ARCHIVES
Vol.
OF BIOCHEMISTRY
285, No. 1, February
AND
BIOPHYSICS
15, pp. 60-63,
1991
Phosphate, Not Superoxide Dismutase, Facilitates Electron Transfer from Ferrous Salts to Cytochrome Wayne
F. Beyer,
Jr. and Irwin
Department
of Biochemistry,
Received
9, 1990; and in revised
July
Fridovich
Duke University
form
c’
October
Medical
Center, Durham,
North
Carolina
27710
19, 1990
Peterson and Eaton (1989, Biochem. Biophys. Res. Commun. 165, 164-167) reported that the copperand zinc-containing, but not the manganese-containing, superoxide dismutase catalyzes the reduction of cytochrome c by ferrous salts. This activity, erroneously attributed to the enzyme, is now shown to have been due to inorganic phosphate. 0 1991 Academic Press, Inc.
Superoxide dismutases (SODS)’ are a family of enzymes which provide a defense against oxygen toxicity by catalyzing the dismutation of 0; into dioxygen plus hydrogen peroxide (1). SODS containing manganese, or iron, or copper plus zinc, have been isolated. All of these enzymes catalyze the same reaction and do so with comparable efficiency. Deletion of SOD, in Escherichia coli, has been shown to decrease tolerance for 02, and for compounds which mediate the univalent reduction of O2 (2), and to increase spontaneous mutagenesis under aerobic, but not under anaerobic, conditions (3). Restoration of SOD, by insertion of the corresponding gene on a plasmid, restored the normal phenotype (4). This was the case even when the SOD restored was of a different type from that deleted. Thus the human Cu,ZnSOD’ complemented FeSOD and MnSOD deficiency in E. coli (4), a tobacco MnSOD complemented a deficiency of MnSOD in a yeast and in E. coli (5), and a Bacillus stearothermophilus MnSOD complemented a deficiency of Cu,ZnSOD in a yeast (6). These results establish that the several members of the SOD family provide the same protective effect in uiuo, just as they catalyze the same reaction in vitro. i This work was supported by grants from the Council for Tobacco Research-U.S.A., Inc., The American Cancer Society, the National Science Foundation, and the National Institutes of Health. * Abbreviations used: SODS, superoxide dismutases; CuZnSOD, the copperand zinc-containing superoxide dismutase; BESOD, bovine erythrocyte superoxide dismutase; BSA, bovine serum albumin.
Peterson and Eaton (7), in apparent contradiction of this view, recently reported that Cu,ZnSOD, but not MnSOD, accelerated the reduction of cytochrome c by Fe(R). They concluded that the dismutation of 0; might be only one of the activities of SODS; stating, “While it has long been felt that SOD is an antioxidant enzyme, functioning to remove superoxide, recent studies suggest that this view may be simplistic.” We now demonstrate that the activity, attributed by Peterson and Eaton to Cu,ZnSOD, was due to inorganic phosphate, which was present as a major contaminant of the commercial Cu,ZnSOD, but not of the MnSOD, which they used in their work. MATERIALS
AND
METHODS
Absorbance measurements were taken at room temperature (-2225’C) on either Beckman DU-70 or Hitachi 100-80 spectrophotometers. The assay wavelength (550 nm) was verified by location of the maximum absorbance of dithionite-reduced horse heart cytochrome c. Reactions were monitored for 2 min. The following reagents were used as obtained from Sigma Chemical Co. (St. Louis, MO): horse heart cytochrome c (type III) (Sigma 2506, Lot 118F-72053, bovine serum albumin (Sigma A-4378, Lot 125F-9350), fatty acid free (less than 0.005%) bovine serum albumin (Sigma A-0281, Lot 87F-9375), bovine erythrocyte superoxide dismutase (Sigma S2515, Lot lOOF-9300). Bovine erythrocyte superoxide dismutase was also obtained as a generous gift from Chemie Grunenthal (Lot 163-3). Bovine liver catalase (crystalline suspension in water) was obtained from Boehringer-Mannheim. The suspended crystals were pelleted by centrifugation and washed three times by resuspension in a loo-fold volume excess of water. The crystals were pelleted and finally dissolved in 50 mM Tris-HCl buffer, pH 7.40. Potassium phosphate salts (AR Grade) KpHPOl * 3H20 and KHzPOl were from Mallinckrodt. Tris (crystallized free base) was from Boehringer-Mannheim and HCl (AR grade) was from Mallinckrodt. Fe(S0,). 7Hz0 (99.999%) (Lot 594867B) was obtained from AESAR (Johnson, Matthey). Water was deionized and glass distilled. Stock Fe’+ solutions were prepared fresh daily by dissolution of -174 mg FeSO, * 7HxO in 11 ml of Hz0 and acidified (to prevent autoxidation) by the addition of -10-20 pl of 6 N HCl. This stock solution (-57 mM Fe*+) was kept at O’C. Unless stated otherwise protein solutions were prepared by dissolution of the lyophilized powder directly into 50 mM Tris-HCl buffer, pH 7.40. Protein concentrations were determined by uv absorption (8) using a A$TG= 16.4 (catalase), A:% = 6.67 (BSA), A z = 2.53 (Grunenthal BESOD). The protein concentration of Sigma BESOD was determined by the Lowry 0003.9861/91
60
Copyright All
rights
0
1991 of reproduction
by
Academic in any
form
$3.00
Press, Inc. reserved.
