ANALYTICAL
RIOCHEMISTRY
Superoxide
Salt
(1978)
Dismutase: A Direct, Continuous Using the Oxygen Electrode M.J.
Charles
86, 561-573
Resc~arch
Centw.
Received
MARSHALL Robert
March
Jonr~ Shropshirr.
Linear Assay
AND M. WORSFOLD & Agnes HutIt Englund
17. 1977: accepted
Orthopuc~dic
December
Hospitcrl,
Oswrstry.
5. 1977
A continuous method for the assay of superoxide dismutase activity which gives a linear recording of the reaction whose slope is a linear function of the amount of enzyme added is described. An oxygen electrode is used to measure the consumption of oxygen which results from the photochemical generation of superoxide from dissolved oxygen followed by its dismutation to oxygen and hydrogen peroxide. Excess superoxide is scavenged by a tetrazolium salt which oxidizes it back to oxygen.
The instability of the superoxide anion has until now led to the use of indirect and somewhat unsatisfactory assay procedures for superoxide dismutase. As reviewed by Fridovich (1) it is usual to assay the enzyme by observing its inhibition of a reaction in which superoxide is a transient intermediate. Superoxide may be generated by xanthine oxidase (2), from reduced phenazine methosulfate (3), photochemically (4), or from potassium superoxide (5). The indicating reactants which have been used include cytochrome c (2), nitro blue tetrazolium (3,4), and hydroxylamine (6). Various autoxidation reactions in which superoxide is generated during the reaction and is also involved in its propagation have also been used (7,g). All these methods suffer from nonlinearity, lack of precision, and vulnerability to interference by a wide range offactors, the effects of which may well go undetected and. in any case, are difficult to interpret or quantify. Rigo et ul. (9,lO) have reported a polarographic technique in which a dropping mercury electrode is used as both source and indicator of O,concentration. This method does not yield a direct measure of enzyme activity, however, and this type of apparatus is not widely used in biochemistry laboratories. The use of pulse radiolysis to generate superoxide, combined with photometric measurement of its concentration. allows the reaction to be followed directly (I l), but this apparatus is even less widely available and would appear to be applicable only to relatively high concentrations of purified enzyme. 561
OOO?-2697i78/086?-0~61~0~.00!0 CopyrIght ‘i‘ 1978 hy Academic Prrsr. Inc. All nght? of reproduction m any form rrwrvrd.
562
MARSHALL
AND
WORSFOLD
Photometric measurement of [OJ has also been combined with generation of 02- from potassium superoxide (12) which has led to an assay (13) with good precision and potentially wide application, although this presumably will be limited by the very small optical density changes and the nonlinear record. A recent paper by Misra and Fridovich (14) describes a method involving the partial inactivation of peroxidase by O,-. These authors suggest that the “positive” aspect of this assay overcomes objections to earlier ones which involved the inhibition of an indicator reaction. However, their method is, in reality, only “positive” as the result of the inhibition of an inhibition (a double “negative”) and suffers all the disadvantages, including limitation of useful range, which they ascribe to “negative” (inhibition) assays, excepting only the slope of the graph on which the results are plotted. We report here a simple, rapid method for the direct assay of superoxide dismutase. It yields a continuous record of the extent ofthe reaction, with a slope which is a linear function of enzyme activity. Each part of the reaction sequence is easily and quickly monitored so that interference by extraneous substances is easily recognized and accounted for. Crude (and therefore minimally altered) tissue extracts can readily be assayed. Superoxide anion is generated photochemically from riboflavin and TEMED (cf. 4) and continuously scavenged by reduction of NBT. This sequence involves no net oxygen consumption. Dismutase activity leads to a net uptake of oxygen which is measured directly with a simple Clark-type oxygen electrode (14). METHODS
Extracts of solid tissues were usually prepared by homogenizing with an Ultra-Turrax disintegrator in 4 vol of water, and centrifuging at 25,000 rpm for 1 hr. When supernatants were to be stored, glycerol was added to 33% and the solution was stored in the liquid state at -20°C. Samples of 5 to 25 ~1 were usually used for the SOD assay. Erythrocyte hemolysates were prepared by lysis in 5 vol of water after washing the cells twice with 5 vol of normal saline. Superoxide dismutase from Sigma Chemical Co. (Sigma SOD) was used as a reference activity where indicated, at a concentration of 20 pg/ml in Tris-HCl buffer (pH 8.0). Superoxide dismutase assay was carried out in an oxygen electrode cell with polarizing circuit and magnetic stirrer, manufactured by Rank Bros., Bottisham, Cambs. This cell is made of Perspex with a Clark-type electrode in the detachable base and provision for water circulation through the hollow walls. The top of the cell is closed by a pierced plunger which allows volume adjustment, exclusion of air and injection of small volumes of additives. Usual working volume was 3 ml and a temperature of 25°C. Polarizing voltage was 0.6 V and the output (about 10 mV at 2 kR) was
ASSAY
OF SUPEROXIDE
DISMUTASE
563
recorded on a chart recorder at 2 mV FSD with appropriate zero suppression. The cell was placed at the centre of a 22-W toroidal lamp of the daylight fluorescent type, 8 in. in diameter. In early experiments, a black paper hood was placed over the electrode vessel when measuring the “dark” current. Later, a hinged wooden box was constructed both to support the lamp and to exclude light. The standard incubation medium (pH 8.9, unless stated) contained Tris-HCl (66 mM), tetramethylethylenediamine (TEMED) (9 mM), EDTA (0.1 mM), and bovine plasma albumin (33 pg/ml) (Armour). Nitro blue tetrazolium (NBT) (0.1 mM), riboflavin (20 PM), and catalase (330 U/ml) (Boehringer) were added to the stock buffer to be used at the beginning of each day. To overcome the poor solubility and the instability of riboflavin (in water), a stock solution of 200 PM riboflavin in 5 mM KOH was made up and frozen in lo-ml portions. One of these was thawed and used for each day’s work, and then discarded. The riboflavin concentration is chosen by experiment according to the illumination system in use and the range of enzyme activities to be assayed. In early experiments, catalase from BDH (330 EUlml) was used, but one batch of this had a significant SOD activity which led to high blanks. Catalase was stored in 50% glycerol (50,000 U/ml) at -20°C. The purpose ofthe EDTA was to overcome the SOD-like activity of transition metal ion contamination. The albumin contributes to the stability of the linear reaction, probably by inhibiting the coprecipitation of SOD with the formazan. Its concentration may be increased to at least 100 pglml without interfering with the reaction. and this may be helpful if a highly purified enzyme is being assayed. This medium was allowed to equilibrate in the thermostatted cell (open to the air) for about 10 min; then the stopper was inserted to exclude air from the medium. The dark output was followed for a few minutes, the enzyme sample was injected. and the output was followed for a further short period until it was stable. The light was then switched on, and the rate of change of the output due to SOD activity was recorded. The activity with illumination but without enzyme may also be recorded at the beginning of the same run (see Fig. 1). but as explained later this is better done in a separate run to avoid excessive formazan accumulation. RESULTS
The record ofa typical assay is shown in Fig. 1. except that the prolonged “light blank” (section X .-.-. X) would not normally be included in the same run, since it leads to heavy formazan precipitation. It is evident that rapid oxygen consumption takes place only in the presence of enzyme and with the light switched on. Boiled enzyme is without activity. Cyanide (0.5 mM) inhibits the reaction, and it should be noted here that the true
564
MARSHALL
AND
WORSFOLD
*---.x FIG. conditions
I. Tracing
of oxygen
as described
under
electrode Methods.
