ANALYTICAL

BIOCHtMISTR\r

153, 536-541

(1986)

A Sensitive
Spectrophotometric
Superoxide
Dismutase
FRANCEXO

PAOLETTI,

Method
for the Determination
Activity in Tissue Extracts

DONATELLA
AND ANNA

ALDINUCCI,
CAPARRINI

Received October

ALESSANDRA

of

MOCALI,

3. 1985

Superoxide dismutase (EC I. IS. I. I) has been assayed by a spectrophotometric method based
on the inhibition
of a superoxide-driven
N.ADH oxidation.
The assay consists ofa purely chemical
reaction sequence which involves
EDTA.
Mn(II).
mercaptoethanol.
and molecular
oxygen.
re-

quiring neither auxiliary enzymes nor sophisticated equipment. The method is very flexible and
rapid and is applicable with high sensitivity
to the determination
of both pure and crude superoxide
dismutase
preparations.
The decrease of the rate of NADH
oxidation
is a function
of enzyme
concentration.
and saturation
levels are attainable.
Fifty percent inhibition.
corresponding
to one
unit ofthe enzyme, is produced
by approximately
I5 ng of pure superoxide
dismutase.
Experiments
on rat liver cytosol have shown the specificity
of the method for superoxide
dismutase.
Moreover.
common
cellular components
do not interfere
with the measurement.
except for hemoglobin
when present at relatively
high concentrations.
The assay is performed
at physiological
pH and
is unaffected
by catalase.
15 1986 Academtc Press. Inc
KEY WORDS: superoxide
dismutase;
spectrophotometric
determination:
chemical
assay: superoxide:
NADH
oxidation:
metal complex.

Superoxide dismutase (SOD) (EC 1.15.1.1)

is a family of metalloenzymes which is known
to accelerate spontaneous dismutation of the
superoxide radical to hydrogen peroxide and
molecular oxygen (1). SOD is widely distributed among aerobically living organisms and
has been inferred to play an important role in
controlling superoxide levels in cellular compartments (2,3).
The direct measurement of SOD activity (47) is possible but its application is hampered
by the requirement of special apparatus not
commonly available in the typical laboratory.
The other methods employed for enzyme determination are indirect and rely on the ability
of SOD to inhibit a superoxide-driven reaction. The extent to which SOD reduces the
rate of that reaction is taken as a measure of
enzymic activity. Either enzymatic or nonenzymatic systems are used for the generation
Abbreviations
used: SOD. superoxide
Dea. triethanolamine-diethanolamine;
tetrazolium.
0003-2697/86

$3.00

Copyright 2 1986 by Academic Press. Inc

All rights of reproductmn in any form reserved.

of superoxide (see Refs. (8-9) for review): detection is then accomplished by luminometric
( 10). polarographic
(1 l), or calorimetric
( 1,12,13) techniques, depending on the different approaches and experimental requirements.
Notwithstanding
the large number of
methods available, the need still exists to increase the specificity. accuracy, and simplicity
of the assay. In this paper we describe a spectrophotometric
method for SOD determination based on a purely chemical reaction sequence which eventually leads to NADH oxidation. This procedure, involving stable and
inexpensive reagents. allows a rapid and highly
sensitive measurement of SOD activity in pure
and crude enzyme preparations, with negligible interference by cellular components.
MATERIALS