PHOSPHATE
SPEEDS
REDUCTION
OF
CYTOCHROME
c BY
61
FE(H)
0.040 / ,:’
0 032-
24
46
I
I
72
96
120
0
24
46
Seconds
72
/.‘.
96
120
Seconds
FIG. 1. Effects of various agents on the rate of ferricytochrome c reduction. The reaction volume was 3 ml and the buffer was 50 mM Tris-HCl, pH 7.40. Each reaction contained 20 pM ferricytochrome c. Line 1, 18 pM Fe’+ alone; line 2, 18 pM Fe*+ + 100 pg/ml BSA; line 3, 18 pM Fe*+ + 100 pg/ml BSA + 634 pg (2300 SOD units) of Grunenthal BESOD; line 4,18 pM Fe’+ + 100 kg/ml BSA + 519 pg (830 SOD units) of Sigma BESOD.
Effect of Sigma BESOD, Sigma BESOD (Cl0 desalted) and Grunenthal BESOD on the rate of ferricytochrome c reduction. Each reaction (3 ml) contained 20 fiM ferricytochrome c, 18 pM Fez+ and 100 rg/ml BSA (plus the specified addition) in 50 mM Tris-HCl, pH 7.40, buffer. Line 1,634 pg (2300 SOD units) of Grunenthal BESOD; line 2, no addition; line 3, 480 pg (823 SOD units) of Sigma BESOD (Cl0 desalted); line 4, 519 pg (830 SOD units) Sigma BESOD.
method (9) using BSA as a standard. Superoxide dismutase activity was measured as previously described (10). Cytochrome c was found to be 99.7% ferric cytochrome c by dithionite reduction using EE:‘h, = 2.99 x 104 M-l cm-l and *@+-Fe8+ = 2.1 X lo4 M-’ cm-’ (11). The concen550 nm tration of cytochrome c in each assay refers to ferricytochrome c. Each reaction (-3.0 ml) was initiated by the addition of Fe’+ to the cuvette containing the indicated components and the Asso nm was monitored continuously using either a strip chart recorder (Hitachi 100-80) or storage in the computer microprocessor memory (DU-70) for later routing to a HP7475 plotter. Buffer exchange for Sigma BESOD was performed by placing 100 pl (-5 mg) of stock Sigma BESOD in a Cl0 centricon microconcentrator (Amicon), diluting to 2.5 ml with 50 mM Tris-HCl, pH 7.4, buffer, concentrating to -100 ~1 and repeating this dilution/ concentration for a total of three times. The ultrafiltrate from the first dilution (100 ~1-2.5 ml)) was saved, the others were discarded. During the course of this study, we determined that the reaction rate was remarkably sensitive to the presence of phosphate (vide infra); thus to optimize reproducible rates, cuvettes required rigorous washing and flushing with Hz0 between reactions to remove trace amounts of phosphate salts.
When the Sigma SOD was freed of its low molecular weight components, by repeated dilution with Tris buffer followed by centrifugal ultrafiltration, it lost its ability to augment the reduction of cytochrome c by Fe(II), although it did not lose its SOD activity. This result, shown in Fig. 2, established that a low molecular weight contaminant was responsible for the activity which Peterson and Eaton had attributed to SOD. This conclusion was buttressed by the observation, shown in Fig. 3, that the ultrafiltrate of the Sigma SOD was able to hasten the reduction of cytochrome c by Fe(B). This ultrafiltrate was devoid of SOD activity. Sigma’s label on their lyophilized Cu,ZnSOD indicated that it contained 2-5s (w/w) potassium phosphate salts.
FIG. 2.
