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Lucigenin (Bis-N-methylacridinium) as a Mediator of Superoxide Anion Production
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 337, No. 1, January 1, pp. 115–120, 1997 Article No. BB979766
Lucigenin (Bis-N-methylacridinium) as a Mediator of Superoxide Anion Production1 Stefan I. Liochev and Irwin Fridovich2 Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
Received July 8, 1996, and in revised form October 14, 1996
2/
Lucigenin (bis-N-methylacridinium) (Luc ) has frequently been used for the luminescent detection of O0 2 . In fact, the pathway leading to this luminescence requires univalent reduction of Luc2/ to LucH•/ followed by formation of an unstable dioxetane by reac•/ tion of LucH•/ with O0 2 . It is now shown that LucH 2/ rapidly autooxidizes and so produces O0 . Luc can 2 thus mediate O0 2 production in systems, such as glucose plus glucose oxidase, in which there is ordinarily 2/ no O0 luminescence can thus be 2 production. Luc used as the basis for assaying superoxide dismutase activity but should not be used for measuring, or even detecting, O0 q 1997 Academic Press, Inc. 2 .
The nature of the process leading to Luc2/ luminescence suggests that it could not be used as a reliable measure of O20 concentration, because reduction of Luc2/ to LucH•/ is also a prerequisite for luminescence. Nevertheless, the desire for a convenient measure of O20 , which could be applied to biological systems, has led to frequent use of Luc2/ for this purpose (6–20). A thorough study of the behavior of Luc2/ in enzymic and biological systems appeared both timely and desirable in order to forestall further misuse of this method. We now report results indicating that Luc2/, like paraquat, can mediate the production of O20 and thus its luminescence is not a reliable indicator of the presence of O20 . MATERIALS AND METHODS
The xanthine oxidase reaction was seen to elicit the luminescence of Luc2/,3 over 35 years ago and a role for undefined free radicals was postulated (1, 2). Further study of this process exposed the likely involvement of both LucH•/ and O20 in the luminescent process (3). More recently the chemistry of Luc2/ luminescence was reviewed and the process stated to depend upon reaction of LucH•/ with O20 to yield a dioxetane, whose decomposition yields the electronically excited Nmethyl acridone whose return to ground state is accompanied by emission of light (4). This scheme is in full accord with the results of early electrochemical investigations (5). 1 This work was supported by grants from the Council for Tobacco Research, U.S.A., Inc. (2871AR2), the U.S. Army Medical Research (Contract DAMD17-95-C-5065), and the National Institutes of Health (PO1 HL31992-11A1). 2 To whom correspondence should be addressed. Fax: (919) 6848885. 3 Abbreviations used: XO, xanthine oxidase; Luc2/, lucigenin; LucH•/, univalently reduced lucigenin; LucH2 , divalently reduced lucigenin; SOD, superoxide dismutase; DMSO, dimethyl sulfoxide; PQ2/, paraquat; NBT, nitroblue tetrazolium.
Ferricytochrome c (type III), paraquat, NADH (grade III), xanthine, lipoamide dehydrogenase from torula yeast (type IV), and glucose oxidase from Aspergillus niger were from Sigma. Glucose oxidase (grade I) and catalase were from Boehringer-Mannheim; glucose and H2O2 were from Mallinkrodt; Luc2/ was from Aldrich; 18-Crown6 was from Kodak; Cu,ZnSOD was from Diagnostic Data Inc.; KO2 was from Alfa; and both sodium vanadate and dimethyl sulfoxide were from Fisher. Xanthine oxidase from bovine cream (21) was prepared by R. D. Wiley. Light emission was followed with a Turner Designs Model 20E luminometer set to a delay time of 5 s and integrate times of 10 s. A standard 14C source of 1.22 1 105 dpm, in scintillation fluid, gave a 10-s integral of luminescence of between 140 and 148 over the course of this work. All measurements were at room temperature (Ç237C). One unit of XO is that amount per 1 ml which generated 1 mM O0 2 /min under the conditions of the SOD assay (22). Anaerobic reactions were followed in a cuvette which permitted sweeping the reaction mixture with N2 (23). When KO2 was used as a source of O0 2 , it was dissolved to 50 mM in dry DMSO containing 100 mM 18-Crown-6. This stock was diluted, immediately prior to use, with dry DMSO, to make a working solution, which was injected into the buffered solution of Luc2/ in the luminometer.