dismutdse:
TeaNBT. nitro blue

AND

METHODS

Chemicals. Reduced adenine dinucleotide

(P-NADH,
536

disodium salt) was obtained from

SUPEROXIDE

DISMUTASE

Boehringer-Mannheim
(West Germany),
while MnClz . 2H20, ethylenedinitrilotetraacetic acid (EDTA), and 2-mercaptoethanol were
supplied by Merck. Darmstad (West Germany). Pure SOD preparations were obtained
from Diagnostic Data Inc. (beef erythrocytes,
3300 U/mg protein) and from Sigma Chemical
Company (human erythrocytes, ca. 2500 U/
mg protein). Catalase (beef liver. 350 mg/ml
ammonium sulfate suspension) was provided
by Boehringer-Mannheim.
All other chemicals
were analytical grade and used without further
purification.
Eqrr;pmenr. Assays were performed with a
Gilford spectrophotometer (Model 350) connected to a recorder.
Rcqynts l~inrlsolutions. All solutions were
made up with deionized or well-aerated distilled water. according to the following scheme.
( 1) Triethanolamine-diethanolamine
(Tea-Dea) buffer. 100 mM each, pH 7.4. Dilute 14.9 g Tea. 10.5 g Dea. and ca. 13.8 ml
of 37%, HCI up to 1 liter with water, taking
care to maintain the pH around 7.4-7.5.
(3) NADH, 7.5 mM. For 100 assays, dissolve 20 mg of reduced nucleotide (disodium
salt) in 4 ml of water.
(3) EDTA/MnCI,,
100 mM/50 mM. Make
a stock solution of EDTA. 0.3 M (i.e., dissolve
5.85 g of EDTA-acid in a final volume of 100
ml. adjusting the pH to around 7 with molar
NaOH solution) and of MnCL2. 0.1 M (by dissolving 1.62 g of MnC12. 7Hz0 in 100 ml water). The mixture of equal volumes of these
two stock solutions yields our third reagent
(EDTA/MnCI:).
(4) Mercaptoethanol,
10 mrvr. Dilute 0.05
ml of concentrated thiol. 14.2 M, up to 7 I ml
with water.
The NADH solution is stable for at least 3
days in the refrigerator. For longer storage.
keep it at ~20C. The solutions of EDTA.
MnCl?, and mercaptoethanol are quite stable,
even at room temperature. for months. Reagent 3, once made up. can be used over a 2week period (see further comments in l.he Results section).

537

DETERMINATION

Preparation qf rat liver c~msoi. For the

measurement of SOD in rat liver, the tissue
extract is first prepared by homogenizing the
liver in 3 vol of Tea-Dea buffer 25 mM, pH
7.4, and then cleared by centrifugation
at
30,000 rpm for 60 min at 4C. The supernatant is dialyzed against cold homogenization
buffer and then employed for enzymatic assays. Protein determination was carried out
by the L.owry method ( 14) on samples dialyzed
against 0.9% NaCl buffered at pH 7.5-8 with
molar NaHCOI solution.

RESULTS

Description oJt/re method. The principle of

this method is based on the oxidation of
NADH, mediated by superoxide radical, in a
purely chemical system recently developed in
our laboratory. Coenzyme oxidation occurs in
the presence of suitable concentrations of
EDTA. Mr? and mercaptoethanol (see below) through a free-radical chain of reactions
involving thiol oxidation and univalent 02 reduction. A detailed explanation of the reaction
mechanism is beyond the scope of the present
paper and will be reported elsewhere (manuscript submitted). The addition of SOD to the
reaction mixture causes a proportionate inhibition, of the rate of NADH oxidation, thus
confirming the involvement of superoxide in
the process and providing the basis for SOD
activity determination.
To perform the assay sequentially add the
following (see Materials and Methods) to a
cuvette: Tea-Dea buffer. 0.800 ml: NADH
solution, 0.040 ml; EDTA/MnC12 solution,
0.035 ml; sample (or water or buffer) 0.100
ml: mix thoroughly and read against air at 340
nm for a stable baseline: then add mercaptoethanol solution. 0.100 ml.
Mix and monitor the decrease in absorbance
using 0.5-I full scale deflection. The final volume in the cuvette is 1.065 ml and the light
path is IO mm.
A typical analysis of SOD activity is shown
in Fig. 1 where the kinetics of NADH oxidation in the absence (control) and in the pres-

538

PAOLETTI
, SgAq

1.4

-.-*-i -

-----__

---_

-.

--_
\

--__

-11

--__

E
c

z
0

1.2

-SOD

'

- - -SOD 3

-.

\
\

\
\

\ \
\

\
'SOD,

\CONIROI

0
0

16

24

INCUBATION (mtnl

FIG. I. Effect of superoxide

dismutase
on the rate of
NADH
oxidation.
Four assays are carried
out simultaneously in the absence (control)
and in the presence of
SOD (sample 1. IO ng; sample 2, 80 ng: sample 3, 380 ng
of pure enzyme from bovine erythrocytes.
Diagnostic
Data
Inc.). Assay mixtures
are prepared as described
in the text
and decreases in absorbance.
at 340 nm. are recorded
for
about 15 min after mercaptoethanol
addition.
The rate of
NADH
oxidation
in the control is ca. 5 nmol per min. at
room temperature.

ence of superoxide dismutase (SODrm3) are

compared by simultaneous assay using a multicell holder.
The reaction is started by mercaptoethanol
and changes in absorbances are recorded for
about 15 min. Rates of NADH oxidation are
initially low, then increase progressively (usually 2-4 min after mercaptoethanol addition)
to yield good linear kinetics (5- 10 min) which
are used for calculations. Under our conditions
Ai1340 over an S-min interval is 0.250 for the
control, while the presence of 10, 80. and 380
ng of SOD in the assay mixture. yields AA
values of 0.150.0.038, and 0.008, respectively.
For calculations the maximal rates obtained
are expressed as a percentage of the control
(ordinate) and plotted against a suitable reference (abscissa). One unit of the enzyme is
the amount of SOD capable of inhibiting by