RESULTS
Effect of BSA and SOD on cytochrome c reduction. Fe(I1) caused a slow reduction of cytochrome c, which was linear during the 2-min period of observation. BSA, at 100 pg/ml accelerated this reaction and addition of Cu,ZnSOD from Sigma at 519 pg/ml, caused a much greater rate of reduction. These conditions mimic those used by Peterson and Eaton and these results agree with theirs. However, as shown in Fig. 1, Cu,ZnSOD from Grunenthal, at 634 Kg/ml, slightly inhibited, rather than accelerated the reduction of cytochrome c. It should be noted that the specific SOD activity of the Grunenthal enzyme was 2.5 times greater than that of the Sigma material. It was thus apparent that an impurity in the Sigma preparation was responsible for its effect.
E c s: Lo
0.024 .A
t
.A
.H
.y’
;; 8
0.016-
“0
24
40
72
96
120
Seconds
FIG. 3.
Stimulation of the rate of ferricytochrome c reduction by the Cl0 ultrafiltrate from Sigma BESOD. Each reaction contained (in 3 ml) 20 pM ferricytochrome c, 18 pM Fe*+, and 100 pg/ml BSA in 50 mM Tris-HCl, pH 7.4, buffer. Line 1, no addition; line 2,50 pi of Cl0 ultrafiltrate, line 3, 100 jd of Cl0 ultrafiltrate.
62
BEYER
AND
The label on the Sigma MnSOD, in contrast, indicated Tris buffer salts rather than phosphate contaminants. Phosphate was thus suspected of being the low molecular weight contaminant responsible for the “activity” of the Cu,ZnSOD. Moreover, phosphate is known to stimulate electron transfer (12) from Fe(I1) to O2 and association of Fe(I1) with the anionic phosphate would eliminate the electrostatic barrier otherwise hindering the reduction of the cationic cytochrome c by a cationic reductant. Figure 4 shows that phosphate did augment the reduction of cytochrome c by Fe(II), while Fig. 5 compares the effects of BSA and of BSA plus Grunenthal SOD, or Sigma SOD before and after ultrafiltration, and after ultrafiltration with phosphate added. From the dose-dependent effect of phosphate, shown in Fig. 4 and the stimulative effect of Sigma Cu,ZnSOD, we calculated the amount of KzHPO, which would have to be present in the SOD to account for its “activity.” The result, which was 3% (w/w), agrees with the supplier’s estimate of 2-5%. Its phosphate content is thus adequate to fully account for its effect on the reduction of cytochrome c by Fe(I1). It should be noted that phosphate was not the only impurity in the Sigma Cu,ZnSOD. There was absorbance at 400 nm suggestive of hemo or flavo enzyme. There was also an absorption maximum at 280 nm indicating extraneous proteins, since the bovine erythrocyte Cu,ZnSOD absorbs maximally at 265 nm because of its lack of tryptophan and paucity of tyrosine (10). Finally, the actual specific activity of the Sigma SOD was -1600 u/mg, rather than the -3000 u/mg claimed on the label. When CaC& was added to 1.6 mM to a solution of the Sigma Cu,ZnSOD in Tris chloride buffer at pH 7.4, a precipitate was seen to form. This precipitate, presumably calcium phosphate, was removed by centrifugation and the supernatant, which retained its SOD activity, had lost its ability to augment the reduction of cytochrome c by Fe(I1). Peterson and Eaton reported a difference between BSA and defatted BSA, both from Sigma, in that the former
100
200 300 JLM Potassium
400 500 Phosphate
600
700
FIG. 4. Effect of potassium phosphate on the rate of ferricytochrome c reduction. Each reaction contained (in 3 ml) 18 MM Fe*+, 100 fig/ml BSA (dialyzed against 50 mM Tris-HCl, pH 7.4, buffer), 20 pM ferriq&chrome c, and the indicated amount of KPi added as KHPPO,/KzPOI, pH 7.4. The buffer was 50 mM Tris-HCl, pH 7.4.
FRIDOVICH
1
2
3
4
5
FIG. 6. Comparative effect of BESOD on the rate of ferricytochrome c reduction. Each reaction contained (in 3 ml) 18 pM Fe’+, 100 pg/ml BSA (dialyzed against 50 mM Tris-HCl, pH 7.4) 20 pM ferricytochrome c. The buffer was 50 mM Tris-HCl, pH 7.4. The additions were (1) no addition, control; (2) 640 pg Grunenthal BESOD (2300 SOD units; (3) 519 pg (830 SOD units) Sigma BESOD; (4) 480 ng (823 SOD units) Sigma BESOD (Cl0 desalted); (5) 82 pM KP,, pH 7.4.