which controls superoxide generation. supernatant) was added where indicated included in the same run as the enzyme
output Points
during marked
assay
of superoxide
ON and
OFF refer
dismutase. to the
light
Assay source
Superoxide dismutase (20 ~1 of a rat muscle (SOD). The light blank (X X) is not routinely assay, as it leads to excessive formazan precipitation.
inhibition is greater than appears in this trace, due to simultaneous inhibition of the catalase which is routinely included to remove hydrogen peroxide. Catalase decreases the observed reaction rates by a factor of 2 since it liberates oxygen from the hydrogen peroxide, but if it is omitted the record becomes nonlinear, presumably due to the instability of the hydrogen peroxide and its toxicity to the enzyme (16,17). After addition of cyanide to the medium containing catalase, the observed reaction rates must be divided by 2 to retain the same stoichiometry. The rate of oxygen consumption is proportional to the amount of enzyme added over a wide range (Fig. 2a) and is quite reproducible (Fig. 2b). The high blank (no enzyme) rate in this figure is due to the use of a catalase preparation (BDH) which contained some SOD activity. The linear dependence of reaction rate on enzyme concentration was confirmed with human hemolysates and with purified SOD preparations (Sigma and Miles Laboratories), as well as with extracts of rat thymus, lung, heart, muscle, and liver. Mixtures of pure SOD with hemolysates gave activities equal to the sum of the individually measured activities, within the linear range of the assay. Substantial variation of the reaction conditions can be tolerated, so long as care is taken to ensure that the enzyme appears to be saturated; that is, that the product of riboflavin concentration and light intensity are such that moderate variations in either do not affect the rate of reaction. It is one of the strengths of this method that the influence of each component of the system can be studied with ease. If NBT is omitted, then all the O,- which is generated must dismutate, whether or not enzyme is added, and therefore, the oxygen consumption rate is equal to half the rate of superoxide production (or one-quarter in the presence of catalase). The rate then becomes an almost linear function of
ASSAY
01 a
OF SUPEROXIDE
a
40
10 Muscle
FIG.
under
2a. Linearity Methods.
565
DISMUTASE
of SOD assay. Aliquots
supernatant,
of a rat muscle
PI
supernatant.
assayed
as described
1 Enzyme
0 ’ b
5
15
Added
PI
t 25
FIG. 2b. Repeatability of SOD assay. A mouse muscle supernatant, containing 33% glycerol, was stored at -20°C. Three different portions were assayed each day for 5 days, as described under Methods. Each point is the mean of five daily measurements with standard deviations as indicated. The catalase preparation used in this experiment contained some SOD activity, which is the cause of the high reaction rate with no enzyme added.
riboflavin concentration (Fig. 3. curve 5). In the presence of NBT, but without enzyme, the oxygen consumption rate is a measure of the spontaneous dismutation rate under conditions close to those of the assay. This varies somewhat with the riboflavin concentration, and hence, with the rate of O,- generation (Fig. 3, curve 1). The dismutation catalyzed by superoxide dismutase or by simple tissue
566
MARSHALL
AND
WORSFOLD
FIG. 3. Superoxide generation rate, and catalyzed and spontaneous dismutation rates as a function of riboflavin concentration. Conditions as described in Methods. except as indicated below. Curve 1: no additions, spontaneous dismutation rate. This blank has been subtracted from the measured rates to obtain curves 2.3, and 4. Curve 2: 5 ~1 of Sigma SOD (20 &ml). Curve 3: 10 ~1 of Sigma SOD. Curve 4: 0.25 PM of Cu*+ (EDTA omitted). Curve 5: NBT omitted. Oxygen consumption rate equals one-quarter of the superoxide generation rate.
extracts shows a completely different dependence on riboflavin concentration. At low riboflavin levels, virtually all the O,- generated is dismutated by the enzyme, while at higher levels the enzyme-catalyzed reaction becomes saturated and depends only on the amount of enzyme added (Fig. 3, curves 2 and 3). This contrasts with the catalysis of dismutation by copper ions (Fig. 3, curve 4) which, like the spontaneous reaction, is almost proportional to riboflavin concentration. In order to vary the rate of superoxide generation without altering the chemical concentration of the medium, we varied the light intensity at a constant riboflavin level in one set of experiments (Fig. 4). This was done by covering up parts of the circular light for the lower points on the curves or by adding an extra lamp for the higher points. As we were unable to predict or measure the light intensity inside the cell, the rate of superoxide generation was measured in a separate experiment for each arrangement of the light by measuring oxygen consumption in the absence of NBT. Since we have shown that O,production is almost proportional to riboflavin concentration at a fixed light intensity (Fig. 3), the ordinates of Figs. 3 and 4 are comparable.
ASSAY
Rateof
Production
OF SUPEROXIDE
of 0; hUmin?
567
DISMUTASE
160
FIG. 4. Comparison of copper-catalyzed and SOD-catalyzed dismutation as a function of superoxide generation rate. The rate of generation of O,- was varied by altering the light intensity, and measured at each level of illumination in a separate experiment. by omission of NBT (see Fig. 3. curve 5). An extra lamp was added to obtain the highest points. Other conditions as described under Methods except that albumin was omitted from the copper-catalyzed reaction. Curve 1: blank (no addition). Curve 2: 0.125 FM 01” (no EDTA). Curve 3: 5 ~1 of Sigma SOD. The blank has been subtracted from curves 2 and 3.
Figure 5 shows the variation of O,- generation rate (curve 1) and of spontaneous dismutation rate (curve 2) with pH. The spontaneous dismutation rate falls with increasing pH. For this reason, a pH of 8.9 was chosen as the best compromise between a low blank rate and maximum O,flux, bearing in mind the desirability of working as close to “physiological” pH as possible. In order to establish the relationship of our method to that of Beauchamp and Fridovich (4). we made an estimate of the amount of formazan
FIG. 5. Rate of generation of superoxide pH. Conditions as under Methods. except was omitted.
