ANALYTICAL
BIOCHEMISTRY
75, 356-362 (1976)
A Sensitive Assay for Superoxide Dismutase the Autoxidation of 6-Hydroxydopamine
Based on
RICHARD E. HEIKKILA AND FELICITAS CABBAT The Mount
Sinai
School of Medicine, Department 100th Street, New York, New
of Neurology, York 10029
Fifth
Avenue
at
Received October 6, 1975; accepted May 13, 1976 A simple, sensitive spectrophotometric assay system for superoxide dismutase (SOD) has been developed. This assay is based on the inhibitory effects of SOD on the initial rate of 6-hydroxydopamine autoxidation. The inhibition of 6-hydroxydopamine autoxidation was virtually linear to an SOD concentration of approximately 100 ng of SOD/ml (about a 50% inhibition at 100 @ml: there was a greater inhibition at higher SOD concentrations). With this assay system it was determined that SOD levels in rat brain. liver, and spinal cord were 84, 660, and 56 pg of SOD/g of tissue, respectively. These results agree very well with results obtained by other assays.
Superoxide dismutase (SOD) is a ubiquitous protein whose enzymatic activity was first described by McCord and Fridovich (1). SOD catalyzes the dismutation of the superoxide radical (0,) to hydrogen peroxide (H,O,) and 0,. Note that in this reaction one molecule of O.,- is oxidized to O2 while the other molecule is reduced to H,O,: 20,-
+ 2H+ + H,O,
+ 0,.
In the present study we have developed a sensitive, simple assay system for SOD based on the inhibitory effects of SOD on the spontaneous autoxidation of 6-hydroxydopamine. This assay is similar in many respects to assay systems dependent upon the inhibitory effects of SOD on the basecatalyzed autoxidation of epinephrine (2) or pyrogallol(3) and should prove to be a useful tool. MATERIALS
AND METHODS
Buffer and tissue preparation. A 0.05 M sodium phosphate buffer at pH 7.4 containing 10e4 M EDTA was used in all experiments. The brain, liver, or spinal cord was rapidly removed from male Sprague-Dawley rats (150-200 g) after decapitation. The tissues were briefly stored in ice-cold 0.9% saline (w/v), weighed, and then homogenized at room temperature in 9 vol of the above medium with seven strokes in glass homogenizing tubes fitted with a Teflon pestle. These tissue homogenates were then centrifuged at 4°C for 10 min at 700g in a Sorvall RC-2B refrigerated 356 Copyright All rights
0 1976 by Academx Prez\. Inc. of reproduction in any form rexwed.
SIMPLE
ASSAY
%
FOR
.20
-
I5
-
IO
-1
SUPEROXIDE
CONTROL
DISMUTASE
357
0-O
e-0-
: G > ii 4.
/‘, ,.“.sOD
/
E s 2
/.*
-
o---o’--” (200
nq/ml)
,o.‘,* In the present study, brain and liver homogenates, like SOD itself, inhibited in a dose-dependent fashion the absorbance increase at 490 nm of autoxidizing 6-OHDA (Figs. 2 and 3). The lack of inhibitory effect on 6-OHDA autoxidation of boiled brain or boiled liver indicates that the agent in these tissues responsible for slowing down 6-OHDA autoxidation was most likely an enzyme.3 Note that the liver was considerably more potent than the brain as an inhibitor of the color formation (Fig. 2). It has been established by previously published techniques, among them the epinephrine-adrenochrome assay system (7) as well as an immunological technique (9), that mammalian liver contains considerably more SOD than does brain. These inhibitory effects of brain and liver on the autoxidation of 6-OHDA were completely prevented by preincubation with the copperbinding agent diethyldithiocarbamate (5) or by preincubation with cyanide and were partially prevented by preincubation with azide (all lop3 M). Similar results were obtained with pure SOD, a copper-containing enzyme. Diethyldithiocarbamate treatment also has been shown (5) to prevent completely the inhibitory actions of brain, liver, and pure SOD in several other SOD assay systems, namely epinephrine autoxidation (2), pyrogallol autoxidation (3), cytochrome c reduction (l), and nitroblue tetrazolium reduction (10). Additionally, the time course for inactivation ofbrain, liver, and SOD itself by diethyldithiocarbamate was similar: A 1.5hr preincubation with diethyldithiocarbamate prior to the assay completely prevented * In the course of the present experiments it was found that catalase (Worthington Biochemicals, 39,132 unitsimg) at 13 1, 262, and 656 ngiml had no effect on the autoxidation of 6-hydroxydopamine. This indicates that the H,O, coming from 6-hydroxydopamine (maximum final concentration = 10ml M) plays no role in determining the initial rate of autoxidation of 6hydroxydopamine. * Horseradish peroxidase, another enzyme which decomposes H,O, (Sigma, 3000 units/ml), up to 100 rig/ml had no effect on the autoxidation rate of 6-OHDA. However, at higher concentrations, horseradish peroxidase actually stimulated the autoxidation of 6-OHDA. This finding suggests that tissues with a very high peroxidase content might interfere with the 6-OHDA assay for SOD. Similar findings have previously been reported for the pyrogallol assay (3). 3 In separate experiments, SOD (IO &ml), brain (100 mg/ml), and liver (12.5 mg/ml) were heated in the phosphate buffer at 75°C for 10 min. Aliquots (100 ~1) were removed and assayed for SOD activity by the 6-OHDA assay and by the epinephrine-adrenochrome assay (2). Under these experimental conditions the brain showed a 3 1 2 10% loss in activity in the 6-OHDA system and a 42 2 3% loss in the epinephrine-adrenochrome system; the liver showed losses of 34 + 8 and 37 ? 12%. respectively: and pure SOD showed losses of 32 t 10 and 25 f 7. respectively (mean values + SD for six separate experiments run in triplicate). Thus the agent in brain and liver responsible for slowing down 6-OHDA autoxidation was, like pure SOD, relatively heat stable. It was previously reported (12) that SOD is quite stable to heat inactivation.
SIMPLE
ASSAY
FOR
SUPEROXIDE
DISMUTASE
361
the slowing down of 6-OHDA autoxidation by the tissues and SOD, while there were no effects at zero time (no preincubation) and intermediate effects at intermediate time periods (5). All of these above data, particularly the data with diethyldithiocarbamate, strongly suggest that the agent in tissues responsible for the inhibition of 6-OHDA color formation by brain and liver is indeed SOD. With the above in mind, we have developed an assay system for SOD based on the autoxidation of 6-OHDA. In this assay 6-OHDA is both the source of O,- as well as the detection system for 03-. In the assay described by Misra and Fridovich (2), epinephrine similarly served as both the source and detector for 02- while pyrogallol had the same function in the assay of Marklund and Marklund (3). The 6-OHDA assay takes a very short time (initial rates obtained in 15 set, Figs. 2 and 3, Table 1). This allows one to analyze a great number of samples in a minimum of time. For example, we routinely assay the SOD content of brain and liver from up to 12 experimental animals in triplicate (72 individual determinations) in a 3-hr period. This assay system is also reproducible: SOD standards assayed over a several-month interval have given very similar inhibitory effects. In large measure, reproducibility may be due to the large absorbance increase given by 6-OHDA at 490 nm in comparison to tissue blanks. For example, the system described gives an absorbance increase of approximately 0.1 OD unit/l5 see at pH 7.4 compared to representative blanks of 0.025 OD unit for brain and 0.01 OD unit for liver at concentrations of 0.5 and 0.1 mgml, respectively (see Fig. 2). In contrast, the epinephrine-adrenochrome assay system (2) gives an absorbance increase of only 0.025 OD unit/min at pH 10.2, while the pyrogallol assay (3) gives an absorbance increase of only 0.007 unit/min at pH 7.9. The desirability of high absorbance increases