependent losses by extrapolation to zero time, are shown in Table II.

Content of Cu++ and .%*--Double integration of the electron paramagnetic resonance signal indicated 2.04 moles of Cu++ per 31,100 g of enzyme. Atomic absorption spectroscopy in- dicated 1.93 moles of Cu++ and 1.80 moles of Zn++ per 31,100 g of superoxide dismutase.

DISCUSSION

The molecular properties of superoxide dismutase appear to have been rigidly preserved during the evolution of eucaryotes. Thus, the enzyme from N. crassa is similar to that from bovine tissues (2, 3) and from garden peas (5) with respect to molecular weight, quaternary structure, metal content, visible, ultraviolet, and electron paramagnetic resonance spectra, amino acid com- position, and enzymatic activity. In addition, the Neurospora enzyme, like the bovine enzyme, survived an unusual purification procedure which included the use of a chloroform-ethanol step to denature extraneous proteins, followed by the salting out of an ethanol-rich phase. During this step both the bovine and the Neurospora enzymes migrated into the supernatant organic phase and could be recovered therefrom by precipitation with cold acetone. It may, perhaps, be anticipated that all eucaryotes contain superoxide dismutase whose properties are similar to those already found for the enzymes from the cow (2, 3), the garden peas (5) and N. crassa, whereas all procaryotes will be found to contain the distinct manganese-containing enzyme already demonstrated in Escherichiu coli (4). Isolation of this enzyme from additional sources is already under way in order to test the validity of this generalization.

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