and its spontaneous dismutation as a function of that no enzyme was added. and in curve 1. NBT
568
MARSHALL
AND
WORSFOLD
produced in the presence of different amounts of enzyme. The reaction was followed polarographically as usual for 2 min; then the light was switched off and a sample was withdrawn and its optical density was measured at 540 nm. The molar concentration of formazan was calculated from a separate calibration, carried out with NADH as the reducing agent, coupled with phenazine methosulfate. This method is not completely quantitative, as the absorption of light by the formazan is influenced by other components of the medium. and also becomes nonlinear at higher concentrations of formazan. such as are generated when dismutase activity is low. A further complication is the affinity of the formazan for the plastic reaction vessel. Despite these problems there is an inverse relationship between oxygen consumption and formazan production (Fig. 6). The oxygen curve follows the amount of added enzyme much more faithfully than does the formazan color, however, especially at low enzyme levels. It must be noted that the superoxide flux which is optimal for the polarographic method is too high for satisfactory operation of the formazan method, since formazan production very quickly exceeds the linear range of optical density. It is for this reason that the reaction was stopped at 2 min. Similarly, the high enzyme levels needed to depress formazan production significantly at these short incubation times are outside the linear range of the polarographic method. The SOD activities of some tissue extracts, measured by our method, are listed in Table 1. The mean activity of human hemolysates, equivalent to 271 p.g/g of hemoglobin may be compared with 210 pg/g found by Concetti 40Total
1
Oxygen
N > 2
Consumed
0 / Enzyme
2lo
40
Added
60
PI
60
FIG. 6. Inhibition of formazan production by SOD compared with oxygen consumption. Conditions as under Methods, except that the light was switched off after 2 min. and a portion of the reaction mixture was removed. Formazan production was calculated from its optical density at 540 nm.
ASSAY
OF SUPEROXIDE TABLE
SUPEROXIDE DISMUTASE PREPARATIONS
Source
of enzyme
” Based
on the sample
from
I
ACTIVITY OF Two COMMERCIAL AND SOME T~ssut EXTRACTS
(pm01
Miles Laboratories (bovine erythrocyte) Sigma (bovine erythrocyte) Rat Muscle (pectoralis) Spleen Pancreas Kidney Heart Testis Liver Lung Thymus Rat tumours (16) Human erythrocytes (5 I) Cultured rat tumour cells Cultured human umbilical cord (endothelial cells) Cultured guinea pig kidney
Activity of O,-/mitt)
SOD
Reference unit
259 238
mg mg
I? 30 15 51 35 76 I I5 48 II Z-16 70.1 ? I3 (SD) 0.09
g g g g g g g g g g
0.50 0.01 Miles
569
DISMUTASE
wet wet wet wet wet wet wet wet wet wet
SOD” (CL@
( 1000) 920 wt wt wt wt wt wt wt wt wt wt
g Hb IO” cells
46 116 97 197 I35 100 444 I85 41 8-61 271 0.35
IO” cells IO” cells
1.9 0.04
Laboratories.
et al. (18). Among many possible reasons for the difference, explanation lies in the relative purities of the SOD standards.
a likely
DISCUSSION Many authors have complained ofthe lack of agood assay for superoxide dismutase (I) which has impeded progress in this increasingly active field. The belief is widespread that because the substrate O,- is unstable and because the enzyme-catalyzed reaction is the same as the spontaneous one, no continuous direct assay is possible. However, if the steady-state concentration of O,- can be maintained above the saturation level of the enzyme, and if the product of its reaction can be measured without interference by the generating or scavenging system, then a direct assay is feasible. The usual tactic has been to divert, by the dismutase activity, part of an intermediate pool of O,- from its reaction with a chromogenic scavenger and to measure the suppression of the latter reaction. This measurement of the O,--scavenger reaction is unnecessary if the dismutation can be followed directly. The superoxide anion can act either as a reducing agent or as an oxidising
570
MARSHALL
AND
WORSFOLD
agent, or it can oxidize and reduce itself (dismutate). oxidizing agent, it is itself reduced, e.g.,
When it acts as an
H+ + O,- + RH + R + H,O,
111
If the source of O,- is dissolved oxygen: 02 + e -
121
o*-,
reaction [l] leads to a net consumption of oxygen in the system. When O,acts as a reducing agent, as in its reaction with NBT: N-N-R, R -d! 3
A+-R,
N-N-R, ’H
+ H+ + 20,- + R -8 3
lN/
\
+ 20,
[31
N=N-R,
then there is no net oxygen consumption. All the O,- is converted back to oxygen. The intermediate course is the dismutation reaction: O,- + 02- + 2H+ -+ H,O,
+ 0,.
[41
Here, half of the O,- is captured as H,O,. Even if the H202 is decomposed, for instance by catalase. 2H,O,
-+ 2H,O + 02,
[51
there is still a net conversion of 1 in 4 mol of the O,- (and hence, of the original 0,) to water. Therefore, in a system in which reactions [2], [3], and [4] are proceeding, the net oxygen consumption is a direct measure of the extent of reaction [4]. If catalase is added, so that reaction [5] proceeds rapidly, then the same is true but the oxygen consumption is halved. This oxygen consumption will be unaffected by side reactions or by alternative pathways of scavenger (NBT) reduction so long as these are not so active as to reduce the O,- concentration below the apparent saturation level for superoxide dismutase (see below), and so long as there is no alternative oxygen-consuming system. Both of these possibilities can be controlled and measured. Fried et al. (19) measured oxygen consumption by the xanthine oxidase-tetrazolium system and found that consumption was increased by an inhibitor of tetrazolium reduction which they prepared from liver and which is now known to be superoxide dismutase. A similar observation was made by Fridovich (20) using cytochrome c as the electron acceptor. None of these workers appears to have used the phenomenon as an assay for SOD. We chose photochemical production of O,- because it avoids other oxygen-consuming reactions. An oxidizing (reducible) scavenger must be used, so that there is no net oxygen consumption in the absence of
ASSAY
OF SUPEROXIDE
DISMUTASE
571
dismutation, and its affinity for O,- must be such as to allow a dismutase-saturating steady-state concentration of O,- to exist at reasonable rates of O,- generation. Its reduction should preferably be irreversible. NBT fulfils these conditions and has the advantage that its behavior is well known. It has the disadvantage of yielding a colored reduced form, however, which means that interference with efficient photochemical generation of O,- could be a problem. The problem would be most likely to occur when only small SOD activities are being measured, while the O,- generation rate is large. We have observed that the oxygen consumption curve in the presence of low enzyme activities tends to flatten out earlier than with large amounts of enzyme, and this effect appeared to coincide with the heavy precipitation of formazan. The effect is much more marked when purified enzyme preparations are used, however, and is abolished by the addition of a small concentration of albumin. We therefore conclude that it may be due to the coprecipitation of SOD with the formazan, and that this is a nonspecific reaction which is prevented in the presence of a competing protein. The “substantivity” of formazans for protein is well known in histochemistry. The general criteria for optimum assay conditions for our method may be summarized as follows: 1. O,- flux at a level which maintains O,- at SOD-saturating levels over the widest possible range of SOD activities, but does not consume substrate at too great a rate (or yield undesirable products too quickly, e.g., formazan, H,O,). 