ARCHIVES
OF BIOCHEMISTRY
The Mechanism
AND
202-205
1’72,
(1976)
of the Activity-Dependent Oxidase ’ ELLEN
Department
BIOPHYSICS
of Biochemistry,
K. HODGSON
AND
Luminescence
IRWIN
of Xanthine
FRIDOVICH
Duke University Medical Center, Durham, Received June 30, 1975
North
Carolina
27710
The weak luminescence that accompanies the aerobic xanthine oxidase reaction is inhibited by superoxide dismutase, by catalase, and by scavengers of hydroxyl radicals. It is also entirely dependent upon the presence of carbonate. It thus appears that the O,and H202 produced during the aerobic action of xanthine oxidase interact to generate OH. which, in turn, reacts with carbonate to yield the carbonate radical (CO,-). The species that is directly responsible for light emission appears to be produced by a dimerization of carbonate radicals, since the light intensity was a function of the square of the carbonate concentration. The data provide no reason to suppose that the lightemitting species is singlet oxygen.
Xanthine oxidase causes a weak luminescence while catalyzing the aerobic oxidation of its substrates (1, 2). Arneson (3) observed that this luminescence was inhibited by catalase or by superoxide dismutase and deduced that both O,- and H,O, were essential intermediates in the lightproducing process. Since it had previously been suggested (4-6) that singlet oxygen could emit in the visible by the formation of dimers, followed by pooling and simultaneous emission of excitation energy, Arneson (3) concluded that the emitting species in the xanthine oxidase reaction was singlet oxygen produced by the following reactions. O,- + H,O, OH. + O,-
+ OH-
+ OH. + 0,
+ OH- + O,*.
[Al
PI
More recently Stauff and co-workers (7) have shown that the luminescence accompanying the oxidation of H,O, by periodate, previously attributed to singlet oxygen, is actually dependent upon carbonate. They suggested that the actual emitting species was produced by a reaction involving carbonate radicals. We have reinvesti1 This work was supported in full by research grants, No. GM-10287 from the National Institutes of Health, Bethesda, Md., and No. RDRP-IP-12410-L from the United States Army Research Office, Durham, N. C. 0 1976 by Academic Press, Inc. of reproduction in any form reserved.
MATERIALS
AND
METHODS
Xanthine oxidase was prepared from cream by a procedure that avoids proteolysis (8). Catalase was from Sigma Chemical Company and was freed of contaminating superoxide dismutase (9) by repeated washing on an XMlOOA Diaflo ultrafiltration membrane obtained from the Amicon Corporation. Catalase activity was assayed at 25°C by the method of Beers and Sizer (101. The manganese-containing superoxide dismutase was prepared from Escherichia coli as previously described (11). The acetaldehyde used was freshly distilled daily. All other materials used were reagent grade. Luminescence measurements were made with either a Nuclear Chicago Mark I liquid scintillation counter or a Packard TriCarb scintillation spectrometer, Model 3003. In either case the coincidence circuit was turned off and the signal from a control vial was subtracted from the signal given by the luminescent reaction. RESULTS
The Dependence upon Carbonate When 4 x lo-' M xanthine oxidase catalyzed the oxidation 202
Copyright All rights
gated the production of light during the xanthine oxidase reaction and have demonstrated that carbonate is essential for this luminescence. The actions of catalase, superoxide dismutase and scavengers of hydroxyl radical were also reinvestigated, and the results lead to a reaction scheme that accounts for the properties of this system.
of 10 mM acetaldehyde
LUMINESCENCE
AND
at pH 10.0 the intensity of luminescence was found to increase as the concentration of the carbonate buffer was raised. This is shown in Fig. 1. The total light emitted during the first 40 min of the reaction, estimated by integrating the curves in Fig. 1, was a linear function of the square of the concentration of carbonate and it extrapolated to zero luminescence in the absence of carbonate. This is shown in Fig. 2. It follows that carbonate is an essential reactant in the process leading to the actual emitting species and that two carbonates are somehow cooperatively involved in the production of that species. The Effects of Catalase Dismutase
XANTHIIW 5
r
2
and Superoxide
It has already been reported (3) that superoxide dismutase and catalase inhibit
.
25
30
35
40
FIG. 1. Effect ofcarbonate on luminescence. Reaction mixtures contained 0.01 M acetaldehyde, 4 x lo-’ M xanthine oxidase, 1 x 10e4 M EDTA and the indicated concentrations of sodium carbonate, all buffered at pH 10.0 and at 8°C. Xanthine oxidase was the last component added and the total reaction volume was 3 ml.
