Determination of the Activity Forms of Erythrocyte V. M. Institute
MISHIN,
N. N.
of Electrophoretically Superoxide Dismutase
VOLSKY,
AND
V. V.
Separable
LYAKHOWCH
of Erperimcntul and Clinical Medicine, Academy of Medical Sciences of the USSR S’berian Branch, Novosibirsk 630091, USSR Received January 19, 1977
The quantitative method for determining activities of elxtrophoretically separable forms of superoxide dismutase is presented. The m&hod consists of SOD separation on polyacrylamide gels, elution of SOD forms from the gels, and determination of inhibition activities of eluates with nitro blue tetrazolium reduction. It is shown that SOD partially purified from bovine erythrocytes sqaratrs into three fractions with different O,- .-trapping activities.
INTRODUCTION The multiple forms of superoxide dismutasc (SOD, EC 1.15.1.1) separated electrophoretically on polyacrylamide gel have been fomld in all human tissues (Beckman et al., 1973), in beef liver (Fran@ 1973), in plants (Puget and Michelson, 1974), and in microorganisms ( Marmocchi et al., 1974). Because of the important biological significance of SOD (Fridovich, 1972), we tried to determine the relative activities of the eIectrophoretically separable forms of this enzyme. There have been a few reports on this subject. Bohnenkamp and Weser (1975) estimated the SOD activities by the densitometry of the achromatic zones which appeared after staining using the light + riboflavin + nitro Mue tetrazolium (NBT) system. Beckman et al. (1973) used the visual method of determining the activities of SOD forms after separation on polyacrylamide gel and staining. The shortcomings of the densitometric method as well as those of the visual method are well known ( Maurer, 1968) . In this report we discuss a method for determining the relative activities of SOD forms based on enzyme separation on polyacrylamide gel, subsequent elution of the separate fractions, and spectrophotometric determination of the eluate activities. MATERIALS
AND METHODS
The material used in the study was bovine erythrocyte SOD prepared according to McCord and Fridovich ( 1969) but omitting DEAE-cellulose chromatography. The resulting activities of the samples were usually 500-550 units/mg of protein according to McCord and Fridovich (1969). Polyacrylamide gel electro34 0014-4800/78/0281-0034$02.00/O Copyright All rights
0 1978 by Academic Press. Inc. of reproduction in any form reserved.
ACTIVITY
OF
SUPEROXIDE
DISMUTASE
35
FORMS
phoresis was performed according to Davis (1964) in Reanal apparatus (Hungary) at 3 mA/tube. Determination of SOD activities and gel staining were performed according to Nishikimi et al. (1972) with minor modifications. The reduction of NBT by OZY, generated by reoxidation of phenazine metasulfate (PMS), has been used in assays of the activities of initial SOD samples as well as in those of gel eluates. The medium consisted of 150 nmole of NBT, 0.7 nmole of PMS, and 234 nmole of NADH in 0.017 M pyrophosphate buffer, pH 8.3. The final volume was 3.0 ml at 25°C. Under these conditions, when the inhibitor was omitted, the OD change at 560 nm was O.OS/min. The reaction was linear during 4-5 min after the short lag phase. The inhibition was determined twice with the addition of appropriate sample solution with the inhibitor. The 5070 inhibition was estimated by the graphic method (Misra and Fridovich, 1971). After electrophoresis the gels were immersed for 20 min in 0.017 M pyrophosphate buffer, pH 8.3, containing 1.2 mM NBT and 0.028 mM PMS. After being washed with water, the gels were immersed in the same buffer containing 0.15 mM NADH only. The contrasting achromatic zones were achieved in about 60 min while soaking the gels. From the unstained gels, small pieces (2-3 mm in thickness) were cut out corresponding to the achromatic zones on the stained gels. These pieces were homogenized in 0.017 M pyrophosphate buffer, pH 8.3 (total volume 2.25 ml). The homogenates were left standing overnight at 4°C and then were centrifugated at 8OOg for 30 min. The clear supernatant was used for determination of activity as described above. RESULTS
AND
DISCUSSION
After electrophoresis on polyacrylamide gel and staining, three achromatic zones were obtained which we defined as SODr, SODZ, and SODS, counting from the anode side. The corresponding dark zones were obtained after staining the gel with amido black (Fig. 1) .