2. Affinity of the O,- scavenger should be such as to keep the O,- level in the range over which spontaneous dismutation is relatively slow, but SOD is saturated with O,-. 3. Spontaneous dismutation rates as low as possible. This condition is approached by raising the pH. 4. The H,O, formed by dismutation should be either completely stable or completely decomposed for reproducible stoichiometry. Since H,O, is toxic to many proteins, including SOD, and since catalase will be an uncontrolled component of many tissue extracts, we routinely include an excess of catalase in the assay mixture. It must be freshly diluted, as it is unstable. Some commercial samples of catalase contain a little SOD activity, which leads to increased blanks. This is easily tested and corrected by adjusting the amount of catalase or by using a different source of the enzyme. 5. The instrumentation should be sensitive and quiet. Only relatively small amounts of oxygen are consumed during an assay. and uptake may be slow. This condition is not an extreme one, and we use one of the cheapest commercially available oxygen electrodes with an ordinary chart recorder; but it is convenient to use a 2- to IO-fold scale expansion with appropriate zero suppression. Some attention must be given to quiet, uniform stirring of
572
MARSHALL
AND
WORSFOLD
the electrode vessel; here again our electrode is not of the most satisfactory design from this point of view, in that the electrode and stirrer are both at the bottom of the cell. A cell with an overhead electrode and stirring from beneath, might be better; as would a glass cell (for rapid temperature equilibration) rather than the acrylic plastic one we used. The output of the oxygen electrode is very temperature-sensitive at this degree of scale expansion. The affinity of formazan for the plastic is also detrimental. A great advantage of this assay is that interference at any point in the system can be monitored easily. In a single run, one can carry out a dark blank (no O,- generation) without and with enzyme: and add an internal enzyme standard to check for inhibitors or other interfering material in the sample. Known or putative inhibitors or stimulators can be added at any stage, and their instantaneous, transient or delayed effects on both the SOD activity or the individual nonenzymic stages of the reaction can be observed. The effects of inhibitors on the catalase must be born in mind, however, especially in the case of cyanide and similar complexing agents. Catalase is even more sensitive to cyanide than is SOD, so that an apparent increase in activity may be observed if small amounts of cyanide are added to the standard system, especially with cyanide-insensitive SOD preparations. The two effects can be distinguished by omitting the catalase from the reaction mixture, but the assay then becomes quantitatively less reliable. A feature of the present technique is that the rate of generation of O,appears not to be critical and need not be constant or free from interference so long as it is high enough to maintain a SOD-saturating pool of O,-. Methods which rely on measurement of an oxidised or reduced scavenger for O,- are extremely vulnerable to perturbations of the O,- flux. A disadvantage of our method is that multiple samples cannot be assayed simultaneously, which reduces its value as a screening test for large numbers of samples. Throughout this discussion we have spoken of, and assumed, saturation kinetics for SOD. Pulse radiolysis studies, however, have failed to detect any evidence of a Michaelis complex or saturation of the enzyme. Rotilio et al. (11) and Bannister et al. (21) state that the K, must be greater than 0.5 mM if it exists at all. Nonetheless, the results of the experiments depicted by Figs. 3 and 4 would seem to imply the saturation of the enzyme with substrate and we are unable to explain the discrepancy. It is remarkable that the copper-catalyzed dismutation rate shows no saturation so long as catalase is present, which argues against an artifact of the method being responsible for the apparent saturation of the enzyme. The pulse radiolysis studies of Fielden’s group were done under very different conditions from those reported here. In particular, very high concentrations of purified enzyme were used; under such conditions, Michaelis-Menten kinetics might not be expected to be obeyed. The significantly large molarity of the enzyme (11, Fig. 1) compared with that of
ASSAY
OF SUPEROXIDE
DISMUTASE
573
the substrate, must also mean that steady-state conditions of enzyme turnover could not be established until quite late in the reaction. Measurements of initial velocities, therefore, might be expected to indicate second-order kinetics, as a reflection of the reaction of enzyme with substrate, rather than the steady-state conditions of continuous turnover of enzyme. Since complete details of the means of derivation of the kinetic data from the oscilloscope traces have not been given, it would be fruitless to discuss the other possible reasons for the discrepancy. The study of competitive inhibition of SOD by Forman and Fridovich (22) does not demonstrate directly the absence of the saturation of the enzyme, but simply estimates the presumed second-order rate constant on the assumption that the enzyme is not saturated. The question of the true kinetics of the enzyme is not crucial to the usefulness of our assay method. It is sufficient for practical purposes that, for a wide range of enzyme activities. a level of signal is produced which is a linear function of the amount of added enzyme: it is reproducible and it is insensitive to moderate variation of the reaction conditions. REFERENCES 1. Fridovich, I. (1975) Annu. Rev. Biochem. 44, 147-159. 2. McCord, J. M., and Fridovich. I. (1969) J. Bid. Chem. 244, 6049-6055. 3. Nishikimi. M., Rao. N. A.. and Yagi. K. (1972) Biociwm. Bioph.vs. Res. Cornmtrn. 46, 849-854. 4. Beauchamp. C.. and Fridovich, I. (1971) And. Biochern. 44, 276-287. 5. Henry. L. E. A.. Halliwell. B., and Hall. D. 0. (1976) Feb.? Left. 66, 303-306. 6. Elstner, E. F., and Heupel, A. (1976) Anal. Biochem. 70, 616-620. 7. Misra. H. P.. and Fridovich. I. (1972) J. Bid. Chrm. 247, 3170-3175. 8. Marklund. S.. and Marklund. G. (1974) Eur. J. B&hem. 47, 469-474. 9. Rigo, A., Tomat, R., and Rotilio. Ci. (1974)EI~~rrorrnu/~r. Chem. Inrerfac. Electrochem, 57,
IO. Il. 12. 13. 14. 1. 16. 17.
18. 19. 20. 21. 22.
291-296.
Rigo, A., Viglino, P., and Rotilio, G. (1975) Anal. Biocham. 68, l-8. Rotilio. G., Bray, R. C., and Fielden. E. M. (1972)Biochim. Biophys. Actn 268,605-609. McClune, G. J.. and Fee, J. A. (1976) Febs Lrtt. 67, 294-298. Marklund. S. (1976) J. Bid. Clzem. 251, 7504-7507. Misra, H. P., and Fridovich. I. (1977) Anul. Biochem. 79, 553-560. Clark, L. C. (1956) Trans. Amer. Sot. Artijciul Itzternal Orgarzs 2, 41-46. Bray. R. C.. Cockle, S. A.. Fielden. E. M.. Roberts, P. B., Rotilio. G., and Calabrese. L. ( 1974) Biochrm. J. 139, 43-48. Hodgson. E. K., and Fridovich, I. (1975) Biochrm. 14, 5294-5299. Concetti. A.. Massei, P.. Rotilio. G.. Brunori. M.. and Rachmilewitz. E. A. (1976)5. Lob. C/in. Med. 87, 1057-1064. Fried. R., Fried, L. W.. and Babin. D. (1970) Eur. J. Biochern. 16, 399-406. Fridovich. I. (1970)5. Bid. C/fern. 245, 4053-4057. Bannister. J. V., Bannister, W. H.. Bray, R. C.. Fielden. E. M.. Roberts, P. B.. and Rotilio. G. (1973) FEBS Lrtf 32, 303-306. Forman. H. J.. and Fridovich. I. (1973) Arch. Biochrm. Bioplzys. 158, 396-400.