4
6 8 [Corbmatcj2 MxlO'
10
12
4
FIG. 2. Integrated luminescence as a function of the square of carbonate concentration. The total light emitted during the first 40 min of reaction was estimated by integrating the curves in Fig. 1. The relative areas under these curves are presented here as a function of the square of the carbonate concentration.
xanthine oxidase luminescence. It seemed important to repeat these observations because it is now known that preparations of catalase are often contaminated with superoxide dismutase (9) and that the copper- and zinc-containing superoxide dismutases can themselves react with H,O, and generate a luminescence (12). These sources of ambiguity can easily be circumvented by purifying the catalase and by using the bacterial manganese-superoxide dismutase that does not react with H,O,. When these precautions were taken, both catalase and superoxide dismutase were again found to inhibit the luminescence powerfully, in full agreement with the earlier report (3). These results are shown in Fig. 3. Inhibition Radical
20 Mlnufer
203
OXIDASE
by Scavengers of Hydroxyl
Arneson (3) had noted that xanthine oxidase emitted more light when acting on acetaldehyde than when acting on xanthine. It appeared possible that this was due to an inhibitory effect of xanthine and/or urate. Accordingly, these compounds, as well as others, which would be expected to effectively scavenge OH., were
HODGSON AND FRIDOVICH
204
way of a dimerization process and this would serve to explain the second-order dependence of the light on the carbonate concentration. The dependence upon carbonate does in any case indicate that singlet oxygen need not be considered as the emitting species in the case under consideration. It must now be asked whether other cases of weak luminescence, such as that associated with phagocytosis (20) or microsomal lipid peroxidation (21) etc., not also show a dependence upon
might
carbon-
40FIG. 3. Effects of catalase and superoxide dismutase on luminescence. Reaction mixtures contained 0.01 M acetaldehyde, 5.4 x 1OWM xanthine oxidase, 1 x 1O-4M EDTA, 0.3 M sodium carbonate buffered at pH 10.2 and approximately l-2°C. Additional components were: (1) None; (2) 420 units of catalase/ml; and (3) 3 pg of superoxide dismutase/ml. Xanthine oxidase was the last component added.
35-
tested and were found to inhibit the xanFigure 4 illusthine oxidase luminescence. trates these results. The profound inhibition caused by 0.01 mM xanthine or urate explains why Arneson (3) observed more intense luminescence when he used acetaldehyde in place of xanthine. DISCUSSION
The inhibition of luminescence by catalase, superoxide dismutase, and hydroxyl radical scavengers and the dependence upon carbonate can all be accommodated by the following reaction scheme: O,- + H,Oz -+ OH- + OH. + Oz OH, + C032- -+ OH- + CO37 2C03- + x -+ hv + products.
[Al Bl [Cl
Xanthine oxidase is known to produce O,and HzO, when acting aerobically on its substrates (13-151, and O,- and H,O, react as in [Al to yield the hydroxyl radical (1618). The hydroxyl radical reacts with carbonate to yield carbonate radicals (19), and two carbonate radicals are proposed to react to produce the light-emitting species. Indeed, Stauff et al. (7) have proposed that the carbonate radical might yield light by
OO
I 5
I IO
I 15
I 20
FIG. 4. Effect of various compounds on luminescence. Reaction mixtures contained 0.01 M acetaldehyde, 1.3 x 10e6 M xanthine oxidase, 1 x 1O-4 M EDTA and 0.05 M sodium carbonate buffered at pH 10.2 and 8°C. Additional components were: (1) None; (2) 0.01 M aside; (3) 1% ethanol; (4) 1 x 1OP M xanthine; (5) 1 X lo+ M urate; and (6) 0.05 M formate. Xanthine oxidase was the last component added.
LUMINESCENCE
AND
ate or bicarbonate. The assumption that singlet oxygen is the source of such weak luminescences must now be reexamined. REFERENCES 1. STAUFF, J., SCHMIDKUNZ, H., AND HARTMANN, G. (1963) Nature (London) 198, 281-282. 2. STAUFF, J., AND Wow, H. (196412. Naturforsch. 19B, 87-96. 3. ARNESON, R. M. (1970)Arch. Biochem. Biophys. 136, 352-360. 4. KHAN, A. U., AND KASHA, M. (1964) Nature (Lo&on) 204,241-243. 5. ARNOLD, ,J. S., BROWNE, R. J., AND OGRYZU), E. A. (1965) Photo&em. Photobiol. 4,963-969. 6. KHAN, A. U., AND KASHA, M. (1966) J. Amer. Chem. Sot. 88, 1574-1576. 7. STAUFF, (J., SANDERS, U., AND JAESCHKE, W. (1973) ,in Chemiluminescence and Bioluminescence (Cormier, M. J., Hercules, D. M., and Lee, J., eds), pp. 131-140, Plenum Press, New York. 8. WAUD, W. R., BRADY, F. O., WILEY, R. D., AND RAJAGOPALAN, K. V. (1975) Arch. Biochem. Biophys. 169, 695-701.