RESULTS
Enzymatic reduction of Luc2/. The reduction of Luc2/ by XO plus NADH can be followed at 367 nm, at which wavelength the NADH makes only a small 115
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FIG. 2. Effect of Luc2/ on the oxidation of NADH. Reaction mixtures contained 50 mM NADH in 50 mM sodium phosphate buffer at pH 7.3. Reactions were initiated with 25 U/ml of XO. Where indicated Luc2/ was added to 20 mM, vanadate to 0.4 mM, and Cu,ZnSOD to 5 mg/ml.
FIG. 1. The reduction of Luc2/ by XO / NADH. Reaction mixtures contained 20 mM NADH and 20 mM Luc2/ in 50 mM potassium phosphate, pH 7.8. Reactions were started by adding 100 U/ml of XO. Line 1, aerobic; line 2, anaerobic.
contribution to the absorbance. Line 1 in Fig. 1 demonstrates only a slow decrease of A367 nm under aerobic conditions and most of that can be attributed to NADH oxidation, while line 2 shows a rapid and accelerating reduction of Luc2/ under anaerobic conditions. The acceleration shown by line 2 undoubtedly corresponds with the exhaustion of residual O2 . A similar result was reported earlier in which case xanthine was the reducing substrate (3) and it was noted that LucH2 did not readily autooxidize. We can conclude that XO can reduce Luc2/ univalently and that LucH•/ readily autooxidizes, in which case it must reduce O2 to O20 . That Luc2/ does mediate O20 production in the XO plus NADH reaction mixture was shown in two ways. One of these exploited the ability of O20 / vanadate to initiate a free radical chain oxidation of NADH (24– 28) and the other the ability of O20 to reduce cytochrome c (22, 29). Line 1 in Fig. 2 demonstrates that the rate of NADH oxidation by XO was increased by Luc2/ and that subsequent addition of vanadate caused a further marked increase, which was obliterated by SOD. In line 2 the Luc2/ was added after the vanadate and, as expected, vanadate speeded the oxidation of NADH by XO and Luc2/ caused a further augmentation. Luc2/
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also augmented the reduction of cytochrome c by XO plus NADH, but this reduction was only partially inhibitable by SOD. Figure 3 demonstrates that the susceptibility to inhibition by SOD increased as the con-
FIG. 3. Reduction of cytochrome c by XO in the presence of Luc2/. Reaction mixtures contained 0.15 mM NADH, 33 mM Luc2/, the indicated concentration of cytochrome c, and 8.3 U/ml of XO in 50 mM potassium phosphate buffer at pH 7.8. Line 1, the rate of reduction of cytochrome c; line 2, the percentage inhibition caused by 5 mg/ml of SOD.
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FIG. 4. The reduction of cytochrome c by glucose oxidase. Reaction mixtures contained 1.0 mM glucose and 10 mM cytochrome c in 50 mM Tris–chloride at pH 9.5. Line 1, reaction started with 0.25 mg/ ml glucose oxidase (Boehringer) and 10 mg/ml SOD added at the arrow; line 2, reaction started with 0.10 mg/ml glucose oxidase and 20 mM Luc2/ added at the arrow.