RIOCHEMISTRY
Superoxide
Salt
(1978)
Dismutase: A Direct, Continuous Using the Oxygen Electrode M.J.
Charles
86, 561-573
Resc~arch
Centw.
Received
MARSHALL Robert
March
Jonr~ Shropshirr.
Linear Assay
AND M. WORSFOLD & Agnes HutIt Englund
17. 1977: accepted
Orthopuc~dic
December
Hospitcrl,
Oswrstry.
5. 1977
A continuous method for the assay of superoxide dismutase activity which gives a linear recording of the reaction whose slope is a linear function of the amount of enzyme added is described. An oxygen electrode is used to measure the consumption of oxygen which results from the photochemical generation of superoxide from dissolved oxygen followed by its dismutation to oxygen and hydrogen peroxide. Excess superoxide is scavenged by a tetrazolium salt which oxidizes it back to oxygen.
The instability of the superoxide anion has until now led to the use of indirect and somewhat unsatisfactory assay procedures for superoxide dismutase. As reviewed by Fridovich (1) it is usual to assay the enzyme by observing its inhibition of a reaction in which superoxide is a transient intermediate. Superoxide may be generated by xanthine oxidase (2), from reduced phenazine methosulfate (3), photochemically (4), or from potassium superoxide (5). The indicating reactants which have been used include cytochrome c (2), nitro blue tetrazolium (3,4), and hydroxylamine (6). Various autoxidation reactions in which superoxide is generated during the reaction and is also involved in its propagation have also been used (7,g). All these methods suffer from nonlinearity, lack of precision, and vulnerability to interference by a wide range offactors, the effects of which may well go undetected and. in any case, are difficult to interpret or quantify. Rigo et ul. (9,lO) have reported a polarographic technique in which a dropping mercury electrode is used as both source and indicator of O,concentration. This method does not yield a direct measure of enzyme activity, however, and this type of apparatus is not widely used in biochemistry laboratories. The use of pulse radiolysis to generate superoxide, combined with photometric measurement of its concentration. allows the reaction to be followed directly (I l), but this apparatus is even less widely available and would appear to be applicable only to relatively high concentrations of purified enzyme. 561
OOO?-2697i78/086?-0~61~0~.00!0 CopyrIght ‘i‘ 1978 hy Academic Prrsr. Inc. All nght? of reproduction m any form rrwrvrd.
562
MARSHALL
AND
WORSFOLD
Photometric measurement of [OJ has also been combined with generation of 02- from potassium superoxide (12) which has led to an assay (13) with good precision and potentially wide application, although this presumably will be limited by the very small optical density changes and the nonlinear record. A recent paper by Misra and Fridovich (14) describes a method involving the partial inactivation of peroxidase by O,-. These authors suggest that the “positive” aspect of this assay overcomes objections to earlier ones which involved the inhibition of an indicator reaction. However, their method is, in reality, only “positive” as the result of the inhibition of an inhibition (a double “negative”) and suffers all the disadvantages, including limitation of useful range, which they ascribe to “negative” (inhibition) assays, excepting only the slope of the graph on which the results are plotted. We report here a simple, rapid method for the direct assay of superoxide dismutase. It yields a continuous record of the extent ofthe reaction, with a slope which is a linear function of enzyme activity. Each part of the reaction sequence is easily and quickly monitored so that interference by extraneous substances is easily recognized and accounted for. Crude (and therefore minimally altered) tissue extracts can readily be assayed. Superoxide anion is generated photochemically from riboflavin and TEMED (cf. 4) and continuously scavenged by reduction of NBT. This sequence involves no net oxygen consumption. Dismutase activity leads to a net uptake of oxygen which is measured directly with a simple Clark-type oxygen electrode (14). METHODS
Extracts of solid tissues were usually prepared by homogenizing with an Ultra-Turrax disintegrator in 4 vol of water, and centrifuging at 25,000 rpm for 1 hr. When supernatants were to be stored, glycerol was added to 33% and the solution was stored in the liquid state at -20°C. Samples of 5 to 25 ~1 were usually used for the SOD assay. Erythrocyte hemolysates were prepared by lysis in 5 vol of water after washing the cells twice with 5 vol of normal saline. Superoxide dismutase from Sigma Chemical Co. (Sigma SOD) was used as a reference activity where indicated, at a concentration of 20 pg/ml in Tris-HCl buffer (pH 8.0). Superoxide dismutase assay was carried out in an oxygen electrode cell with polarizing circuit and magnetic stirrer, manufactured by Rank Bros., Bottisham, Cambs. This cell is made of Perspex with a Clark-type electrode in the detachable base and provision for water circulation through the hollow walls. The top of the cell is closed by a pierced plunger which allows volume adjustment, exclusion of air and injection of small volumes of additives. Usual working volume was 3 ml and a temperature of 25°C. Polarizing voltage was 0.6 V and the output (about 10 mV at 2 kR) was
ASSAY
OF SUPEROXIDE
DISMUTASE
563
recorded on a chart recorder at 2 mV FSD with appropriate zero suppression. The cell was placed at the centre of a 22-W toroidal lamp of the daylight fluorescent type, 8 in. in diameter. In early experiments, a black paper hood was placed over the electrode vessel when measuring the “dark” current. Later, a hinged wooden box was constructed both to support the lamp and to exclude light. The standard incubation medium (pH 8.9, unless stated) contained Tris-HCl (66 mM), tetramethylethylenediamine (TEMED) (9 mM), EDTA (0.1 mM), and bovine plasma albumin (33 pg/ml) (Armour). Nitro blue tetrazolium (NBT) (0.1 mM), riboflavin (20 PM), and catalase (330 U/ml) (Boehringer) were added to the stock buffer to be used at the beginning of each day. To overcome the poor solubility and the instability of riboflavin (in water), a stock solution of 200 PM riboflavin in 5 mM KOH was made up and frozen in lo-ml portions. One of these was thawed and used for each day’s work, and then discarded. The riboflavin concentration is chosen by experiment according to the illumination system in use and the range of enzyme activities to be assayed. In early experiments, catalase from BDH (330 EUlml) was used, but one batch of this had a significant SOD activity which led to high blanks. Catalase was stored in 50% glycerol (50,000 U/ml) at -20°C. The purpose ofthe EDTA was to overcome the SOD-like activity of transition metal ion contamination. The albumin contributes to the stability of the linear reaction, probably by inhibiting the coprecipitation of SOD with the formazan. Its concentration may be increased to at least 100 pglml without interfering with the reaction. and this may be helpful if a highly purified enzyme is being assayed. This medium was allowed to equilibrate in the thermostatted cell (open to the air) for about 10 min; then the stopper was inserted to exclude air from the medium. The dark output was followed for a few minutes, the enzyme sample was injected. and the output was followed for a further short period until it was stable. The light was then switched on, and the rate of change of the output due to SOD activity was recorded. The activity with illumination but without enzyme may also be recorded at the beginning of the same run (see Fig. 1). but as explained later this is better done in a separate run to avoid excessive formazan accumulation. RESULTS
The record ofa typical assay is shown in Fig. 1. except that the prolonged “light blank” (section X .-.-. X) would not normally be included in the same run, since it leads to heavy formazan precipitation. It is evident that rapid oxygen consumption takes place only in the presence of enzyme and with the light switched on. Boiled enzyme is without activity. Cyanide (0.5 mM) inhibits the reaction, and it should be noted here that the true
564
MARSHALL
AND
WORSFOLD
*---.x FIG. conditions
I. Tracing
of oxygen
as described
under
electrode Methods.
