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Superoxide Dismutase Beauchamp1971
Superoxide Dismutase Beauchamp1971
Superoxide Dismutase:
Improved Assays and an Assay Applicable to Acrylamide Gels1
‘The work reported here was supported in full by Grant GM-10287 from the
National Institutes of Health, Bethesda, Maryland.
‘Predoctoral trainee of the National Institutes of Health.
276
ASSAYS OF SUPEROXIDE DISMUTASE 277
upon reoxidation in air (8) and that 02- reduces nitro blue tetrazolium
(11,12) suggested an assay for superoxide dismutase which could be
applied both in free solution and in polyacrylamide gels and which could
be made independent of other enzymes or proteins. The assays
developed,
utilized photochemical events to generate O,- and used nitro blue tetra-
zolium to detect this radical. Superoxide dismutase inhibited the formation of
the blue formazan and could be quantitated on this basis. When the
reactions were performed on acrylamide gels, superoxide dismutase
signaled its location by causing achromatic zones on otherwise uniformly
blue gels. Nitro blue tetraeolium also proved to be a useful detector of O,-
generated by the xanthine oxidase system and provided the basis for an
assay which was free of interference by other catalytic activities and which
could be used to assay for superoxide dismutase in crude extracts.
RESULTS
032-
5 028-
2
In
'0 024-
:
I I I I I I
0 I n?h~ne 03xidase4 x510’] 6 7
Xa [hi
4 8 12 16 20 24 28 32
Superoxide Dismutase (nanograms/ml)
bovine heart gave multiple bands of protein (SC) but only one band of
superoxide dismutase activity (2b), which coincided with the band pro-
duced by the enzyme when purified (2a) from this source. Crude extracts
of bovine brain (set 3)) bovine lung (set 4)) or bovine erythrocytes (set
5) similarly exhibited multiple protein bands but only one zone of superoxide
dismutase activity. The diffuseness of the superoxide dis-mutase zone in gel (5b)
was caused by the presence of massive amounts of hemoglobin which, although
devoid of superoxide dismutase activity,
ASSAYS OF SUPEROXIDE DISMUTASE
I a b c Za’b c
3 a b 4a b 5a bc
FIG. 5. Activity and protein stains applied to polyacrylamide elcctrophoretograms
of several crude extracts and purified preparations of superoxide dismutase. In all
cases samples were applied to the gels and electrophoreais was performed before
the staining procedures were used: (la) 1.4 fig E. coli superoxide dismutase, stained
for activity; (lb) 7.5 ~1 crude extract E. co& stained for activity; (1~) 15 pl crude
extract E. coli, stained for protein; (2a) 0.07 pg purified bovine heart superoxide
dismutase stained for activity; (2b) 1.0 pl crude extract bovine myocardium,
stained for activity; (2~) 5.0 pl crude extract bovine myocardium, stained for
protein; (3a) 1.0 pl crude extract bovine lung, stained for activity; (3b) 12.5 ~1
crude extract bovine lung, stained for protein; (4a) 1.0 ~1 crude extract bovine
brain, stained for activity; (4b) 15 ~1 crude extract of bovine brain, stained for
protein; (5a) 0.049 pg purified bovine erythrocyte superoxide dismutase, stained
for activity; (5b) 1.0 ~1 bovine hemolyzate, stained for activity ; (5~) 3 ~1 bovine
hemolyzate, stained for protein.
284 BEAUCHAMP AND FRIDOVICH
did modify the color of the blue formazan deposited in the gel, and this
resulted in an apparent achromatic zone in black and white photographs.
The superoxide dismutase bands obtained with extracts of bovine heart,
lung, brain, and erythrocytes all- migrated identically under these
con-ditions. This result suggests their identity. Bovine heart and
erythrocyte superoxide dismutase have already been found to be
identical by several criteria (2) as have the corresponding proteins
from human brain, liver, and erythrocytes (17).