enhanced the reduction of cytochrome c by Fe(B) while the latter did to a lesser extent. They attributed this difference to an electron transport function by the ?r electrons of polyunsaturated fatty acids bound to the BSA. We verified this difference, but noted that dialysis of BSA against Tris buffer, at pH 7.4, eliminated its activity. It follows that here too Peterson and Eaton were misled by a low molecular weight contaminant, probably phosphate. In our experiments, examining the effect of Cu,ZnSOD and of phosphate, we used their conditions and added these components to reaction mixtures containing BSA. This was not necessary, however, since phosphate stimulated Fe2+-dependent cytochrome c reduction in the absence of BSA. In addition, bovine liver catalase (100 pg, 4000 units) had no effect on the rate of Fe2+-mediated ferricytochrome c reduction, suggesting that H202 was not involved. DISCUSSION Investigators using commercial preparations of enzymes at high concentration must always be wary of artifacts due to impurities. This applies with special force when their studies lead to claims of new activities for well-studied enzymes. In the case of the work by Peterson and Eaton (7) the activity which they attributed to Sigma Cu,ZnSOD was due to inorganic phosphate in the lyophilized material. This must also have been the case for the activity they saw with BSA. Accordingly, this “activity” was eliminated by cycles of dilution and ultrafiltration or by dialysis or precipitation with Ca(I1); was present in an ultrafiltrate; and could be restored by adding back KzHPOl. The amount of phosphate required to restore the original level of “activity” was within the range of phosphate contents stated on the supplier’s label. The Sigma defatted BSA and MnSOD, being free of phosphate, did not exhibit this “activity.” Peterson and Eaton did attempt to correlate the ability to catalyze the reduction of cytochrome c by Fe(I1) with
PHOSPHATE
SPEEDS
REDUCTION
the SOD activity of the Cu,ZnSOD. They used diethyldithiocarbamate to inactivate the Sigma Cu,ZnSOD and saw a loss of the “activity” they were following. Unfortunately they never considered a low molecular weight contaminant, such as phosphate, and so did not suspect that gel-exclusion chromatography, in a Tris chlorideequilibrated column, would remove the DDC and also the phosphate. In the discussion section of their paper Peterson and Eaton (7) make a few statements which misrepresent the current knowledge of the function of SOD. These erroneous statements bear correction. They thus state that, “Bacteria do not require SOD for aerobic survival and growth,” and they quote Carlioz and Touati (2) as support for this assertion. Carlioz and Touati actually came to the opposite conclusion, because they noted that E. coli mutants lacking SOD exhibited a conditional sensitivity toward Oz. One basis of the 02-dependent nutritional requirements imposed by lack of SOD, which they observed, has since been elucidated (13). Peterson and Eaton (7) also stated that “. . . the presence of increased amounts of SOD may actually cause a paradoxical increase in the sensitivity of bacteria to oxidant challenge.” It must be noted that the work they quote in support of this assertion could not be repeated by other workers (14) and has been contradicted by recent work of others (15). It is interesting that the group now reporting that a defect in SOD production imparts enhanced sensitivity toward paraquat (15) is the same as that which previously reported the contradictory result (16). Note added in proof. Peterson and Eaton (17) have recently reported that catalase facilitates electron transfer from Fe(I1) to cytochrome c. It appears very likely, given the inactivity of our phosphate-free solutions of catalase, that this is another case of attributing to an enzyme an effect actually due to an impurity, such as inorganic phosphate.
OF
CYTOCHROME
c BY
63
FE(II)
REFERENCES 1. Fridovich, 2. Carlioz, 3. Farr, USA
I. (1989)
J. Biol.
A., and Touati, S. B., D’Ari, 83.8268-8272.
Chem.
264,
D. (1986)
EMBO
R., and Touati,
4. Natvig, D. O., Imlay, K., Touati, Biol. C&m. 262, 14,697-14,701.
7761-7764. J. 5,623-630.
D. (1986)
Proc. Natl.
D., and Hallewell,
Acad.
Sci.
R. A. (1987)
J.
5. Bowler, C., Alliotte, T., Van Den Bulcke, M., Bauw, G., Vandekerckhove, J., Van Montagu, M., and Inz’e, D. (1989) Proc. Natl. Acad. Sci. USA 86,3237-3241. 6. Bowler, C., Van Kaer, L., Van Camp, D., and Dhaese, P. (1990) J. Bacterial. 7. Peterson, Commun. 8. Fasman, Biology,
D. A., and Eaton,
W., Van Montagu, 172, 1539-1546.
J. W. (1989)
Biophys.
Res.
166,164-167. G. (Ed.) (1976) Handbook of Biochemistry 3rd ed., Vol. 2 CRC Press, Cleveland, OH.