which controls superoxide generation. supernatant) was added where indicated included in the same run as the enzyme
output Points
during marked
assay
of superoxide
ON and
OFF refer
dismutase. to the
light
Assay source
Superoxide dismutase (20 ~1 of a rat muscle (SOD). The light blank (X X) is not routinely assay, as it leads to excessive formazan precipitation.
inhibition is greater than appears in this trace, due to simultaneous inhibition of the catalase which is routinely included to remove hydrogen peroxide. Catalase decreases the observed reaction rates by a factor of 2 since it liberates oxygen from the hydrogen peroxide, but if it is omitted the record becomes nonlinear, presumably due to the instability of the hydrogen peroxide and its toxicity to the enzyme (16,17). After addition of cyanide to the medium containing catalase, the observed reaction rates must be divided by 2 to retain the same stoichiometry. The rate of oxygen consumption is proportional to the amount of enzyme added over a wide range (Fig. 2a) and is quite reproducible (Fig. 2b). The high blank (no enzyme) rate in this figure is due to the use of a catalase preparation (BDH) which contained some SOD activity. The linear dependence of reaction rate on enzyme concentration was confirmed with human hemolysates and with purified SOD preparations (Sigma and Miles Laboratories), as well as with extracts of rat thymus, lung, heart, muscle, and liver. Mixtures of pure SOD with hemolysates gave activities equal to the sum of the individually measured activities, within the linear range of the assay. Substantial variation of the reaction conditions can be tolerated, so long as care is taken to ensure that the enzyme appears to be saturated; that is, that the product of riboflavin concentration and light intensity are such that moderate variations in either do not affect the rate of reaction. It is one of the strengths of this method that the influence of each component of the system can be studied with ease. If NBT is omitted, then all the O,- which is generated must dismutate, whether or not enzyme is added, and therefore, the oxygen consumption rate is equal to half the rate of superoxide production (or one-quarter in the presence of catalase). The rate then becomes an almost linear function of
ASSAY
01 a
OF SUPEROXIDE
a
40
10 Muscle
FIG.
under
2a. Linearity Methods.
565
DISMUTASE
of SOD assay. Aliquots
supernatant,
of a rat muscle
PI
supernatant.
assayed
as described
1 Enzyme
0 ’ b
5
15
Added
PI
t 25
FIG. 2b. Repeatability of SOD assay. A mouse muscle supernatant, containing 33% glycerol, was stored at -20°C. Three different portions were assayed each day for 5 days, as described under Methods. Each point is the mean of five daily measurements with standard deviations as indicated. The catalase preparation used in this experiment contained some SOD activity, which is the cause of the high reaction rate with no enzyme added.
riboflavin concentration (Fig. 3. curve 5). In the presence of NBT, but without enzyme, the oxygen consumption rate is a measure of the spontaneous dismutation rate under conditions close to those of the assay. This varies somewhat with the riboflavin concentration, and hence, with the rate of O,- generation (Fig. 3, curve 1). The dismutation catalyzed by superoxide dismutase or by simple tissue
566
MARSHALL
AND
WORSFOLD
FIG. 3. Superoxide generation rate, and catalyzed and spontaneous dismutation rates as a function of riboflavin concentration. Conditions as described in Methods. except as indicated below. Curve 1: no additions, spontaneous dismutation rate. This blank has been subtracted from the measured rates to obtain curves 2.3, and 4. Curve 2: 5 ~1 of Sigma SOD (20 &ml). Curve 3: 10 ~1 of Sigma SOD. Curve 4: 0.25 PM of Cu*+ (EDTA omitted). Curve 5: NBT omitted. Oxygen consumption rate equals one-quarter of the superoxide generation rate.
extracts shows a completely different dependence on riboflavin concentration. At low riboflavin levels, virtually all the O,- generated is dismutated by the enzyme, while at higher levels the enzyme-catalyzed reaction becomes saturated and depends only on the amount of enzyme added (Fig. 3, curves 2 and 3). This contrasts with the catalysis of dismutation by copper ions (Fig. 3, curve 4) which, like the spontaneous reaction, is almost proportional to riboflavin concentration. In order to vary the rate of superoxide generation without altering the chemical concentration of the medium, we varied the light intensity at a constant riboflavin level in one set of experiments (Fig. 4). This was done by covering up parts of the circular light for the lower points on the curves or by adding an extra lamp for the higher points. As we were unable to predict or measure the light intensity inside the cell, the rate of superoxide generation was measured in a separate experiment for each arrangement of the light by measuring oxygen consumption in the absence of NBT. Since we have shown that O,production is almost proportional to riboflavin concentration at a fixed light intensity (Fig. 3), the ordinates of Figs. 3 and 4 are comparable.
ASSAY
Rateof
Production
OF SUPEROXIDE
of 0; hUmin?
567
DISMUTASE
160
FIG. 4. Comparison of copper-catalyzed and SOD-catalyzed dismutation as a function of superoxide generation rate. The rate of generation of O,- was varied by altering the light intensity, and measured at each level of illumination in a separate experiment. by omission of NBT (see Fig. 3. curve 5). An extra lamp was added to obtain the highest points. Other conditions as described under Methods except that albumin was omitted from the copper-catalyzed reaction. Curve 1: blank (no addition). Curve 2: 0.125 FM 01” (no EDTA). Curve 3: 5 ~1 of Sigma SOD. The blank has been subtracted from curves 2 and 3.
Figure 5 shows the variation of O,- generation rate (curve 1) and of spontaneous dismutation rate (curve 2) with pH. The spontaneous dismutation rate falls with increasing pH. For this reason, a pH of 8.9 was chosen as the best compromise between a low blank rate and maximum O,flux, bearing in mind the desirability of working as close to “physiological” pH as possible. In order to establish the relationship of our method to that of Beauchamp and Fridovich (4). we made an estimate of the amount of formazan
FIG. 5. Rate of generation of superoxide pH. Conditions as under Methods. except was omitted.