in relationship to blank values is a very important consideration, particularly in tissues where the SOD content is relatively low. It should be emphasized that low absorbance increases are adequate only in situations where a blank is low or negligible, e.g., in studies with purified SOD. In separate studies we have found that the epinephrine-adrenochrome assay and the pyrogallol assay, both run as described (2,3) except at pH 7.4 in the 0.05 M phosphate buffer with 1O-4 M EDTA, have very low absorbance increases (less than 0,005 absorbance unit/min). Thus it appears that the 6-OHDA assay described in the present study is preferable to these other two autoxidation assays at pH 7.4. (It should be emphasized that other assays based on the O,--mediated reductions, for example, either cytochrome c reduction ( 1) or nitroblue tetrazolium reduction (IO), may be run at physiological pH). A distinct disadvantage of the 6-OHDA system compared to the other two assays based on autoxidation is the fact that the absorbance increases of both the pyrogallol and the epinephrine systems are linear for longer time periods. However, the 6-OHDA assay system can
362
HEIKKILA
AND CABBAT
be adapted, and experiments can be done with other experimental conditions. For example, we have done similar experiments at pH 6.5 with 2 x 10m4M 6-OHDA (all other conditions identical). With these modifications, the autoxidation rate of 6-OHDA is slower and nearly linear up to 30 set (5).4 In conclusion, the 6-OHDA assay system appears to be well suited for the assay of SOD in different tissues. Data obtained with this system agree very closely, both qualitatively and quantitatively, with data obtained by other means. For example, Crapo and Tierney (7), using the epinephrineadrenochrome assay, reported the ratio of SOD in rat liver compared to brain to be 7.89, while Albergoni et al. (8), using a nitroblue tetrazolium reduction assay, found a ratio of 7.0. In the present study a ratio of 7.86 is obtained (Table 1, 660 pg of SOD/g of liver and 84 pg of SOD/g of brain). Additionally, the 660 pg of SOD/g of liver found in the present study agrees reasonably well with the SOD pg of SOD/g of liver reported by McCord and Salin (11). In light of its consistency with other methods and its simplicity, as well as its capacity to be run at pH 7.4, the 6-OHDA assay system for SOD should prove to be a useful technique for the determination of levels of this very important enzyme in diverse biological specimens. ACKNOWLEDGMENTS This work was supported by the Clinical Research Center for Parkinson’s Diseases and NS 05 184.
and Allied
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
McCord, J. M.. and Fridovich, I. (1969). /. Biol. Chem. 244, 6049-6055. Misra, H. P., and Fridovich. I. (1972)J. Biol. Chem. 247, 3170-3175. Marklund, S., and Marklund, G. (1974) Eur. J. Biochem. 47, 469-474. Heikkila, R. E., and Cohen, G. (1973) Science 181, 456-457. Heikkila, R. E., Cabbat, F., and Cohen, G. (1976) J. Biol. Chem. 251, 2182-2185. Asada, K., Takahashi, M., and Nagate, M. (1974) Agr. Biol. Chem. 38, 471-473. Crapo, J. D., and Tiemey, D. F. (1974) Amer. J. Physiof. 226, 1402-1403. Albergoni, V., Cassini, A., Favero, A., and Rocco. G. P. (1975) Biochem. Pharmacol. 24,1131-1133. Hartz, J. W., Funakoshi, S., and Deutsch, H. F. (1973) Clin. Chim. Actn 46, 125- 132. Beauchamp, C., and Fridovich, I. (1971) Anal. Biochem. 44, 276-287. McCord, J. M., and Salin, M. L. (1975) in Erythrocyte Structure and Function (Brewer, G., ed.), pp. 731-746, Alan R. Liss, New York. McCord, J. M., and Fridovich, I. (1969) J. Biol. Chem. 244, 6056-6063.
4 With blood, a stimulatory factor (perhaps peroxidase activity) which promoted the autoxidation of 6-OHDA was discovered at pH 6.5 (5); this condition, however, did not exist at pH 7.4. Thus, the 6-OHDA assay is not suitable for blood at pH 6.5.