9. Lowry, 0. H., Rosebrough, N. J., Farr, J.Biol.Chem. 193,265-275. 10. McCord,
Biochem.
M., Inz’e,
J. M., and Fridovich,
and Molecular
A. L., Randall,
I. (1969)
R. J. (1951)
J. Biol. Chem.
244,
6049-
6055. 11. Massey,
V. (1959)
12. Lambeth, Biochim.
D. O., Ericson, Biophys. Acta
13. Kuo,
Biochim.
C. F., Mashino,
Biophys.
Acta
G. R., Yorek,
34, 255.
M. A., and Ray, P. D. (1982)
719, 501-508. T.,
and Fridovich,
I. (1987)
J. Biol.
Chem.
262,4724-4727. 14. Hassan,
H. M. (1989)
15. Bloch, C. A., Thorne, mun. 57,2141-2148. 16. Bloch,
Adv.
Genetics
26,65-97.
G. M., and Ausubel,
C. A., and Ausubel,
17. Peterson, D. A., and Eaton, Suppl. 1, 182 Abstr #19.22.
F. M. (1986)
F. M. (1989)
J. Bacterial.
J. W. (1990)
Free
Infect.
Im-
168, 796-798.
Rad. Biol.
Med.
9,
Vol.
OF BIOCHEMISTRY
285, No. 1, February
AND
BIOPHYSICS
15, pp. 60-63,
1991
Phosphate, Not Superoxide Dismutase, Facilitates Electron Transfer from Ferrous Salts to Cytochrome Wayne
F. Beyer,
Jr. and Irwin
Department
of Biochemistry,
Received
9, 1990; and in revised
July
Fridovich
Duke University
form
c’
October
Medical
Center, Durham,
North
Carolina
27710
19, 1990
Peterson and Eaton (1989, Biochem. Biophys. Res. Commun. 165, 164-167) reported that the copperand zinc-containing, but not the manganese-containing, superoxide dismutase catalyzes the reduction of cytochrome c by ferrous salts. This activity, erroneously attributed to the enzyme, is now shown to have been due to inorganic phosphate. 0 1991 Academic Press, Inc.
Superoxide dismutases (SODS)’ are a family of enzymes which provide a defense against oxygen toxicity by catalyzing the dismutation of 0; into dioxygen plus hydrogen peroxide (1). SODS containing manganese, or iron, or copper plus zinc, have been isolated. All of these enzymes catalyze the same reaction and do so with comparable efficiency. Deletion of SOD, in Escherichia coli, has been shown to decrease tolerance for 02, and for compounds which mediate the univalent reduction of O2 (2), and to increase spontaneous mutagenesis under aerobic, but not under anaerobic, conditions (3). Restoration of SOD, by insertion of the corresponding gene on a plasmid, restored the normal phenotype (4). This was the case even when the SOD restored was of a different type from that deleted. Thus the human Cu,ZnSOD’ complemented FeSOD and MnSOD deficiency in E. coli (4), a tobacco MnSOD complemented a deficiency of MnSOD in a yeast and in E. coli (5), and a Bacillus stearothermophilus MnSOD complemented a deficiency of Cu,ZnSOD in a yeast (6). These results establish that the several members of the SOD family provide the same protective effect in uiuo, just as they catalyze the same reaction in vitro. i This work was supported by grants from the Council for Tobacco Research-U.S.A., Inc., The American Cancer Society, the National Science Foundation, and the National Institutes of Health. * Abbreviations used: SODS, superoxide dismutases; CuZnSOD, the copperand zinc-containing superoxide dismutase; BESOD, bovine erythrocyte superoxide dismutase; BSA, bovine serum albumin.