centration of cytochrome c decreased. This can be understood on the basis of a competition between cytochrome c and O2 for reaction with LucH•/. At high cytochrome c direct reduction of cytochrome c by LucH•/ predominates, while at low cytochrome c autooxidation of LucH•/ with production of O20 becomes more important and then much of the cytochrome c reduction is due to O20 . It can be estimated that the rate constant for the direct reduction of cytochrome c by LucH•/ is 50- to 60-fold greater than for the reduction by O2 by LucH•/. Lipoamide dehydrogenase has been reported to produce O20 , as deduced from the SOD-inhibitable reduction of cytochrome c (30) and by spin trapping (31). Since this enzyme, in the presence of NADH plus O2 , also caused the luminescence of Luc2/ (30), it appeared likely that it could also catalyze the reduction of Luc2/ to LucH•/. In that case Luc2/ should increase the rate of reduction of cytochrome c and this reduction should be inhibitable by SOD to an extent which became greater as the concentration of cytochrome c diminished. This was the case with lipoamide dehydrogenase (data not shown). Luminescence elicited by glucose oxidase. Given that Luc2/ can be univalently reduced and can then give rise to O20 by autooxidation, it follows that any enzyme capable of reducing Luc2/ to LucH•/ should elicit an SOD-inhibitable luminescence. This would be the case even if there were no production of O20 by the enzyme when Luc2/ was not present. Glucose oxidase has been shown not to produce O20 during the oxidation of glucose (32, 33) and it is capable of directly reducing NBT2/ (33). Figure 4 (line 1) demonstrates that glucose
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oxidase plus glucose is able to reduce cytochrome c and that this inhibition was not inhibitable by SOD, while line 2 shows that Luc2/ speeds the reduction of cytochrome c by this system. This indicates that this enzyme too can reduce Luc2/ to LucH•/ which then reduces cytochrome c. The reduction of Luc2/ to LucH2 by glucose oxidase could also be followed at 367 nm when O2 was excluded (data not shown). That glucose oxidase plus glucose can elicit the luminescence of Luc2/ is shown by line 1 in Fig. 5, while line 2 shows that this luminescence was largely inhibitable by SOD. It should be noted that SOD did not inhibit completely and moreover that luminescence in the presence of SOD increased with time. H2O2 would be expected to accumulate in these reaction mixtures and it can act as both an oxidant and a reductant, the latter action being more pronounced at elevated pH. Indeed, we observed that H2O2 per se elicited the chemiluminescence of lucigenin at pH 9.5 but not at pH 7 (data not shown). A similar effect has been previously reported (34). It should be noted that commercial sources of glucose oxidase may be contaminated with SOD. Thus, we noted that a sample purchased from Sigma contained 6.5 units (22) of SOD per milligram of protein and since
FIG. 5. Luc2/ luminescence elicited by the glucose oxidase reaction. Reaction mixtures contained 0.75 mM glucose, 0.25 mg/ml glucose oxidase (Boehringer), and 0.1 mM Luc2/ in 50 mM Tris–chloride at pH 9.5. Line 1, no SOD; line 2, 10 mg/ml SOD present.
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reduced by XO. However, the relative reactivities of PQ•/ and LucH•/ with O2 and cytochrome c differ. Thus, PQ•/ reduces O2 much more rapidly than cytochrome c, with the consequence that the reduction of cytochrome c by the XO reaction remains inhibitable by SOD in the presence of PQ2/. In contrast, LucH•/ reduces cytochrome c more rapidly than O2 , with the consequence that cytochrome c reduction by the XO reaction is made relatively insensitive to SOD by the presence of Luc2/. Since Luc2/ and PQ2/ must be in competition for reduction by xanthine oxidase, we can expect that PQ2/ will make cytochrome c reduction in the presence of Luc2/ more sensitive to inhibition by SOD. Figure 7 demonstrates this effect. Thus, line 1 shows that Luc2/ speeds cytochrome c reduction by xanthine oxidase plus NADH and that the inhibitory effect of SOD was increased by subsequent additions of PQ2/. In line 2 the order of addition is different and PQ2/ added after Luc2/ is seen to further enhance the rate of cytochrome c reduction, which is then sensitive to inhibition by SOD. DISCUSSION FIG. 6. Luc2/ luminescence elicited by KO2 . 0.2 ml of 0.5 mM KO2 , 1.0 mM 18-Crown-6 in dry DMSO was injected into 0.2 ml of 0.2 mM Luc2/ in 50 mM Tris–Cl. Line 1, no SOD; line 2, 10 mg/ml SOD present in the Tris–Cl.