FIG. (A)
1. Polyacrylamide Staining for SOD
gel electrophoregrams activity; (B) staining
(scheme) for protein.
of SOD
from
bovine
erythrocytes.
36
MISHIN,
1
VOLSKY,
AND
LYAKHOVICH
++ . 50 SOD
100 total
eluate
150 in ~1
200
FIG. 2. The inhibition activity ratio of SOD before and after-electrophoresis. ( 0-0 ) Inhibition activity of SOD without electrophoresis; ( m---m) inhibition activity of SOD after electrophoresis; (A---A) inhibition activity of control gel eluates (without SOD).
In experiments in which 44.6 units of SOD were separated and the three zones revealed were eluted, it was found that practically the whole activity of SOD eluted from the gel. Moreover, the sum of the activities eluted from the three zones exceeded that initially applied on the gel. This can be explained partly by the fact that the control gel eluates (that is, without application of SOD) resulted in some inhibition of the NBT reduction (Fig. 2). In separate experiments we have found that ammonium persulfate (2.5 mg/ml) completely inhibits the reduction of NBT by NADH + PMS and xanthine + xanthine oxidase systems,
300
SOD
FIG. 3. The electrophoresis
sample
ratio of the inhibition activity of SOD depending on the quantity of applied
in
pl
before ( O-O enzyme.
) and
after
( M-B
)
ACTIVITY
OF
SUPEROXIDE
DISMUTASE
TABLE Activities
of Electrophoretically
Separable 96 units
applied
Activity eluted from the gel (units) SOD, SODI SOD, SOD3
+ SODz
133.1 It 19.7 7.2 f 1.4
+ SOD3
29.6 f 96.3 f
3.1 15.2
37
FORMS
I Forms
of SOD
on t,he gel Percentage of total activity
100
from
Bovine
388 units activity from
applied
eluted the gel (units)
327.8
5.0 * 1 23.0 f 5 72.0 f 5
Erythrocytes
f
9.3 f
25.0
0.1
54.5 rt 8.8 264.0 f 22.5
on the gel Percentage of total act,ivity
100 3.0 f 17.0 f 80.0 f
0 3 7
but has no effect on NADH + PMS + cytochrome c and xanthine + xanthine oxidase + cytochrome c systems nor on the autooxidation of adrenalin at alkaline pH. Thus, the slight contamination of eIuates with ammonium persulfate can be accounted for by direct interaction with NBT. Nevertheless, we believe that the inhibitory action of ammonium persulfate is not essential to the determination of the relative activities of SOD forms since the activity ratios of SOD bef’ore electrophoresis to SOD in the eluates did not change with changes in the amount of SOD applied (Fig. 3). As can also be seen from Fig. 3, enzyme elution from the gels proceeded successfully to completion over the wide range of SOD quantities applied on the gel. Based on the above experiments the activity of each SOD form was determined. The results for two concentrations of SOD are presented in Table 1. It should be noted that the results presented here reflect the activity ratios of the individual samples of SOD, because the activity ratios obtained can change slightly with different preparations of SOD. It is of interest that in all SOD preparations studied, the activity values for electrophoretically separable forms of SOD did not coincide with the intensity of staining for protein of each band (compare Fig. 1 and Table 1). It can be seen that the amount of protein in SOD2 band is equal to or greater than that in SOD3 band, while its activity is much less than that of SODS. This can be explained by the following assumptions: (1) The SOD, band may include some other nonactive proteins with very close electrophoretic properties; (2) the activity of the SOD2 form was destroyed partially during the enzyme purification; (3) the specific activity of SOD2 is less than that of SODS. The reason for this is under further investigation. REFERENCES BECKMAN,
different 33%345.