Peterson and Eaton (7), in apparent contradiction of this view, recently reported that Cu,ZnSOD, but not MnSOD, accelerated the reduction of cytochrome c by Fe(R). They concluded that the dismutation of 0; might be only one of the activities of SODS; stating, “While it has long been felt that SOD is an antioxidant enzyme, functioning to remove superoxide, recent studies suggest that this view may be simplistic.” We now demonstrate that the activity, attributed by Peterson and Eaton to Cu,ZnSOD, was due to inorganic phosphate, which was present as a major contaminant of the commercial Cu,ZnSOD, but not of the MnSOD, which they used in their work. MATERIALS
AND
METHODS
Absorbance measurements were taken at room temperature (-2225’C) on either Beckman DU-70 or Hitachi 100-80 spectrophotometers. The assay wavelength (550 nm) was verified by location of the maximum absorbance of dithionite-reduced horse heart cytochrome c. Reactions were monitored for 2 min. The following reagents were used as obtained from Sigma Chemical Co. (St. Louis, MO): horse heart cytochrome c (type III) (Sigma 2506, Lot 118F-72053, bovine serum albumin (Sigma A-4378, Lot 125F-9350), fatty acid free (less than 0.005%) bovine serum albumin (Sigma A-0281, Lot 87F-9375), bovine erythrocyte superoxide dismutase (Sigma S2515, Lot lOOF-9300). Bovine erythrocyte superoxide dismutase was also obtained as a generous gift from Chemie Grunenthal (Lot 163-3). Bovine liver catalase (crystalline suspension in water) was obtained from Boehringer-Mannheim. The suspended crystals were pelleted by centrifugation and washed three times by resuspension in a loo-fold volume excess of water. The crystals were pelleted and finally dissolved in 50 mM Tris-HCl buffer, pH 7.40. Potassium phosphate salts (AR Grade) KpHPOl * 3H20 and KHzPOl were from Mallinckrodt. Tris (crystallized free base) was from Boehringer-Mannheim and HCl (AR grade) was from Mallinckrodt. Fe(S0,). 7Hz0 (99.999%) (Lot 594867B) was obtained from AESAR (Johnson, Matthey). Water was deionized and glass distilled. Stock Fe’+ solutions were prepared fresh daily by dissolution of -174 mg FeSO, * 7HxO in 11 ml of Hz0 and acidified (to prevent autoxidation) by the addition of -10-20 pl of 6 N HCl. This stock solution (-57 mM Fe*+) was kept at O’C. Unless stated otherwise protein solutions were prepared by dissolution of the lyophilized powder directly into 50 mM Tris-HCl buffer, pH 7.40. Protein concentrations were determined by uv absorption (8) using a A$TG= 16.4 (catalase), A:% = 6.67 (BSA), A z = 2.53 (Grunenthal BESOD). The protein concentration of Sigma BESOD was determined by the Lowry 0003.9861/91
60
Copyright All
rights
0
1991 of reproduction
by
Academic in any
form
$3.00
Press, Inc. reserved.
PHOSPHATE
SPEEDS
REDUCTION
OF
CYTOCHROME
c BY
61
FE(H)
0.040 / ,:’
0 032-
24
46
I
I
72
96
120
0
24
46
Seconds
72
/.‘.
96
120
Seconds
FIG. 1. Effects of various agents on the rate of ferricytochrome c reduction. The reaction volume was 3 ml and the buffer was 50 mM Tris-HCl, pH 7.40. Each reaction contained 20 pM ferricytochrome c. Line 1, 18 pM Fe’+ alone; line 2, 18 pM Fe*+ + 100 pg/ml BSA; line 3, 18 pM Fe*+ + 100 pg/ml BSA + 634 pg (2300 SOD units) of Grunenthal BESOD; line 4,18 pM Fe’+ + 100 kg/ml BSA + 519 pg (830 SOD units) of Sigma BESOD.
Effect of Sigma BESOD, Sigma BESOD (Cl0 desalted) and Grunenthal BESOD on the rate of ferricytochrome c reduction. Each reaction (3 ml) contained 20 fiM ferricytochrome c, 18 pM Fez+ and 100 rg/ml BSA (plus the specified addition) in 50 mM Tris-HCl, pH 7.40, buffer. Line 1,634 pg (2300 SOD units) of Grunenthal BESOD; line 2, no addition; line 3, 480 pg (823 SOD units) of Sigma BESOD (Cl0 desalted); line 4, 519 pg (830 SOD units) Sigma BESOD.
method (9) using BSA as a standard. Superoxide dismutase activity was measured as previously described (10). Cytochrome c was found to be 99.7% ferric cytochrome c by dithionite reduction using EE:‘h, = 2.99 x 104 M-l cm-l and *@+-Fe8+ = 2.1 X lo4 M-’ cm-’ (11). The concen550 nm tration of cytochrome c in each assay refers to ferricytochrome c. Each reaction (-3.0 ml) was initiated by the addition of Fe’+ to the cuvette containing the indicated components and the Asso nm was monitored continuously using either a strip chart recorder (Hitachi 100-80) or storage in the computer microprocessor memory (DU-70) for later routing to a HP7475 plotter. Buffer exchange for Sigma BESOD was performed by placing 100 pl (-5 mg) of stock Sigma BESOD in a Cl0 centricon microconcentrator (Amicon), diluting to 2.5 ml with 50 mM Tris-HCl, pH 7.4, buffer, concentrating to -100 ~1 and repeating this dilution/ concentration for a total of three times. The ultrafiltrate from the first dilution (100 ~1-2.5 ml)) was saved, the others were discarded. During the course of this study, we determined that the reaction rate was remarkably sensitive to the presence of phosphate (vide infra); thus to optimize reproducible rates, cuvettes required rigorous washing and flushing with Hz0 between reactions to remove trace amounts of phosphate salts.