most of this was inhibitable by 1.0 mM CN0 it was probably due to a Cu,ZnSOD. Boehringer glucose oxidase was much purer, having a higher glucose oxidase activity and only Ç0.5 U/mg of SOD. Can O20 reduce Luc2/? The foregoing demonstrates that several enzymes, and even H2O2 at pH 9.5, can reduce Luc2/ to LucH•/; that LucH•/ can autooxidize with the formation of O20 ; and that SOD-inhibitable luminescence will then be observed. It remained to be seen whether O20 per se can reduce Luc2/. This was tested by rapidly mixing 0.25 mM KO2 , solubilized in dry DMSO, with 0.5 mM 18-Crown-6 (35), with Luc2/ buffered at pH 7.8. Line 1 in Fig. 6 demonstrates that this admixture was followed by an intense and rapidly declining luminescence, while line 2 shows the profound inhibition caused by SOD. This indicates that O20 can reduce Luc2/, in agreement with the results of earlier electrochemical studies (5). There was a relatively small luminescence that decayed slowly and that was due to H2O2 produced by the spontaneous dismutation of O20 . This H2O2-dependent luminescence at pH 7.8 was inhibited by catalase and was largely dependent on the simultaneous presence of DMSO. Luc2/ and paraquat: Similarities and differences. Both paraquat (PQ2/) and Luc2/ can be univalently
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Although the luminescence of Luc2/ depends upon O and thus is inhibitable by SOD, it cannot be considered a measure of O20 . This the case because Luc2/ can be reduced univalently by diverse enzymes including XO, glucose oxidase, dihydrolipoamide dehydrogenase, liver aldehyde oxidase (3), and bacterial dehydroorotate dehydrogenase (3). The LucH•/ so produced then 0 2
FIG. 7. Reduction of cytochrome c by XO: competition between Luc2/ and PQ2/. Reaction mixtures contained 10 mM cytochrome c, 0.1 mM NADH, and 15 U/ml of XO. Where indicated additional components were added as follows: Luc2/ to 40 mM, SOD to 10 mg/ml, and PQ2/ as indicated.
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reduces O2 to O20 which can react with LucH•/ to give the dioxetane, whose decomposition is responsible for luminescence (4). H2O2 can weakly elicit Luc2/ luminescence at pH 9.5 and this probably depends upon HO20 acting as a reductant for Luc2/. When that happens the immediate products are LucH•/ and O20 , the prerequisites for luminescence. At pH 7.8 H2O2 did not elicit Luc2/ luminescence unless DMSO was present and we can propose that the Luc2/ reductants generated in that case were derived from the oxidation of DMSO by H2O2 . It should be noted that dilution of the Luc2/ in phosphate buffer with an equal volume of DMSO caused an increase in pH from 7.8 to 8.3 due to the effect of diminished dielectric constant on the pKa of the phosphate buffer. Raising the pH by this amount in the absence of DMSO did not cause significant luminescence from the H2O2 plus Luc2/ mixture. We have seen that KO2 per se causes the luminescence of Luc2/, which implies that O20 can reduce Luc2/. This is, in effect, the reverse of the autooxidation of LucH•/. However, in enzymatic systems, this does not happen to a detectable degree. Thus, the reduction of Luc2/ to LucH2 by XO plus xanthine (3), or by XO plus NADH (this study), could be seen anaerobically but not aerobically. This implies that the autooxidation of LucH•0 was effectively irreversible. The following scheme of reactions serves to clarify the reactions of Luc2/. Luc2/ / EnzH2 r EnzH• / LucH•/ LucH•/ / EnzH• r LucH2 / Enz LucH•/ / LucH•/ r LucH2 / Luc2/ LucH•/ / Cyt c3/ r Luc2/ / H/ / Cyt c2/ LucH•/ / O2 r Luc2/ / O20 / H/ O uw f. LucH•/ / O20 r Luc{O r light / acridone g. O20 / O20 / 2H/ r H2O2 / O2 h. O20 / Cyt c3/ r O2 / Cyt c2/ a. b. c. d. e.