G., LUNDGREN, human tissues,
E., their
and TARNVIK, genetic control
A. ( 1973). and
Superoxide dismutase intracellular localization. Hum.
isozymes Hered.
in 23,
W., and WESER, U. ( 1975). Superoxide Dismutase Micro Assay in Biological Material. Hoppe-Seyler’s 2. Ph&ol. Chem. 356, 747-754. II. Method and application to human serum. Ann. DAVIS, B. J. (1964). Disc electrophoresis. N.Y. Acad. Sci. 121, 404-468. FRANTS, R. (1973). On the accordance of two SOD systems in bovine tissues. Acta Acad. Aboensis 33, No. 17. BOHNENKAMP,
38
MISHIN,
VOLSKY,
AND
LYAKHOVICH
FRIWVICH, I. (1972). Superoxide radical and superoxide dismutase. Act. Chem. Res. 5, 321326. MAKMOCCHI, F., VENAHDI, G., CAULINI, G., and ROTILIO, G. (1974). Enzyme activity of superoxide dismutase protomers. FEBS Lett. 44, 337-339. MAUHER, H. R. ( 1968). “Disk-Electrophorese,” p. 102. Walter de Gruyter, Berlin. MCCORD, J. M., and FRIDOXCH, I. ( 1969). Tl re utility of superoxide dismutase in studying free radical reactions. J. Biol. Chcm. 244, 6056-6063. MISRA, H. P., and FRIDOWCH, I. (1971). Th e generation of superoxide radical during the autoxidation of ferredoxins. J. Biol. Chem. 246, 6856-6890. NISIIIKIMI, M., RAO, N. A., and YAGI, K. (1972). The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular osygen. Biochem. Biophys. Res. Commun. 46, 849-853. PUGET, K., and MICHELSON, A. M. (1974). Isolation of a new copper-containing superoxide dismutase bacteriocuprein. Biochn. Biophus. Res. Commun. 58, 830-838.
MISHIN,
N. N.
of Electrophoretically Superoxide Dismutase
VOLSKY,
AND
V. V.
Separable
LYAKHOWCH
of Erperimcntul and Clinical Medicine, Academy of Medical Sciences of the USSR S’berian Branch, Novosibirsk 630091, USSR Received January 19, 1977
The quantitative method for determining activities of elxtrophoretically separable forms of superoxide dismutase is presented. The m&hod consists of SOD separation on polyacrylamide gels, elution of SOD forms from the gels, and determination of inhibition activities of eluates with nitro blue tetrazolium reduction. It is shown that SOD partially purified from bovine erythrocytes sqaratrs into three fractions with different O,- .-trapping activities.
INTRODUCTION The multiple forms of superoxide dismutasc (SOD, EC 1.15.1.1) separated electrophoretically on polyacrylamide gel have been fomld in all human tissues (Beckman et al., 1973), in beef liver (Fran@ 1973), in plants (Puget and Michelson, 1974), and in microorganisms ( Marmocchi et al., 1974). Because of the important biological significance of SOD (Fridovich, 1972), we tried to determine the relative activities of the eIectrophoretically separable forms of this enzyme. There have been a few reports on this subject. Bohnenkamp and Weser (1975) estimated the SOD activities by the densitometry of the achromatic zones which appeared after staining using the light + riboflavin + nitro Mue tetrazolium (NBT) system. Beckman et al. (1973) used the visual method of determining the activities of SOD forms after separation on polyacrylamide gel and staining. The shortcomings of the densitometric method as well as those of the visual method are well known ( Maurer, 1968) . In this report we discuss a method for determining the relative activities of SOD forms based on enzyme separation on polyacrylamide gel, subsequent elution of the separate fractions, and spectrophotometric determination of the eluate activities. MATERIALS
AND METHODS
The material used in the study was bovine erythrocyte SOD prepared according to McCord and Fridovich ( 1969) but omitting DEAE-cellulose chromatography. The resulting activities of the samples were usually 500-550 units/mg of protein according to McCord and Fridovich (1969). Polyacrylamide gel electro34 0014-4800/78/0281-0034$02.00/O Copyright All rights
0 1978 by Academic Press. Inc. of reproduction in any form reserved.