When the Sigma SOD was freed of its low molecular weight components, by repeated dilution with Tris buffer followed by centrifugal ultrafiltration, it lost its ability to augment the reduction of cytochrome c by Fe(II), although it did not lose its SOD activity. This result, shown in Fig. 2, established that a low molecular weight contaminant was responsible for the activity which Peterson and Eaton had attributed to SOD. This conclusion was buttressed by the observation, shown in Fig. 3, that the ultrafiltrate of the Sigma SOD was able to hasten the reduction of cytochrome c by Fe(B). This ultrafiltrate was devoid of SOD activity. Sigma’s label on their lyophilized Cu,ZnSOD indicated that it contained 2-5s (w/w) potassium phosphate salts.
FIG. 2.
RESULTS
Effect of BSA and SOD on cytochrome c reduction. Fe(I1) caused a slow reduction of cytochrome c, which was linear during the 2-min period of observation. BSA, at 100 pg/ml accelerated this reaction and addition of Cu,ZnSOD from Sigma at 519 pg/ml, caused a much greater rate of reduction. These conditions mimic those used by Peterson and Eaton and these results agree with theirs. However, as shown in Fig. 1, Cu,ZnSOD from Grunenthal, at 634 Kg/ml, slightly inhibited, rather than accelerated the reduction of cytochrome c. It should be noted that the specific SOD activity of the Grunenthal enzyme was 2.5 times greater than that of the Sigma material. It was thus apparent that an impurity in the Sigma preparation was responsible for its effect.
E c s: Lo
0.024 .A
t
.A
.H
.y’
;; 8
0.016-
“0
24
40
72
96
120
Seconds
FIG. 3.
Stimulation of the rate of ferricytochrome c reduction by the Cl0 ultrafiltrate from Sigma BESOD. Each reaction contained (in 3 ml) 20 pM ferricytochrome c, 18 pM Fe*+, and 100 pg/ml BSA in 50 mM Tris-HCl, pH 7.4, buffer. Line 1, no addition; line 2,50 pi of Cl0 ultrafiltrate, line 3, 100 jd of Cl0 ultrafiltrate.
62
BEYER
AND
The label on the Sigma MnSOD, in contrast, indicated Tris buffer salts rather than phosphate contaminants. Phosphate was thus suspected of being the low molecular weight contaminant responsible for the “activity” of the Cu,ZnSOD. Moreover, phosphate is known to stimulate electron transfer (12) from Fe(I1) to O2 and association of Fe(I1) with the anionic phosphate would eliminate the electrostatic barrier otherwise hindering the reduction of the cationic cytochrome c by a cationic reductant. Figure 4 shows that phosphate did augment the reduction of cytochrome c by Fe(II), while Fig. 5 compares the effects of BSA and of BSA plus Grunenthal SOD, or Sigma SOD before and after ultrafiltration, and after ultrafiltration with phosphate added. From the dose-dependent effect of phosphate, shown in Fig. 4 and the stimulative effect of Sigma Cu,ZnSOD, we calculated the amount of KzHPO, which would have to be present in the SOD to account for its “activity.” The result, which was 3% (w/w), agrees with the supplier’s estimate of 2-5%. Its phosphate content is thus adequate to fully account for its effect on the reduction of cytochrome c by Fe(I1). It should be noted that phosphate was not the only impurity in the Sigma Cu,ZnSOD. There was absorbance at 400 nm suggestive of hemo or flavo enzyme. There was also an absorption maximum at 280 nm indicating extraneous proteins, since the bovine erythrocyte Cu,ZnSOD absorbs maximally at 265 nm because of its lack of tryptophan and paucity of tyrosine (10). Finally, the actual specific activity of the Sigma SOD was -1600 u/mg, rather than the -3000 u/mg claimed on the label. When CaC& was added to 1.6 mM to a solution of the Sigma Cu,ZnSOD in Tris chloride buffer at pH 7.4, a precipitate was seen to form. This precipitate, presumably calcium phosphate, was removed by centrifugation and the supernatant, which retained its SOD activity, had lost its ability to augment the reduction of cytochrome c by Fe(I1). Peterson and Eaton reported a difference between BSA and defatted BSA, both from Sigma, in that the former
100
200 300 JLM Potassium
400 500 Phosphate
600
700
FIG. 4. Effect of potassium phosphate on the rate of ferricytochrome c reduction. Each reaction contained (in 3 ml) 18 MM Fe*+, 100 fig/ml BSA (dialyzed against 50 mM Tris-HCl, pH 7.4, buffer), 20 pM ferriq&chrome c, and the indicated amount of KPi added as KHPPO,/KzPOI, pH 7.4. The buffer was 50 mM Tris-HCl, pH 7.4.
