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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
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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.
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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.
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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.
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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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