and its spontaneous dismutation as a function of that no enzyme was added. and in curve 1. NBT
568
MARSHALL
AND
WORSFOLD
produced in the presence of different amounts of enzyme. The reaction was followed polarographically as usual for 2 min; then the light was switched off and a sample was withdrawn and its optical density was measured at 540 nm. The molar concentration of formazan was calculated from a separate calibration, carried out with NADH as the reducing agent, coupled with phenazine methosulfate. This method is not completely quantitative, as the absorption of light by the formazan is influenced by other components of the medium. and also becomes nonlinear at higher concentrations of formazan. such as are generated when dismutase activity is low. A further complication is the affinity of the formazan for the plastic reaction vessel. Despite these problems there is an inverse relationship between oxygen consumption and formazan production (Fig. 6). The oxygen curve follows the amount of added enzyme much more faithfully than does the formazan color, however, especially at low enzyme levels. It must be noted that the superoxide flux which is optimal for the polarographic method is too high for satisfactory operation of the formazan method, since formazan production very quickly exceeds the linear range of optical density. It is for this reason that the reaction was stopped at 2 min. Similarly, the high enzyme levels needed to depress formazan production significantly at these short incubation times are outside the linear range of the polarographic method. The SOD activities of some tissue extracts, measured by our method, are listed in Table 1. The mean activity of human hemolysates, equivalent to 271 p.g/g of hemoglobin may be compared with 210 pg/g found by Concetti 40Total
1
Oxygen
N > 2
Consumed
0 / Enzyme
2lo
40
Added
60
PI
60
FIG. 6. Inhibition of formazan production by SOD compared with oxygen consumption. Conditions as under Methods, except that the light was switched off after 2 min. and a portion of the reaction mixture was removed. Formazan production was calculated from its optical density at 540 nm.
ASSAY
OF SUPEROXIDE TABLE
SUPEROXIDE DISMUTASE PREPARATIONS
Source
of enzyme
” Based
on the sample
from
I
ACTIVITY OF Two COMMERCIAL AND SOME T~ssut EXTRACTS
(pm01
Miles Laboratories (bovine erythrocyte) Sigma (bovine erythrocyte) Rat Muscle (pectoralis) Spleen Pancreas Kidney Heart Testis Liver Lung Thymus Rat tumours (16) Human erythrocytes (5 I) Cultured rat tumour cells Cultured human umbilical cord (endothelial cells) Cultured guinea pig kidney
Activity of O,-/mitt)
SOD
Reference unit
259 238
mg mg
I? 30 15 51 35 76 I I5 48 II Z-16 70.1 ? I3 (SD) 0.09
g g g g g g g g g g
0.50 0.01 Miles
569
DISMUTASE
wet wet wet wet wet wet wet wet wet wet
SOD” (CL@
( 1000) 920 wt wt wt wt wt wt wt wt wt wt
g Hb IO” cells
46 116 97 197 I35 100 444 I85 41 8-61 271 0.35
IO” cells IO” cells
1.9 0.04
Laboratories.
et al. (18). Among many possible reasons for the difference, explanation lies in the relative purities of the SOD standards.
a likely
DISCUSSION Many authors have complained ofthe lack of agood assay for superoxide dismutase (I) which has impeded progress in this increasingly active field. The belief is widespread that because the substrate O,- is unstable and because the enzyme-catalyzed reaction is the same as the spontaneous one, no continuous direct assay is possible. However, if the steady-state concentration of O,- can be maintained above the saturation level of the enzyme, and if the product of its reaction can be measured without interference by the generating or scavenging system, then a direct assay is feasible. The usual tactic has been to divert, by the dismutase activity, part of an intermediate pool of O,- from its reaction with a chromogenic scavenger and to measure the suppression of the latter reaction. This measurement of the O,--scavenger reaction is unnecessary if the dismutation can be followed directly. The superoxide anion can act either as a reducing agent or as an oxidising
570
MARSHALL
AND
WORSFOLD
agent, or it can oxidize and reduce itself (dismutate). oxidizing agent, it is itself reduced, e.g.,
When it acts as an
H+ + O,- + RH + R + H,O,
111
If the source of O,- is dissolved oxygen: 02 + e -
121
o*-,
reaction [l] leads to a net consumption of oxygen in the system. When O,acts as a reducing agent, as in its reaction with NBT: N-N-R, R -d! 3
A+-R,
N-N-R, ’H
+ H+ + 20,- + R -8 3
lN/
\
+ 20,
[31
N=N-R,
then there is no net oxygen consumption. All the O,- is converted back to oxygen. The intermediate course is the dismutation reaction: O,- + 02- + 2H+ -+ H,O,
+ 0,.
[41
Here, half of the O,- is captured as H,O,. Even if the H202 is decomposed, for instance by catalase. 2H,O,
-+ 2H,O + 02,
[51
there is still a net conversion of 1 in 4 mol of the O,- (and hence, of the original 0,) to water. Therefore, in a system in which reactions [2], [3], and [4] are proceeding, the net oxygen consumption is a direct measure of the extent of reaction [4]. If catalase is added, so that reaction [5] proceeds rapidly, then the same is true but the oxygen consumption is halved. This oxygen consumption will be unaffected by side reactions or by alternative pathways of scavenger (NBT) reduction so long as these are not so active as to reduce the O,- concentration below the apparent saturation level for superoxide dismutase (see below), and so long as there is no alternative oxygen-consuming system. Both of these possibilities can be controlled and measured. Fried et al. (19) measured oxygen consumption by the xanthine oxidase-tetrazolium system and found that consumption was increased by an inhibitor of tetrazolium reduction which they prepared from liver and which is now known to be superoxide dismutase. A similar observation was made by Fridovich (20) using cytochrome c as the electron acceptor. None of these workers appears to have used the phenomenon as an assay for SOD. We chose photochemical production of O,- because it avoids other oxygen-consuming reactions. An oxidizing (reducible) scavenger must be used, so that there is no net oxygen consumption in the absence of
ASSAY
OF SUPEROXIDE
DISMUTASE
571
dismutation, and its affinity for O,- must be such as to allow a dismutase-saturating steady-state concentration of O,- to exist at reasonable rates of O,- generation. Its reduction should preferably be irreversible. NBT fulfils these conditions and has the advantage that its behavior is well known. It has the disadvantage of yielding a colored reduced form, however, which means that interference with efficient photochemical generation of O,- could be a problem. The problem would be most likely to occur when only small SOD activities are being measured, while the O,- generation rate is large. We have observed that the oxygen consumption curve in the presence of low enzyme activities tends to flatten out earlier than with large amounts of enzyme, and this effect appeared to coincide with the heavy precipitation of formazan. The effect is much more marked when purified enzyme preparations are used, however, and is abolished by the addition of a small concentration of albumin. We therefore conclude that it may be due to the coprecipitation of SOD with the formazan, and that this is a nonspecific reaction which is prevented in the presence of a competing protein. The “substantivity” of formazans for protein is well known in histochemistry. The general criteria for optimum assay conditions for our method may be summarized as follows: 1. O,- flux at a level which maintains O,- at SOD-saturating levels over the widest possible range of SOD activities, but does not consume substrate at too great a rate (or yield undesirable products too quickly, e.g., formazan, H,O,). 