When O2 was present, reactions e together with f would outcompete reactions b and c and would prevent net reduction of Luc2/ to LucH2 . The inhibition of Luc2/ luminescence is a more sensitive assay of SOD activity than is the inhibition of cytochrome c reduction (36). This indicates that the reaction of O20 with cytochrome c (h) must be faster than its reaction with LucH•/ (f), under the specified conditions of the two assays. In contrast, the reduction of cytochrome c by LucH•/ (d) is faster than the autooxidation of LucH•/ (e), which explains why cytochrome c reduction in the XO / NADH / Luc2/ mixture was relatively insensitive to inhibition by SOD. Luc2/, like PQ2/, and NBT2/ (33, 37–39) can undergo
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a cycle of univalent reduction followed by autooxidation. These compounds thus share the capacity to mediate the production of O20 , even when that radical would not have been made in their absence. They can thus serve as the basis of assays for SOD, but cannot be used to measure, or even to reliably detect, O20 . REFERENCES 1. Totter, J R., Medina, V. J., and Scoseria, J. L. (1960) J. Biol. Chem. 235, 238–241. 2. Totter, J. R., DeDugros, E. C., and Riveiro, C. (1960) J. Biol. Chem. 235, 1839–1842. 3. Greenlee, L., Fridovich, I., and Handler, P. (1962) Biochemistry 1, 779–783. 4. Faulkner, K., and Fridovich, I. (1993) Free Radical Biol. Med. 15, 447–451. 5. Legg, K. D., and Hercules, D. M. (1969) J. Am. Chem. Soc. 91, 1902–1907. 6. Minkenberg, I., and Ferber, E. (1984) J. Immunol. Methods 71, 61–67. 7. Stevens, P., and Hong, D. (1984) Microchem. J. 30, 135–146. 8. Afanas’ev, I. B., Dorozhko, A. I., Polozova, N. I., Kuprianova, N. S., Brodskii, A. V., Ostrachovitch, E. A., and Korkina, L. G. (1993) Arch. Biochem. Biophys. 320, 200–205. 9. Allen, R. C. (1986) Methods Enzymol. 133, 449–493. 10. Mer’etey, K., Antal, M., Rozsnyay, Z., Bo¨hm, U., Elekes, E., and Genti, G. (1987) Inflammation 11, 417–425. 11. Gyllenhammar, H. (1987) J. Immunol. Methods 97, 209–213. 12. Storch, J., and Ferber, E. (1988) Anal. Biochem. 169, 262–276. 13. Grunfeld, S., Hamilton, C. A., Mesaros, S., McClain, S. W., Dominiczak, A. F., Bohr, D. F., and Malinski, T. (1995) Hypertension 26, 854–857. 14. Okuda, M., Lee, H. C., and Chance, B. (1992) Circ. Shock 38, 228–237. 15. Samuni, A., Krishna, C. M., Cook, J., Black, C. D. V., and Russo, A. (1991) Free Radical Biol. Med. 10, 305–313. 16. Okuda, M., Lee, H.-C., Chance, B., and Kumar, C. (1992) Free Radical Biol. Med. 12, 271–279. 17. Tosi, M. F., and Hamedaru, A. (1992) Am. J. Clin. Pathol. 97, 566–573. 18. Pagano, P. T., Ito, Y., Tomheim, K., Callop, P. M., Tauber, A. I., and Cohen, R. A. (1995) Am. J. Physiol. 268, H2274–H2280. 19. Carceni, P., Ryu, H. S., van Thiel, D. H., and Borle, A. B. (1995) Biochim. Biophys. Acta 1268, 249–254. 