XANTHINE
OXIDASE
205
9. HALLIWFU, B. (1973) Biochem. J. 135,379-381. 10. BEERS, R. F., JR., AND SIZER, I. W. (1952)5. Biol. Chem. 195,133-140. 11. KEELE, B. B., MCCORD, J. M., AND FRIDOVICH, I. (1970) J. Biol. Chem. 245, 6176-6181. 12. HODGSON, E. K., AND FRIDOVICH, I. (1975) Biochemistry, in press. 13. MCCORD, J. M., AND FRIDOVICH, I. (1968)5. Biol. Chem. 243, 5753-5760. 14. MCCORD, J. M., AND FRIDOVICH, I. (1969) J. Biol. Chem. 244,6049-6055. 15. FRIDOVICH, I. (1970) J. Biol. Chem. 245, 40534057. 16. HABER, F., AND WEISS, J. (1934) Proc. Roy. Sot. Zion&n A147,332-351. 17. BEAUCHAMP, C., AND FRIDOVICH, I. (1970) J. Biol. Chem. 245, 4641-4646. 18. WALLING, C., AND GOOSEN, A. (1973), J. Amer. Chem. Sot. 95, 2987-2991. 19. BEHAR, D., CZAPSKI, G., AND DUCHOVNY, I. (1970) J. Phys. Chem. 74.2206-2210. 20. WEBB, L. S., KEELE, B. B., AND JOHNSTON, R. B. (1974) Infect. Zmmun. 9, 1051-1056. 21. NOGUCHI, T., AND NAKANO, M. (1974) Biochim. Biophys. Actu 368, 446-455.
OF BIOCHEMISTRY
The Mechanism
AND
202-205
1’72,
(1976)
of the Activity-Dependent Oxidase ’ ELLEN
Department
BIOPHYSICS
of Biochemistry,
K. HODGSON
AND
Luminescence
IRWIN
of Xanthine
FRIDOVICH
Duke University Medical Center, Durham, Received June 30, 1975
North
Carolina
27710
The weak luminescence that accompanies the aerobic xanthine oxidase reaction is inhibited by superoxide dismutase, by catalase, and by scavengers of hydroxyl radicals. It is also entirely dependent upon the presence of carbonate. It thus appears that the O,and H202 produced during the aerobic action of xanthine oxidase interact to generate OH. which, in turn, reacts with carbonate to yield the carbonate radical (CO,-). The species that is directly responsible for light emission appears to be produced by a dimerization of carbonate radicals, since the light intensity was a function of the square of the carbonate concentration. The data provide no reason to suppose that the lightemitting species is singlet oxygen.
Xanthine oxidase causes a weak luminescence while catalyzing the aerobic oxidation of its substrates (1, 2). Arneson (3) observed that this luminescence was inhibited by catalase or by superoxide dismutase and deduced that both O,- and H,O, were essential intermediates in the lightproducing process. Since it had previously been suggested (4-6) that singlet oxygen could emit in the visible by the formation of dimers, followed by pooling and simultaneous emission of excitation energy, Arneson (3) concluded that the emitting species in the xanthine oxidase reaction was singlet oxygen produced by the following reactions. O,- + H,O, OH. + O,-
+ OH-
+ OH. + 0,
+ OH- + O,*.
[Al
PI
More recently Stauff and co-workers (7) have shown that the luminescence accompanying the oxidation of H,O, by periodate, previously attributed to singlet oxygen, is actually dependent upon carbonate. They suggested that the actual emitting species was produced by a reaction involving carbonate radicals. We have reinvesti1 This work was supported in full by research grants, No. GM-10287 from the National Institutes of Health, Bethesda, Md., and No. RDRP-IP-12410-L from the United States Army Research Office, Durham, N. C. 0 1976 by Academic Press, Inc. of reproduction in any form reserved.