ET

AL

50% the rate of NADH oxidation observed in

the control.
Culihration cme \tii/l pure SOD. The determination of SOD activity in pure enzyme
preparations from bovine erythrocytes is
shown in Fig. 2. Relative rates of NADH oxidation are reported as a function of the
amount of enzyme in the assay mixture. The
curve thus obtained shows that inhibition is
not directly proportionate to SOD concentration, but rather follows an exponential-like
function. Almost complete saturation levels
(99% inhibition) are obtained with 400 ng of
the enzyme, while the same amount of heated
SOD fails to inhibit the reaction. One unit of
the enzyme corresponds to ca. 15 ng of pure
superoxide dismutase from beef erythrocytes.
Least-square linear regression analysis was
used to obtain a best-fitting curve over the
range 4-40 ng, by transforming SOD values
into logarithms. The equation is ?; = 1 16.638
- 55.619 s; the correlation coefficient Y

= -0.9925; n = 22.
Deterrninution ofSOD in liver euxtructs. To
test the applicability of this method to SOD
determination in tissue extracts, experiments
were carried out using rat liver cytosol as the
sample (Fig. 3). The assay conditions are the
same as described for pure SOD and the percentage of NADH oxidation is reported as a
function of the total proteins in the extract.
The curve obtained is essentially identical to
that shown in Fig. 2 for the purified enzyme
and in this case too, the boiled sample fails to
inhibit NADH oxidation at any rate. Fifty
percent inhibition is produced by approximately 10 pg of liver extract which means an
average of 100 units of SOD per mg protein
of cell cytosol. while the saturation level is
reached with ca. 180-200 yg proteir.
Curve fitting to experimental data is comparable to that described in the comments to
Fig. 2. The linear regression equation, over a
range of 4-40 pg protein, is c = 115.366
- 67.687 s; the correlation coefficient t

= -0.9869; n = 28.
Precautions and optimal conditiom ,fi~r
rnemuwnmt.
EDTA or other chelators for
Mn2+ (not EGTA)

may alter the optimum

SUPEROXIDE

DISMUTASE

539

DETERMINATION

--t-l--c-i-+20
SUPEROXIDE DISMlJiASE lngl

400

40

FIG. 2. Titration
curve with pure superoxide
dismutase.
Increasing
amounts
of SOD (O-400 ng) from
bovine erythrocytes
(Diagnostic
Data Inc.) are added to incubation
mixtures
and assayed for activity
as
previously
reported.
The rate of NADH
oxidation
(g-min linear kinetic)
is expressed as a percentage
of the
control. which is always run in each set of assays. Values (0: n = 27) refer to separate determinations
carned
out individually
by three of us using different
stock solutions
of the enzyme.
Samples containing
inactive
SOD (0). heated at 100C for 2 min. are shown for comparison.

EDTA/Mn+
ratio and affect the rate of reaction. Alternatively, excess of Mn ions in
the sample could slow down the rate of NADH
oxidation. Other divalent cations of the second
transition series, at concentrations comparable
to that of Mn. do not start the reaction, but
may compete for the chelator. In addition,
when the sample contains free thiols, i.e.,
mercaptoethanol,
cysteine and reduced glutathione. faster reaction rates, as compared to
the control. are observed. To avoid all interference by compounds mentioned above.
samples must be dialyzed against suitable me-

0 L+-

++-

dia before the analysis. However, owing to

handle a large number of samples. dialysis
could be conveniently replaced by a rapid desalting through a small Sephadex G-25 (coarse)
column.
For precise calculations, NADH-oxidase
activity, if present in the sample, should be
evaluated before mercaptoethanol
addition
and subtracted from the final rate. Moreover,
because of the high sensitivity of the method.
samples are usually diluted by such a large
factor that NADH-oxidase or any other interfering activity is practically undetectable. In

t~-+--+-~.+.-..++
I6
LIVER

24

--+
32

184

CYTOSOL ,,,g pratml

FIG. 3. Titration
of SOD in rat liver cytosol. The liver extract 1see Materials
and Methods)
is diluted with
100 mM Tea-Dea
buffer. pH 7.4. to concentrations
suitable for the assay. Measurements
are carried out as
described
in the text. Protein content
of the sample is within a range of O-400 pg as determined
by the
Lowry method. Closed dots (0) represent
the average of four separate determinations
and bars (=) indicate
the range of experimental
values. Open squares (0) refer to assays with boiled samples.