FRIDOVICH
1
2
3
4
5
FIG. 6. Comparative effect of BESOD on the rate of ferricytochrome c reduction. Each reaction contained (in 3 ml) 18 pM Fe’+, 100 pg/ml BSA (dialyzed against 50 mM Tris-HCl, pH 7.4) 20 pM ferricytochrome c. The buffer was 50 mM Tris-HCl, pH 7.4. The additions were (1) no addition, control; (2) 640 pg Grunenthal BESOD (2300 SOD units; (3) 519 pg (830 SOD units) Sigma BESOD; (4) 480 ng (823 SOD units) Sigma BESOD (Cl0 desalted); (5) 82 pM KP,, pH 7.4.
enhanced the reduction of cytochrome c by Fe(B) while the latter did to a lesser extent. They attributed this difference to an electron transport function by the ?r electrons of polyunsaturated fatty acids bound to the BSA. We verified this difference, but noted that dialysis of BSA against Tris buffer, at pH 7.4, eliminated its activity. It follows that here too Peterson and Eaton were misled by a low molecular weight contaminant, probably phosphate. In our experiments, examining the effect of Cu,ZnSOD and of phosphate, we used their conditions and added these components to reaction mixtures containing BSA. This was not necessary, however, since phosphate stimulated Fe2+-dependent cytochrome c reduction in the absence of BSA. In addition, bovine liver catalase (100 pg, 4000 units) had no effect on the rate of Fe2+-mediated ferricytochrome c reduction, suggesting that H202 was not involved. DISCUSSION Investigators using commercial preparations of enzymes at high concentration must always be wary of artifacts due to impurities. This applies with special force when their studies lead to claims of new activities for well-studied enzymes. In the case of the work by Peterson and Eaton (7) the activity which they attributed to Sigma Cu,ZnSOD was due to inorganic phosphate in the lyophilized material. This must also have been the case for the activity they saw with BSA. Accordingly, this “activity” was eliminated by cycles of dilution and ultrafiltration or by dialysis or precipitation with Ca(I1); was present in an ultrafiltrate; and could be restored by adding back KzHPOl. The amount of phosphate required to restore the original level of “activity” was within the range of phosphate contents stated on the supplier’s label. The Sigma defatted BSA and MnSOD, being free of phosphate, did not exhibit this “activity.” Peterson and Eaton did attempt to correlate the ability to catalyze the reduction of cytochrome c by Fe(I1) with
PHOSPHATE
SPEEDS
REDUCTION
the SOD activity of the Cu,ZnSOD. They used diethyldithiocarbamate to inactivate the Sigma Cu,ZnSOD and saw a loss of the “activity” they were following. Unfortunately they never considered a low molecular weight contaminant, such as phosphate, and so did not suspect that gel-exclusion chromatography, in a Tris chlorideequilibrated column, would remove the DDC and also the phosphate. In the discussion section of their paper Peterson and Eaton (7) make a few statements which misrepresent the current knowledge of the function of SOD. These erroneous statements bear correction. They thus state that, “Bacteria do not require SOD for aerobic survival and growth,” and they quote Carlioz and Touati (2) as support for this assertion. Carlioz and Touati actually came to the opposite conclusion, because they noted that E. coli mutants lacking SOD exhibited a conditional sensitivity toward Oz. One basis of the 02-dependent nutritional requirements imposed by lack of SOD, which they observed, has since been elucidated (13). Peterson and Eaton (7) also stated that “. . . the presence of increased amounts of SOD may actually cause a paradoxical increase in the sensitivity of bacteria to oxidant challenge.” It must be noted that the work they quote in support of this assertion could not be repeated by other workers (14) and has been contradicted by recent work of others (15). It is interesting that the group now reporting that a defect in SOD production imparts enhanced sensitivity toward paraquat (15) is the same as that which previously reported the contradictory result (16). Note added in proof. Peterson and Eaton (17) have recently reported that catalase facilitates electron transfer from Fe(I1) to cytochrome c. It appears very likely, given the inactivity of our phosphate-free solutions of catalase, that this is another case of attributing to an enzyme an effect actually due to an impurity, such as inorganic phosphate.
OF
CYTOCHROME
c BY
63
FE(II)
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