MATERIALS
AND
METHODS
Xanthine oxidase was prepared from cream by a procedure that avoids proteolysis (8). Catalase was from Sigma Chemical Company and was freed of contaminating superoxide dismutase (9) by repeated washing on an XMlOOA Diaflo ultrafiltration membrane obtained from the Amicon Corporation. Catalase activity was assayed at 25°C by the method of Beers and Sizer (101. The manganese-containing superoxide dismutase was prepared from Escherichia coli as previously described (11). The acetaldehyde used was freshly distilled daily. All other materials used were reagent grade. Luminescence measurements were made with either a Nuclear Chicago Mark I liquid scintillation counter or a Packard TriCarb scintillation spectrometer, Model 3003. In either case the coincidence circuit was turned off and the signal from a control vial was subtracted from the signal given by the luminescent reaction. RESULTS
The Dependence upon Carbonate When 4 x lo-' M xanthine oxidase catalyzed the oxidation 202
Copyright All rights
gated the production of light during the xanthine oxidase reaction and have demonstrated that carbonate is essential for this luminescence. The actions of catalase, superoxide dismutase and scavengers of hydroxyl radical were also reinvestigated, and the results lead to a reaction scheme that accounts for the properties of this system.
of 10 mM acetaldehyde
LUMINESCENCE
AND
at pH 10.0 the intensity of luminescence was found to increase as the concentration of the carbonate buffer was raised. This is shown in Fig. 1. The total light emitted during the first 40 min of the reaction, estimated by integrating the curves in Fig. 1, was a linear function of the square of the concentration of carbonate and it extrapolated to zero luminescence in the absence of carbonate. This is shown in Fig. 2. It follows that carbonate is an essential reactant in the process leading to the actual emitting species and that two carbonates are somehow cooperatively involved in the production of that species. The Effects of Catalase Dismutase
XANTHIIW 5
r
2
and Superoxide
It has already been reported (3) that superoxide dismutase and catalase inhibit
.
25
30
35
40
FIG. 1. Effect ofcarbonate on luminescence. Reaction mixtures contained 0.01 M acetaldehyde, 4 x lo-’ M xanthine oxidase, 1 x 10e4 M EDTA and the indicated concentrations of sodium carbonate, all buffered at pH 10.0 and at 8°C. Xanthine oxidase was the last component added and the total reaction volume was 3 ml.
4
6 8 [Corbmatcj2 MxlO'
10
12
4
FIG. 2. Integrated luminescence as a function of the square of carbonate concentration. The total light emitted during the first 40 min of reaction was estimated by integrating the curves in Fig. 1. The relative areas under these curves are presented here as a function of the square of the carbonate concentration.
xanthine oxidase luminescence. It seemed important to repeat these observations because it is now known that preparations of catalase are often contaminated with superoxide dismutase (9) and that the copper- and zinc-containing superoxide dismutases can themselves react with H,O, and generate a luminescence (12). These sources of ambiguity can easily be circumvented by purifying the catalase and by using the bacterial manganese-superoxide dismutase that does not react with H,O,. When these precautions were taken, both catalase and superoxide dismutase were again found to inhibit the luminescence powerfully, in full agreement with the earlier report (3). These results are shown in Fig. 3. Inhibition Radical
20 Mlnufer
203
OXIDASE
by Scavengers of Hydroxyl
Arneson (3) had noted that xanthine oxidase emitted more light when acting on acetaldehyde than when acting on xanthine. It appeared possible that this was due to an inhibitory effect of xanthine and/or urate. Accordingly, these compounds, as well as others, which would be expected to effectively scavenge OH., were
HODGSON AND FRIDOVICH
204
way of a dimerization process and this would serve to explain the second-order dependence of the light on the carbonate concentration. The dependence upon carbonate does in any case indicate that singlet oxygen need not be considered as the emitting species in the case under consideration. It must now be asked whether other cases of weak luminescence, such as that associated with phagocytosis (20) or microsomal lipid peroxidation (21) etc., not also show a dependence upon
might
carbon-
40FIG. 3. Effects of catalase and superoxide dismutase on luminescence. Reaction mixtures contained 0.01 M acetaldehyde, 5.4 x 1OWM xanthine oxidase, 1 x 1O-4M EDTA, 0.3 M sodium carbonate buffered at pH 10.2 and approximately l-2°C. Additional components were: (1) None; (2) 420 units of catalase/ml; and (3) 3 pg of superoxide dismutase/ml. Xanthine oxidase was the last component added.