20. Scott, M. D., and Eaton, J. W. (1996) Redox Reports 2, 113–119. 21. Waud, W. R., Brady, F. O., Wiley, R. D., and Rajagopalan, K. V. (1975) Arch. Biochem. Biophys. 169, 699-701. 22. McCord, J. M., and Fridovich, I. (1969) J. Biol. Chem. 244, 6049– 6055. 23. Hodgson, E. K., McCord, J. M., and Fridovich, I. (1973) Anal. Biochem. 51, 470–473. 24. Darr, D., and Fridovich, I. (1984) Arch. Biochem Biophys. 232, 562–565. 25. Liochev, S., and Fridovich, I. (1986) Free Radical Biol. Med. 1, 287–292. 26. Liochev, S., and Fridovich, I. (1986) Arch. Biochem. Biophys. 250, 139–145.
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27. Liochev, I., and Fridovich, I. (1988) Arch. Biochem. Biophys. 263, 299–304. 28. Liochev, S. I., and Fridovich, I. (1990) Arch. Biochem. Biophys. 279, 1–7. 29. McCord, J. M., and Fridovich, I. (1968) J. Biol. Chem. 243, 5713– 5760. 30. Grinblat, L., Sreider, C. M., and Stoppani, A. O. (1991) Biochem. Int. 23, 83–92. 31. Bando, Y., and Aki, K. (1991) J. Biochem. (Tokyo) 109, 450–454. 32. Massey, V., Strickland, S., Mayhew, S. G., Howell, L. G., Engel, P. C., Mathews, R. G., Schuman, M., and Sullivan, P. A. (1969) Biochem. Biophys. Res. Commun. 36, 891–897.
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33. Liochev, S. I., and Fridovich, I. (1995) Arch. Biochem. Biophys. 318, 408–410. 34. Gleu, K., and Petsch, W. (1935) Angew. Chem. 48, 57–59. 35. Valentine, J. S., and Cortis, A. B. (1975) J. Am. Chem. Soc. 97, 224–226. 36. Corbisier, P., Houbion, A., and Remacle, J. (1987) Anal. Biochem. 164, 240–247. 37. Epel, B. L., and Neuman, J. (1973) Biochim. Biophys. Acta 325, 520–529. 38. Auclair, C., Torres, M., and Hakim, J. (1978) FEBS Lett. 89, 26– 28. 39. Picker, S. D., and Fridovich, I. (1984) Arch. Biochem. Biophys. 228, 155–158.
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Vol. 337, No. 1, January 1, pp. 115–120, 1997 Article No. BB979766
Lucigenin (Bis-N-methylacridinium) as a Mediator of Superoxide Anion Production1 Stefan I. Liochev and Irwin Fridovich2 Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
Received July 8, 1996, and in revised form October 14, 1996
2/
Lucigenin (bis-N-methylacridinium) (Luc ) has frequently been used for the luminescent detection of O0 2 . In fact, the pathway leading to this luminescence requires univalent reduction of Luc2/ to LucH•/ followed by formation of an unstable dioxetane by reac•/ tion of LucH•/ with O0 2 . It is now shown that LucH 2/ rapidly autooxidizes and so produces O0 . Luc can 2 thus mediate O0 2 production in systems, such as glucose plus glucose oxidase, in which there is ordinarily 2/ no O0 luminescence can thus be 2 production. Luc used as the basis for assaying superoxide dismutase activity but should not be used for measuring, or even detecting, O0 q 1997 Academic Press, Inc. 2 .