Survey of Dyes
A variety of dyeshave been tested for their abilities to generate
02-
when illuminated in the presence of tetramethylethylenediamine and
oxygen. The criterion of O,- production was the ability to cause a photo-
reduction of NBT, which was inhibitable by superoxide dismutase. Thus,
reaction mixtures contained 6.7 X lO+ M or 8.34 x 1O-7 M dye, 1.7 X
1O-4 M NBT, 10e2M
tetramethylethylenediamine, and 0.05 M potassium
phosphate at pH 7.8 and equilibrated with air at
25”. These reaction
mixtures were illuminated for 10 min in the
presence and in the absence
of 1.3 fig superoxide dismutase/ml. All the
dyes tested, whose behavior
is summarized in Table 1, were capable of the
photoproduction of O,-.
DISCUSSION
The assays described for superoxide dismutase utilize NBT as the
detector of 02- and define superoxide dismutase activity in terms of its
ability to inhibit the reduction of NBT due to 02-. The existence of re-
TABLE 1
Dye-Mediated Photoreduction of NBT and Its Inhibition
by Superoxide Dismutase
Cuvets containing 10e2 M tetramethylethylenediamine, 1.7 X 1O-4 M NBT,
and the indicated concentration of dye in 0.05 M potassium phosphate buffer, pH
7.8, at 25” were illuminated for 10 min in the presence and absence of 1.3 rg
superoxide dismutase ml. The change in OD was then read at 560 nm.
AOD/lO min
in presence
Concentration, AOD/lO of superoxide
Dye Dye class M min dismutase
Fluorescein Xanthene 6.67 x 10-C 0.305 0.020
Eosin yellowish Xanthene 6.67 x 10-G 0.203 0.045
Azure C Thiazine 8.34 x lo-’ 0.295 0.018
Methylene blue Thiazine 8.34 x 10-T 0.161 0.013
Proflavin Acridine 6.67 x 10-g 0.331 0.021
Acridine yellow Acridine 6.67 X 10-o 0.205 0.023
ASSAYS OF SUPEROXIDE DIBMCTASE 285
SUMMARY
Nitro blue tetrazolium has been used to intercept
O,- generated en-
zymically or photochemically. The reduction of NBT by 02- has been
utilized as the basis of assays for superoxide dismutase, which exposes
its presence by inhibiting the reduction of NBT. Superoxide dismutase could
thus be assayed either in crude extracts or in purified protein fractions. The
assays described are sensitive to rig/ml levels of super-oxide dismutase and
were applicable in free solution or on polyacrylamide gels. The staining
procedure for localizing superoxide dismutase on poly-
acrylamide electrophoretograms has been applied to extracts
obtained from a variety of sources. E. coli has been found to contain
two superoxide dismutases whereas bovine heart, brain, lung, and
erthrocytes contain only one.
REFERENCES
1. McCo~o, J. M., AND FBIDOVICH, I., J. Biol. Chem. 244, 6049 (1969).
2. KEELE, B. B., JR., MCCOBD, J. M., AND FBIDOVICH, I., J. Biol. Chem. 246, 2875
(1971).
3. KEELE, B. B., JR., MCCORD, J. M., AND FRIDOVICH, I., J. Biol. Chem. 245, 6176
(1970).
4. McCo~o, J. M., KEELE, B. B., JR., AND FBIDOVICH,I., Proc. Nat. Acad. Sci. U, S,, 68,
1024 (1971).
5. MCCOBD,J. M., AND FBIDOVICH, I., J. Biol. Chem. 244, 6056 (1969).
6. McCOBD, J. M., AND FFCIDOVICH, I., J. Bid. Chem. 245, 1374 (1970).
7. BEAUCHAMP, C., AND FRIDOVICH, I., J. Biol. Chem. 245, 4641 (1970).
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287
ASSAYS OF SUPEROXIDE DISMUTASE