BIOCHEMISTRY
75, 356-362 (1976)
A Sensitive Assay for Superoxide Dismutase the Autoxidation of 6-Hydroxydopamine
Based on
RICHARD E. HEIKKILA AND FELICITAS CABBAT The Mount
Sinai
School of Medicine, Department 100th Street, New York, New
of Neurology, York 10029
Fifth
Avenue
at
Received October 6, 1975; accepted May 13, 1976 A simple, sensitive spectrophotometric assay system for superoxide dismutase (SOD) has been developed. This assay is based on the inhibitory effects of SOD on the initial rate of 6-hydroxydopamine autoxidation. The inhibition of 6-hydroxydopamine autoxidation was virtually linear to an SOD concentration of approximately 100 ng of SOD/ml (about a 50% inhibition at 100 @ml: there was a greater inhibition at higher SOD concentrations). With this assay system it was determined that SOD levels in rat brain. liver, and spinal cord were 84, 660, and 56 pg of SOD/g of tissue, respectively. These results agree very well with results obtained by other assays.
Superoxide dismutase (SOD) is a ubiquitous protein whose enzymatic activity was first described by McCord and Fridovich (1). SOD catalyzes the dismutation of the superoxide radical (0,) to hydrogen peroxide (H,O,) and 0,. Note that in this reaction one molecule of O.,- is oxidized to O2 while the other molecule is reduced to H,O,: 20,-
+ 2H+ + H,O,
+ 0,.
In the present study we have developed a sensitive, simple assay system for SOD based on the inhibitory effects of SOD on the spontaneous autoxidation of 6-hydroxydopamine. This assay is similar in many respects to assay systems dependent upon the inhibitory effects of SOD on the basecatalyzed autoxidation of epinephrine (2) or pyrogallol(3) and should prove to be a useful tool. MATERIALS
AND METHODS
Buffer and tissue preparation. A 0.05 M sodium phosphate buffer at pH 7.4 containing 10e4 M EDTA was used in all experiments. The brain, liver, or spinal cord was rapidly removed from male Sprague-Dawley rats (150-200 g) after decapitation. The tissues were briefly stored in ice-cold 0.9% saline (w/v), weighed, and then homogenized at room temperature in 9 vol of the above medium with seven strokes in glass homogenizing tubes fitted with a Teflon pestle. These tissue homogenates were then centrifuged at 4°C for 10 min at 700g in a Sorvall RC-2B refrigerated 356 Copyright All rights
0 1976 by Academx Prez\. Inc. of reproduction in any form rexwed.
SIMPLE
ASSAY
%
FOR
.20
-
I5
-
IO
-1
SUPEROXIDE
CONTROL
DISMUTASE
357
0-O
e-0-
: G > ii 4.
/‘, ,.“.sOD
/
E s 2
/.*
-
o---o’--” (200
nq/ml)
,o.‘,* In the present study, brain and liver homogenates, like SOD itself, inhibited in a dose-dependent fashion the absorbance increase at 490 nm of autoxidizing 6-OHDA (Figs. 2 and 3). The lack of inhibitory effect on 6-OHDA autoxidation of boiled brain or boiled liver indicates that the agent in these tissues responsible for slowing down 6-OHDA autoxidation was most likely an enzyme.3 Note that the liver was considerably more potent than the brain as an inhibitor of the color formation (Fig. 2). It has been established by previously published techniques, among them the epinephrine-adrenochrome assay system (7) as well as an immunological technique (9), that mammalian liver contains considerably more SOD than does brain. These inhibitory effects of brain and liver on the autoxidation of 6-OHDA were completely prevented by preincubation with the copperbinding agent diethyldithiocarbamate (5) or by preincubation with cyanide and were partially prevented by preincubation with azide (all lop3 M). Similar results were obtained with pure SOD, a copper-containing enzyme. Diethyldithiocarbamate treatment also has been shown (5) to prevent completely the inhibitory actions of brain, liver, and pure SOD in several other SOD assay systems, namely epinephrine autoxidation (2), pyrogallol autoxidation (3), cytochrome c reduction (l), and nitroblue tetrazolium reduction (10). Additionally, the time course for inactivation ofbrain, liver, and SOD itself by diethyldithiocarbamate was similar: A 1.5hr preincubation with diethyldithiocarbamate prior to the assay completely prevented * In the course of the present experiments it was found that catalase (Worthington Biochemicals, 39,132 unitsimg) at 13 1, 262, and 656 ngiml had no effect on the autoxidation of 6-hydroxydopamine. This indicates that the H,O, coming from 6-hydroxydopamine (maximum final concentration = 10ml M) plays no role in determining the initial rate of autoxidation of 6hydroxydopamine. * Horseradish peroxidase, another enzyme which decomposes H,O, (Sigma, 3000 units/ml), up to 100 rig/ml had no effect on the autoxidation rate of 6-OHDA. However, at higher concentrations, horseradish peroxidase actually stimulated the autoxidation of 6-OHDA. This finding suggests that tissues with a very high peroxidase content might interfere with the 6-OHDA assay for SOD. Similar findings have previously been reported for the pyrogallol assay (3). 3 In separate experiments, SOD (IO &ml), brain (100 mg/ml), and liver (12.5 mg/ml) were heated in the phosphate buffer at 75°C for 10 min. Aliquots (100 ~1) were removed and assayed for SOD activity by the 6-OHDA assay and by the epinephrine-adrenochrome assay (2). Under these experimental conditions the brain showed a 3 1 2 10% loss in activity in the 6-OHDA system and a 42 2 3% loss in the epinephrine-adrenochrome system; the liver showed losses of 34 + 8 and 37 ? 12%. respectively: and pure SOD showed losses of 32 t 10 and 25 f 7. respectively (mean values + SD for six separate experiments run in triplicate). Thus the agent in brain and liver responsible for slowing down 6-OHDA autoxidation was, like pure SOD, relatively heat stable. It was previously reported (12) that SOD is quite stable to heat inactivation.
SIMPLE
ASSAY
FOR
SUPEROXIDE
DISMUTASE
361
the slowing down of 6-OHDA autoxidation by the tissues and SOD, while there were no effects at zero time (no preincubation) and intermediate effects at intermediate time periods (5). All of these above data, particularly the data with diethyldithiocarbamate, strongly suggest that the agent in tissues responsible for the inhibition of 6-OHDA color formation by brain and liver is indeed SOD. With the above in mind, we have developed an assay system for SOD based on the autoxidation of 6-OHDA. In this assay 6-OHDA is both the source of O,- as well as the detection system for 03-. In the assay described by Misra and Fridovich (2), epinephrine similarly served as both the source and detector for 02- while pyrogallol had the same function in the assay of Marklund and Marklund (3). The 6-OHDA assay takes a very short time (initial rates obtained in 15 set, Figs. 2 and 3, Table 1). This allows one to analyze a great number of samples in a minimum of time. For example, we routinely assay the SOD content of brain and liver from up to 12 experimental animals in triplicate (72 individual determinations) in a 3-hr period. This assay system is also reproducible: SOD standards assayed over a several-month interval have given very similar inhibitory effects. In large measure, reproducibility may be due to the large absorbance increase given by 6-OHDA at 490 nm in comparison to tissue blanks. For example, the system described gives an absorbance increase of approximately 0.1 OD unit/l5 see at pH 7.4 compared to representative blanks of 0.025 OD unit for brain and 0.01 OD unit for liver at concentrations of 0.5 and 0.1 mgml, respectively (see Fig. 2). In contrast, the epinephrine-adrenochrome assay system (2) gives an absorbance increase of only 0.025 OD unit/min at pH 10.2, while the pyrogallol assay (3) gives an absorbance increase of only 0.007 unit/min at pH 7.9. The desirability of high absorbance increases in