ACTIVITY
OF
SUPEROXIDE
DISMUTASE
35
FORMS
phoresis was performed according to Davis (1964) in Reanal apparatus (Hungary) at 3 mA/tube. Determination of SOD activities and gel staining were performed according to Nishikimi et al. (1972) with minor modifications. The reduction of NBT by OZY, generated by reoxidation of phenazine metasulfate (PMS), has been used in assays of the activities of initial SOD samples as well as in those of gel eluates. The medium consisted of 150 nmole of NBT, 0.7 nmole of PMS, and 234 nmole of NADH in 0.017 M pyrophosphate buffer, pH 8.3. The final volume was 3.0 ml at 25°C. Under these conditions, when the inhibitor was omitted, the OD change at 560 nm was O.OS/min. The reaction was linear during 4-5 min after the short lag phase. The inhibition was determined twice with the addition of appropriate sample solution with the inhibitor. The 5070 inhibition was estimated by the graphic method (Misra and Fridovich, 1971). After electrophoresis the gels were immersed for 20 min in 0.017 M pyrophosphate buffer, pH 8.3, containing 1.2 mM NBT and 0.028 mM PMS. After being washed with water, the gels were immersed in the same buffer containing 0.15 mM NADH only. The contrasting achromatic zones were achieved in about 60 min while soaking the gels. From the unstained gels, small pieces (2-3 mm in thickness) were cut out corresponding to the achromatic zones on the stained gels. These pieces were homogenized in 0.017 M pyrophosphate buffer, pH 8.3 (total volume 2.25 ml). The homogenates were left standing overnight at 4°C and then were centrifugated at 8OOg for 30 min. The clear supernatant was used for determination of activity as described above. RESULTS
AND
DISCUSSION
After electrophoresis on polyacrylamide gel and staining, three achromatic zones were obtained which we defined as SODr, SODZ, and SODS, counting from the anode side. The corresponding dark zones were obtained after staining the gel with amido black (Fig. 1) .
FIG. (A)
1. Polyacrylamide Staining for SOD
gel electrophoregrams activity; (B) staining
(scheme) for protein.
of SOD
from
bovine
erythrocytes.
36
MISHIN,
1
VOLSKY,
AND
LYAKHOVICH
++ . 50 SOD
100 total
eluate
150 in ~1
200
FIG. 2. The inhibition activity ratio of SOD before and after-electrophoresis. ( 0-0 ) Inhibition activity of SOD without electrophoresis; ( m---m) inhibition activity of SOD after electrophoresis; (A---A) inhibition activity of control gel eluates (without SOD).
In experiments in which 44.6 units of SOD were separated and the three zones revealed were eluted, it was found that practically the whole activity of SOD eluted from the gel. Moreover, the sum of the activities eluted from the three zones exceeded that initially applied on the gel. This can be explained partly by the fact that the control gel eluates (that is, without application of SOD) resulted in some inhibition of the NBT reduction (Fig. 2). In separate experiments we have found that ammonium persulfate (2.5 mg/ml) completely inhibits the reduction of NBT by NADH + PMS and xanthine + xanthine oxidase systems,
300
SOD
FIG. 3. The electrophoresis
sample
ratio of the inhibition activity of SOD depending on the quantity of applied
in
pl
before ( O-O enzyme.
