Vol.

THE JOURNAL
OF BIOLOGICAL
CHEMISTRY
247, No. 11, Issue of June 10, PD. 34103414,
Printed
in U.S.A.

The Purification
from Neurospora

1972

and Properties
crassa*

of Superoxide

Dismutase

(Received for publication,

HARA
From

P. MISRA

AND

the Department

IRWIN

of Biochemistry,

Duke

Universzty

Soluble extracts of Neurospora

crassa contain a single,
electrophoretically distinct, superoxide dismutase. This
enzyme has been isolated and has been found to be a bluegreen, 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 composedof 2 subunits of equal
size joined only by noncovalent interractions. The Neurospora enzyme contains two Cu++ and two Zn++ per molecule. 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 oxygenmetabolizing
organisms and has been proposed to be an important 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-containing 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 obtained from bovine erythrocytes.
We will now describe the
purification
and properties of the superoxide dismutase from
Neurospora crassa.
AND

Center, Durham,

Carolina 277iO

North

terms of its ability to inhibit the superoxide-mediated

reduction 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 peroxidation 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 adrenochrome (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

METHODS

in full by Research Grant GM-10287

of Health.

Medical

DL-epinephrine,
cytochrome c (type III), and xanthine were
products
of Sigma.
Microgranular
diethylaminoethyl
cellulose (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 proteolytic enzymes (6). Superoxide
dismutase was assayed in
* This work was supported
from the National Institutes

9, 197 2)

FRIDOVICH

SUMMARY

MATERIALS

February

.Q

IO

PO

30

40
50
60
Fraction
Number

70

80

90

100

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

H. P. Misra

Issue of June 10, 197%

and I. Fridovich

3411

TABLE
I
PuriJication

Fraction

Total
protein

Volume

Total units
of enzyme

Specific
activity

Fold
purification

Cield

.%
Soluble extract
Tsuchihashi supernate.
Ethanolic phase
Acetone precipi.
tate
Final product.

224,000

44*

1.0

6,520
1,850

8,150
1,800

652,000
468,000

80
260

1.8
5.9

100
72

120
10

320
36

245,000
95,400

766
2,650

17.4
60.2

38
14

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.

00

2700

2900

3100

GOUSS

3300

350 '0

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
300

400

500

600

700

800

nanometers

FIG. 2. Absorption
spectrum of superoxide dismutase in the
The enzyme was at 19.15 mg per ml in 6.65 M potassium
visible.
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-

nanometers
FIG. 3.

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

Xuperoxide Disnzutase from Neurospora crassa

Vol. 247, hTo. 11

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 phosphatebuffer
(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
suspensionwas vigorously stirred for 2 hours at room temperature. 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 phaseswereallowedto separatefor 30 min,
and the upper phasewas then collected and clarified by centrifugation at 13,000 x g for 15 min. All subsequentsteps
were performed at 0 -+ 4. The organic phasewas cooled to
0, and 0.65 volume of acetone, previously chilled to -2O,
was added with vigorous stirring. The precipitate which
formed wasremoved 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 precipitate which then formed was collected by centrifugation
at 13,000 x g for 20 min and wassuspendedin 120ml of 0.005v
potassiumphosphate(pH 7.8) with the aid of a Potter-Elvehjem
homogenizer. Insoluble material was removed by centrifugation, and the clear solution of superoxide dismutasewas dialyzed against several changesof 0.0025M potassiumphosphate
buffer (pH 7.8) and was then adsorbedonto a column (2.5 x 32
cm) of DE-32 which had previously been equilibrated with
this buffer. A linear gradient of potassiumphosphate (0.0025
-+ 0.050 M) at pH 7.8, in a total volume of 1 liter, was then applied and 5-ml fractions were collected. The results of this
chromatographic procedure are shown in Fig. 1. Fractions
having a specific activity in escessof 1500units of superoxide
dismutaseper 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 pooledmaterial was 2,650.
The results of this purification procedure are summarized
in Table I. The protein concentrationsof the relatively crude
fractions obtained prior to column chromatography were determined by the biuret method (10) whereasthe protein concentrations of chromatographicfractions were basedon absorbancein
the short ultraviolet (11). In the previously reported purification of superoxidedismutasefrom bovine tissues(2), the protein
concentrations of relatively crude fractions were based upon
absorbanceat 280 nm. This was also the method used in
surveying the amount of superoxide dismutasepresent in a
variety of microorganisms(1). When the specific activity of
crude soluble extracts of Neurosporawas determined on the
basisof absorbancyat 280 nm, it was found to be 2.4. This is
comparableto the specific activities found for soluble extracts
of other aerobic organisms(1). On this basis, the total purification achieved by the procedure outlined in Table I was
the central