540

PAOLETTI

addition it is worth mentioning that NADPH

reacts as well as NADH in the system (data
not shown) without serving as a substrate for
NADH-oxidase. This fact confirms the flexibility of our method and may turn out very
useful when assaying for SOD in fractions
containing high levels of NADH-oxidase.
Changes of temperature, pH and oxygen
tension in the system may influence the absolute rate of NADH oxidation, but are without effect on the relative degree of inhibition
observed. Each set of assays must refer to its
own control and best measurements are obtained when the values of .&4+,,, of the control.
over an S-min interval, are within the range
of 0.150-0.400. Reactivity of reagent 3 is increased by storage: therefore aged solutions
immediately yield maximal rates of NADH
oxidation without the initial delay shown by
fresh-made preparation of the complex.
Catalase does not interfere with the assay,
while hydrogen peroxide inhibits it at a millimolar concentration level.
DISCUSSION

Any reaction inhibitable by superoxide dismutase could potentially provide the basis for
an indirect assay of the enzyme and, according
to that principle, several methods have been
developed over the years. However. as also
pointed out by Oberley (15). only a few procedures permit the sensitive and reliable determination of enzymatic activity in tissue extracts with low SOD levels.
Our chemical assay seems particularly suitable to that purpose since it allows the measurement of minute amounts of SOD, such as
2 ng, which are far below the detection limit
of most published methods. In addition. we
have found that fifty percent inhibition, i.e.,
one unit of the enzyme, is produced by 15 ng
of pure protein, while values of catalytic activity. as determined by the xanthine-oxidase/
cytochrome
c ( 1), NADH-diaphorase/hydroxylamine ( 13) and xanthine-oxidase/nitro
blue tetrazolium (NBT) ( 12) systems, are ca.
200,626, and 630 ng, respectively. A substantial improvement of the NBT assay has been
obtained by Buettner (16), whose procedure

tT

AL.

yields exactly the same sensitivity reported

here. However. with his method, saturation
levels are not attainable and different values
for half-maximal inhibition are obtained for
pure and crude SOD preparations. These inconveniences are frequently observed in assays
involving the reduction of NBT. cytochrome
(. or other suitable detector and ascribed to
the action of aspecific electron donors on
chromogenic substrates. Our method. on the
contrary, relies on the oxidation of the detector
(NADH in this case) and therefore will not be
affected by the presence of reductants which
are known to occur in tissue extracts ( 12). This
lack of interference is clearly shown by the
fact that calibration curves for pure SOD and
rat liver cytosol are almost identical and saturation levels (99%) are attainable in both
cases. The latter result implies that in our system the same value for catalytic activity is obtained by using either 50% or half-maximal
inhibition for calculation. This avoids the necessity of running a full calibration curve each
time and is of valuable practical importance,
especially when dealing with samples having
low SOD levels. From our data. a specific activity of ca. 66.000 unit/mg protein and ca.
100 unit/mg protein can be calculated for SOD
in pure beef erythrocytes preparations (Diagnostic Data Inc.: see Materials and Methods)
and rat liver cytosol, respectively.
In addition to the experiments with rat liver
cytosol, the present method has been applied
successfully to the measurement of SOD in a
variety of other cell extracts and body fluids
(data not shown). So far, the only major inconvenience encountered comes from hemoglobin; thus, for reliable determination of
SOD in hemolysates. this molecule must be
removed from the sample before assaying.
An important problem in SOD analysis is
the discrimination between the cuprozinc- and
manganese-form of the enzyme. Cyanide. at
concentrations used for inhibiting the cuprozinc enzyme, does not interfere with our assay
and manganese-SOD (Mn-SOD) can be easily
determined by differential measurement. In
addition, this method has proved ofgreat value
in determining traces of Mn-SOD separated

SUPEROXIDE

DISMUTASE

from rat liver cytosol by means of gel filtration

(data not shown). In this regard it is worth
recalling that our assay is carried out at pH
7.4 which favors the detection of Mn-SOD. In
fact, physiological pH is the most suitable for
optimal activity of Mn-SOD, which is not reliably assayed at elevated pH values as required
by other sensitive methods ( 17,18).
On the whole, the present procedure for
SOD determination
involves stable and inexpensive reagents and consists of a single
spectrophotometric step, easily performed on
a time scale of minutes. It appears particularly
suitable for application in the field of biochemistry. plant physiology, and clinical
chemistry.
ACKNOWLEDGMENTS
We would like to thank Professor A. Fonnesu. &airman
of the Institute.
and Dr. V. Boddi for his continuous
suggestions and statistical
analyses.
This research was supported by a grant from the Minister0
della Pubblica Istruzione (6Og).

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541

DETERMINATION

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d Bid

Chc177