35-
tested and were found to inhibit the xanFigure 4 illusthine oxidase luminescence. trates these results. The profound inhibition caused by 0.01 mM xanthine or urate explains why Arneson (3) observed more intense luminescence when he used acetaldehyde in place of xanthine. DISCUSSION
The inhibition of luminescence by catalase, superoxide dismutase, and hydroxyl radical scavengers and the dependence upon carbonate can all be accommodated by the following reaction scheme: O,- + H,Oz -+ OH- + OH. + Oz OH, + C032- -+ OH- + CO37 2C03- + x -+ hv + products.
[Al Bl [Cl
Xanthine oxidase is known to produce O,and HzO, when acting aerobically on its substrates (13-151, and O,- and H,O, react as in [Al to yield the hydroxyl radical (1618). The hydroxyl radical reacts with carbonate to yield carbonate radicals (19), and two carbonate radicals are proposed to react to produce the light-emitting species. Indeed, Stauff et al. (7) have proposed that the carbonate radical might yield light by
OO
I 5
I IO
I 15
I 20
FIG. 4. Effect of various compounds on luminescence. Reaction mixtures contained 0.01 M acetaldehyde, 1.3 x 10e6 M xanthine oxidase, 1 x 1O-4 M EDTA and 0.05 M sodium carbonate buffered at pH 10.2 and 8°C. Additional components were: (1) None; (2) 0.01 M aside; (3) 1% ethanol; (4) 1 x 1OP M xanthine; (5) 1 X lo+ M urate; and (6) 0.05 M formate. Xanthine oxidase was the last component added.
LUMINESCENCE
AND
ate or bicarbonate. The assumption that singlet oxygen is the source of such weak luminescences must now be reexamined. REFERENCES 1. STAUFF, J., SCHMIDKUNZ, H., AND HARTMANN, G. (1963) Nature (London) 198, 281-282. 2. STAUFF, J., AND Wow, H. (196412. Naturforsch. 19B, 87-96. 3. ARNESON, R. M. (1970)Arch. Biochem. Biophys. 136, 352-360. 4. KHAN, A. U., AND KASHA, M. (1964) Nature (Lo&on) 204,241-243. 5. ARNOLD, ,J. S., BROWNE, R. J., AND OGRYZU), E. A. (1965) Photo&em. Photobiol. 4,963-969. 6. KHAN, A. U., AND KASHA, M. (1966) J. Amer. Chem. Sot. 88, 1574-1576. 7. STAUFF, (J., SANDERS, U., AND JAESCHKE, W. (1973) ,in Chemiluminescence and Bioluminescence (Cormier, M. J., Hercules, D. M., and Lee, J., eds), pp. 131-140, Plenum Press, New York. 8. WAUD, W. R., BRADY, F. O., WILEY, R. D., AND RAJAGOPALAN, K. V. (1975) Arch. Biochem. Biophys. 169, 695-701.
XANTHINE
OXIDASE
205
9. HALLIWFU, B. (1973) Biochem. J. 135,379-381. 10. BEERS, R. F., JR., AND SIZER, I. W. (1952)5. Biol. Chem. 195,133-140. 11. KEELE, B. B., MCCORD, J. M., AND FRIDOVICH, I. (1970) J. Biol. Chem. 245, 6176-6181. 12. HODGSON, E. K., AND FRIDOVICH, I. (1975) Biochemistry, in press. 13. MCCORD, J. M., AND FRIDOVICH, I. (1968)5. Biol. Chem. 243, 5753-5760. 14. MCCORD, J. M., AND FRIDOVICH, I. (1969) J. Biol. Chem. 244,6049-6055. 15. FRIDOVICH, I. (1970) J. Biol. Chem. 245, 40534057. 16. HABER, F., AND WEISS, J. (1934) Proc. Roy. Sot. Zion&n A147,332-351. 17. BEAUCHAMP, C., AND FRIDOVICH, I. (1970) J. Biol. Chem. 245, 4641-4646. 18. WALLING, C., AND GOOSEN, A. (1973), J. Amer. Chem. Sot. 95, 2987-2991. 19. BEHAR, D., CZAPSKI, G., AND DUCHOVNY, I. (1970) J. Phys. Chem. 74.2206-2210. 20. WEBB, L. S., KEELE, B. B., AND JOHNSTON, R. B. (1974) Infect. Zmmun. 9, 1051-1056. 21. NOGUCHI, T., AND NAKANO, M. (1974) Biochim. Biophys. Actu 368, 446-455.