) and
after
( M-B
)
ACTIVITY
OF
SUPEROXIDE
DISMUTASE
TABLE Activities
of Electrophoretically
Separable 96 units
applied
Activity eluted from the gel (units) SOD, SODI SOD, SOD3
+ SODz
133.1 It 19.7 7.2 f 1.4
+ SOD3
29.6 f 96.3 f
3.1 15.2
37
FORMS
I Forms
of SOD
on t,he gel Percentage of total activity
100
from
Bovine
388 units activity from
applied
eluted the gel (units)
327.8
5.0 * 1 23.0 f 5 72.0 f 5
Erythrocytes
f
9.3 f
25.0
0.1
54.5 rt 8.8 264.0 f 22.5
on the gel Percentage of total act,ivity
100 3.0 f 17.0 f 80.0 f
0 3 7
but has no effect on NADH + PMS + cytochrome c and xanthine + xanthine oxidase + cytochrome c systems nor on the autooxidation of adrenalin at alkaline pH. Thus, the slight contamination of eIuates with ammonium persulfate can be accounted for by direct interaction with NBT. Nevertheless, we believe that the inhibitory action of ammonium persulfate is not essential to the determination of the relative activities of SOD forms since the activity ratios of SOD bef’ore electrophoresis to SOD in the eluates did not change with changes in the amount of SOD applied (Fig. 3). As can also be seen from Fig. 3, enzyme elution from the gels proceeded successfully to completion over the wide range of SOD quantities applied on the gel. Based on the above experiments the activity of each SOD form was determined. The results for two concentrations of SOD are presented in Table 1. It should be noted that the results presented here reflect the activity ratios of the individual samples of SOD, because the activity ratios obtained can change slightly with different preparations of SOD. It is of interest that in all SOD preparations studied, the activity values for electrophoretically separable forms of SOD did not coincide with the intensity of staining for protein of each band (compare Fig. 1 and Table 1). It can be seen that the amount of protein in SOD2 band is equal to or greater than that in SOD3 band, while its activity is much less than that of SODS. This can be explained by the following assumptions: (1) The SOD, band may include some other nonactive proteins with very close electrophoretic properties; (2) the activity of the SOD2 form was destroyed partially during the enzyme purification; (3) the specific activity of SOD2 is less than that of SODS. The reason for this is under further investigation. REFERENCES BECKMAN,
different 33%345.
G., LUNDGREN, human tissues,
E., their
and TARNVIK, genetic control
A. ( 1973). and
Superoxide dismutase intracellular localization. Hum.
isozymes Hered.
in 23,
W., and WESER, U. ( 1975). Superoxide Dismutase Micro Assay in Biological Material. Hoppe-Seyler’s 2. Ph&ol. Chem. 356, 747-754. II. Method and application to human serum. Ann. DAVIS, B. J. (1964). Disc electrophoresis. N.Y. Acad. Sci. 121, 404-468. FRANTS, R. (1973). On the accordance of two SOD systems in bovine tissues. Acta Acad. Aboensis 33, No. 17. BOHNENKAMP,
38
MISHIN,
VOLSKY,
AND
LYAKHOVICH
FRIWVICH, I. (1972). Superoxide radical and superoxide dismutase. Act. Chem. Res. 5, 321326. MAKMOCCHI, F., VENAHDI, G., CAULINI, G., and ROTILIO, G. (1974). Enzyme activity of superoxide dismutase protomers. FEBS Lett. 44, 337-339. MAUHER, H. R. ( 1968). “Disk-Electrophorese,” p. 102. Walter de Gruyter, Berlin. MCCORD, J. M., and FRIDOXCH, I. ( 1969). Tl re utility of superoxide dismutase in studying free radical reactions. J. Biol. Chcm. 244, 6056-6063. MISRA, H. P., and FRIDOWCH, I. (1971). Th e generation of superoxide radical during the autoxidation of ferredoxins. J. Biol. Chem. 246, 6856-6890. NISIIIKIMI, M., RAO, N. A., and YAGI, K. (1972). The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular osygen. Biochem. Biophys. Res. Commun. 46, 849-853. PUGET, K., and MICHELSON, A. M. (1974). Isolation of a new copper-containing superoxide dismutase bacteriocuprein. Biochn. Biophus. Res. Commun. 58, 830-838.