6. Polyacrylamide gel electrophoresisof Neurosporaextract (upper set) and of Neurospora superoxide dismutase (lower
FIG.

set).

In each set, the outer gels were stained for protein,

whereas

gel was stained for enzymatic

activity.

The following

amountsof 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

Issue of June 10, 1972

and I. Fridovich

3413
TABLE

Amino

11

acid analysis

Amino acid

Lysine......................................
Histidine ...................................
Arginine ....................................
Aspartic acid. ..............................
Threonine..................................

12
11
9
36
26

Serine ......................................

14

Glutamic acid...............................
Proline .....................................
Glycine .....................................
Alanine.....................................
Half-cystine ................................
Valine .....................................
Methionine .................................
Isoleucine...................................
Leucine.....................................
Tyrosine ....................................
Phenylalanine...............................

20
14
39
20
3
22

Total numberof residues

....................
Total residuesX 120........................

13
11
2
6
258
30,960

a Values are given to the nearestinteger.

llO-fold over the first solubleextract. The specificactivity of
purified superoxidedismutasefrom N. crassais comparableto
that of the enzyme from bovine tissues(2, 3).
Absorption
Spectra-The purified superoxide dismutasewas
blue-greenand exhibited an absorption maximum at 660 nm,
whoseEm was 490. This absorption in the visible region of
the spectrum is shownin Fig. 2. The spectrum of the enzyme
in the ultraviolet region was similar to the absorption spectrum
of phenylalanineand is shownin Fig. 3. This spectrum,which
lacks the 280 nm maximum usually associatedwith proteins,
indicates that the Neurosporasuperoxide dismutase, like the
correspondingbovine enzyme (2, 3), is devoid of tryptophan.
The electron paramagnetic resonancespectrum of Neurospora
superoxidedismutasewas characteristic of Cu*+ and is shown
in Fig. 4. Double integration of this signal indicated 2.04
molesof Cu++ per 31,100g of enzyme. The parametersof the
electron paramagnetic resonancesignal 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 presentsIn 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 slopeof the line in Fig. 5 and assuminga
partial specific volume of 0.73, the molecular weight was
calculatedby the method of Yphantis (12) to be 31,100.
were stained for protein and the central gel was stained for enzymatic activity. The amountof protein that was applied onto
the gelswasasfollows. Upper se6(left to right), 25pg, 100ng, and
20 pg; lowerset (left to right),
10 fig, 80 ng, and 15 Pg. Upper set
Fro. 7. Effect of freezing and thawing
of Neurospora

superoxide

dismutase.

a concentrated solution
Within each set, outer gels

was frozen and thawed at 26 ng per ml; lower

thawed at 2.6 mg per ml.

set was frozen and

3414

Superoxide

Dismutase

Polyacrylamide
Gel Electrophresh-The
crude soluble extract of Neurospora was analyzed by gel electrophoresis
(13),
as was the purified
superoxide dismutase.
Protein was visualized by staining with Amido
black, whereas superoxide
dismutase activity was localized by its ability to prevent the
reduction
of nitroblue
tetrazolium
by photochemically
generated 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 superoxide 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-mercaptoethanol 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 enzyme 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 indicated 1.93 moles of Cu++ and 1.80 moles of Zn++ per 31,100 g
of superoxide dismutase.

from

Neurospora

crassa

Vol. 247, X0.

11

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 composition, 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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