2. Affinity of the O,- scavenger should be such as to keep the O,- level in the range over which spontaneous dismutation is relatively slow, but SOD is saturated with O,-. 3. Spontaneous dismutation rates as low as possible. This condition is approached by raising the pH. 4. The H,O, formed by dismutation should be either completely stable or completely decomposed for reproducible stoichiometry. Since H,O, is toxic to many proteins, including SOD, and since catalase will be an uncontrolled component of many tissue extracts, we routinely include an excess of catalase in the assay mixture. It must be freshly diluted, as it is unstable. Some commercial samples of catalase contain a little SOD activity, which leads to increased blanks. This is easily tested and corrected by adjusting the amount of catalase or by using a different source of the enzyme. 5. The instrumentation should be sensitive and quiet. Only relatively small amounts of oxygen are consumed during an assay. and uptake may be slow. This condition is not an extreme one, and we use one of the cheapest commercially available oxygen electrodes with an ordinary chart recorder; but it is convenient to use a 2- to IO-fold scale expansion with appropriate zero suppression. Some attention must be given to quiet, uniform stirring of
572
MARSHALL
AND
WORSFOLD
the electrode vessel; here again our electrode is not of the most satisfactory design from this point of view, in that the electrode and stirrer are both at the bottom of the cell. A cell with an overhead electrode and stirring from beneath, might be better; as would a glass cell (for rapid temperature equilibration) rather than the acrylic plastic one we used. The output of the oxygen electrode is very temperature-sensitive at this degree of scale expansion. The affinity of formazan for the plastic is also detrimental. A great advantage of this assay is that interference at any point in the system can be monitored easily. In a single run, one can carry out a dark blank (no O,- generation) without and with enzyme: and add an internal enzyme standard to check for inhibitors or other interfering material in the sample. Known or putative inhibitors or stimulators can be added at any stage, and their instantaneous, transient or delayed effects on both the SOD activity or the individual nonenzymic stages of the reaction can be observed. The effects of inhibitors on the catalase must be born in mind, however, especially in the case of cyanide and similar complexing agents. Catalase is even more sensitive to cyanide than is SOD, so that an apparent increase in activity may be observed if small amounts of cyanide are added to the standard system, especially with cyanide-insensitive SOD preparations. The two effects can be distinguished by omitting the catalase from the reaction mixture, but the assay then becomes quantitatively less reliable. A feature of the present technique is that the rate of generation of O,appears not to be critical and need not be constant or free from interference so long as it is high enough to maintain a SOD-saturating pool of O,-. Methods which rely on measurement of an oxidised or reduced scavenger for O,- are extremely vulnerable to perturbations of the O,- flux. A disadvantage of our method is that multiple samples cannot be assayed simultaneously, which reduces its value as a screening test for large numbers of samples. Throughout this discussion we have spoken of, and assumed, saturation kinetics for SOD. Pulse radiolysis studies, however, have failed to detect any evidence of a Michaelis complex or saturation of the enzyme. Rotilio et al. (11) and Bannister et al. (21) state that the K, must be greater than 0.5 mM if it exists at all. Nonetheless, the results of the experiments depicted by Figs. 3 and 4 would seem to imply the saturation of the enzyme with substrate and we are unable to explain the discrepancy. It is remarkable that the copper-catalyzed dismutation rate shows no saturation so long as catalase is present, which argues against an artifact of the method being responsible for the apparent saturation of the enzyme. The pulse radiolysis studies of Fielden’s group were done under very different conditions from those reported here. In particular, very high concentrations of purified enzyme were used; under such conditions, Michaelis-Menten kinetics might not be expected to be obeyed. The significantly large molarity of the enzyme (11, Fig. 1) compared with that of
ASSAY
OF SUPEROXIDE
DISMUTASE
573
the substrate, must also mean that steady-state conditions of enzyme turnover could not be established until quite late in the reaction. Measurements of initial velocities, therefore, might be expected to indicate second-order kinetics, as a reflection of the reaction of enzyme with substrate, rather than the steady-state conditions of continuous turnover of enzyme. Since complete details of the means of derivation of the kinetic data from the oscilloscope traces have not been given, it would be fruitless to discuss the other possible reasons for the discrepancy. The study of competitive inhibition of SOD by Forman and Fridovich (22) does not demonstrate directly the absence of the saturation of the enzyme, but simply estimates the presumed second-order rate constant on the assumption that the enzyme is not saturated. The question of the true kinetics of the enzyme is not crucial to the usefulness of our assay method. It is sufficient for practical purposes that, for a wide range of enzyme activities. a level of signal is produced which is a linear function of the amount of added enzyme: it is reproducible and it is insensitive to moderate variation of the reaction conditions. REFERENCES 1. Fridovich, I. (1975) Annu. Rev. Biochem. 44, 147-159. 2. McCord, J. M., and Fridovich. I. (1969) J. Bid. Chem. 244, 6049-6055. 3. Nishikimi. M., Rao. N. A.. and Yagi. K. (1972) Biociwm. Bioph.vs. Res. Cornmtrn. 46, 849-854. 4. Beauchamp. C.. and Fridovich, I. (1971) And. Biochern. 44, 276-287. 5. Henry. L. E. A.. Halliwell. B., and Hall. D. 0. (1976) Feb.? Left. 66, 303-306. 6. Elstner, E. F., and Heupel, A. (1976) Anal. Biochem. 70, 616-620. 7. Misra. H. P.. and Fridovich. I. (1972) J. Bid. Chrm. 247, 3170-3175. 8. Marklund. S.. and Marklund. G. (1974) Eur. J. B&hem. 47, 469-474. 9. Rigo, A., Tomat, R., and Rotilio. Ci. (1974)EI~~rrorrnu/~r. Chem. Inrerfac. Electrochem, 57,
IO. Il. 12. 13. 14. 1. 16. 17.
18. 19. 20. 21. 22.
291-296.
Rigo, A., Viglino, P., and Rotilio, G. (1975) Anal. Biocham. 68, l-8. Rotilio. G., Bray, R. C., and Fielden. E. M. (1972)Biochim. Biophys. Actn 268,605-609. McClune, G. J.. and Fee, J. A. (1976) Febs Lrtt. 67, 294-298. Marklund. S. (1976) J. Bid. Clzem. 251, 7504-7507. Misra, H. P., and Fridovich. I. (1977) Anul. Biochem. 79, 553-560. Clark, L. C. (1956) Trans. Amer. Sot. Artijciul Itzternal Orgarzs 2, 41-46. Bray. R. C.. Cockle, S. A.. Fielden. E. M.. Roberts, P. B., Rotilio. G., and Calabrese. L. ( 1974) Biochrm. J. 139, 43-48. Hodgson. E. K., and Fridovich, I. (1975) Biochrm. 14, 5294-5299. Concetti. A.. Massei, P.. Rotilio. G.. Brunori. M.. and Rachmilewitz. E. A. (1976)5. Lob. C/in. Med. 87, 1057-1064. Fried. R., Fried, L. W.. and Babin. D. (1970) Eur. J. Biochern. 16, 399-406. Fridovich. I. (1970)5. Bid. C/fern. 245, 4053-4057. Bannister. J. V., Bannister, W. H.. Bray, R. C.. Fielden. E. M.. Roberts, P. B.. and Rotilio. G. (1973) FEBS Lrtf 32, 303-306. Forman. H. J.. and Fridovich. I. (1973) Arch. Biochrm. Bioplzys. 158, 396-400.