relationship to blank values is a very important consideration, particularly in tissues where the SOD content is relatively low. It should be emphasized that low absorbance increases are adequate only in situations where a blank is low or negligible, e.g., in studies with purified SOD. In separate studies we have found that the epinephrine-adrenochrome assay and the pyrogallol assay, both run as described (2,3) except at pH 7.4 in the 0.05 M phosphate buffer with 1O-4 M EDTA, have very low absorbance increases (less than 0,005 absorbance unit/min). Thus it appears that the 6-OHDA assay described in the present study is preferable to these other two autoxidation assays at pH 7.4. (It should be emphasized that other assays based on the O,--mediated reductions, for example, either cytochrome c reduction ( 1) or nitroblue tetrazolium reduction (IO), may be run at physiological pH). A distinct disadvantage of the 6-OHDA system compared to the other two assays based on autoxidation is the fact that the absorbance increases of both the pyrogallol and the epinephrine systems are linear for longer time periods. However, the 6-OHDA assay system can
362
HEIKKILA
AND CABBAT
be adapted, and experiments can be done with other experimental conditions. For example, we have done similar experiments at pH 6.5 with 2 x 10m4M 6-OHDA (all other conditions identical). With these modifications, the autoxidation rate of 6-OHDA is slower and nearly linear up to 30 set (5).4 In conclusion, the 6-OHDA assay system appears to be well suited for the assay of SOD in different tissues. Data obtained with this system agree very closely, both qualitatively and quantitatively, with data obtained by other means. For example, Crapo and Tierney (7), using the epinephrineadrenochrome assay, reported the ratio of SOD in rat liver compared to brain to be 7.89, while Albergoni et al. (8), using a nitroblue tetrazolium reduction assay, found a ratio of 7.0. In the present study a ratio of 7.86 is obtained (Table 1, 660 pg of SOD/g of liver and 84 pg of SOD/g of brain). Additionally, the 660 pg of SOD/g of liver found in the present study agrees reasonably well with the SOD pg of SOD/g of liver reported by McCord and Salin (11). In light of its consistency with other methods and its simplicity, as well as its capacity to be run at pH 7.4, the 6-OHDA assay system for SOD should prove to be a useful technique for the determination of levels of this very important enzyme in diverse biological specimens. ACKNOWLEDGMENTS This work was supported by the Clinical Research Center for Parkinson’s Diseases and NS 05 184.
and Allied
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
McCord, J. M.. and Fridovich, I. (1969). /. Biol. Chem. 244, 6049-6055. Misra, H. P., and Fridovich. I. (1972)J. Biol. Chem. 247, 3170-3175. Marklund, S., and Marklund, G. (1974) Eur. J. Biochem. 47, 469-474. Heikkila, R. E., and Cohen, G. (1973) Science 181, 456-457. Heikkila, R. E., Cabbat, F., and Cohen, G. (1976) J. Biol. Chem. 251, 2182-2185. Asada, K., Takahashi, M., and Nagate, M. (1974) Agr. Biol. Chem. 38, 471-473. Crapo, J. D., and Tiemey, D. F. (1974) Amer. J. Physiof. 226, 1402-1403. Albergoni, V., Cassini, A., Favero, A., and Rocco. G. P. (1975) Biochem. Pharmacol. 24,1131-1133. Hartz, J. W., Funakoshi, S., and Deutsch, H. F. (1973) Clin. Chim. Actn 46, 125- 132. Beauchamp, C., and Fridovich, I. (1971) Anal. Biochem. 44, 276-287. McCord, J. M., and Salin, M. L. (1975) in Erythrocyte Structure and Function (Brewer, G., ed.), pp. 731-746, Alan R. Liss, New York. McCord, J. M., and Fridovich, I. (1969) J. Biol. Chem. 244, 6056-6063.
4 With blood, a stimulatory factor (perhaps peroxidase activity) which promoted the autoxidation of 6-OHDA was discovered at pH 6.5 (5); this condition, however, did not exist at pH 7.4. Thus, the 6-OHDA assay is not suitable for blood at pH 6.5.