The nature of the process leading to Luc2/ luminescence suggests that it could not be used as a reliable measure of O20 concentration, because reduction of Luc2/ to LucH•/ is also a prerequisite for luminescence. Nevertheless, the desire for a convenient measure of O20 , which could be applied to biological systems, has led to frequent use of Luc2/ for this purpose (6–20). A thorough study of the behavior of Luc2/ in enzymic and biological systems appeared both timely and desirable in order to forestall further misuse of this method. We now report results indicating that Luc2/, like paraquat, can mediate the production of O20 and thus its luminescence is not a reliable indicator of the presence of O20 . MATERIALS AND METHODS
The xanthine oxidase reaction was seen to elicit the luminescence of Luc2/,3 over 35 years ago and a role for undefined free radicals was postulated (1, 2). Further study of this process exposed the likely involvement of both LucH•/ and O20 in the luminescent process (3). More recently the chemistry of Luc2/ luminescence was reviewed and the process stated to depend upon reaction of LucH•/ with O20 to yield a dioxetane, whose decomposition yields the electronically excited Nmethyl acridone whose return to ground state is accompanied by emission of light (4). This scheme is in full accord with the results of early electrochemical investigations (5). 1 This work was supported by grants from the Council for Tobacco Research, U.S.A., Inc. (2871AR2), the U.S. Army Medical Research (Contract DAMD17-95-C-5065), and the National Institutes of Health (PO1 HL31992-11A1). 2 To whom correspondence should be addressed. Fax: (919) 6848885. 3 Abbreviations used: XO, xanthine oxidase; Luc2/, lucigenin; LucH•/, univalently reduced lucigenin; LucH2 , divalently reduced lucigenin; SOD, superoxide dismutase; DMSO, dimethyl sulfoxide; PQ2/, paraquat; NBT, nitroblue tetrazolium.
Ferricytochrome c (type III), paraquat, NADH (grade III), xanthine, lipoamide dehydrogenase from torula yeast (type IV), and glucose oxidase from Aspergillus niger were from Sigma. Glucose oxidase (grade I) and catalase were from Boehringer-Mannheim; glucose and H2O2 were from Mallinkrodt; Luc2/ was from Aldrich; 18-Crown6 was from Kodak; Cu,ZnSOD was from Diagnostic Data Inc.; KO2 was from Alfa; and both sodium vanadate and dimethyl sulfoxide were from Fisher. Xanthine oxidase from bovine cream (21) was prepared by R. D. Wiley. Light emission was followed with a Turner Designs Model 20E luminometer set to a delay time of 5 s and integrate times of 10 s. A standard 14C source of 1.22 1 105 dpm, in scintillation fluid, gave a 10-s integral of luminescence of between 140 and 148 over the course of this work. All measurements were at room temperature (Ç237C). One unit of XO is that amount per 1 ml which generated 1 mM O0 2 /min under the conditions of the SOD assay (22). Anaerobic reactions were followed in a cuvette which permitted sweeping the reaction mixture with N2 (23). When KO2 was used as a source of O0 2 , it was dissolved to 50 mM in dry DMSO containing 100 mM 18-Crown-6. This stock was diluted, immediately prior to use, with dry DMSO, to make a working solution, which was injected into the buffered solution of Luc2/ in the luminometer.
RESULTS
Enzymatic reduction of Luc2/. The reduction of Luc2/ by XO plus NADH can be followed at 367 nm, at which wavelength the NADH makes only a small 115
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FIG. 2. Effect of Luc2/ on the oxidation of NADH. Reaction mixtures contained 50 mM NADH in 50 mM sodium phosphate buffer at pH 7.3. Reactions were initiated with 25 U/ml of XO. Where indicated Luc2/ was added to 20 mM, vanadate to 0.4 mM, and Cu,ZnSOD to 5 mg/ml.
FIG. 1. The reduction of Luc2/ by XO / NADH. Reaction mixtures contained 20 mM NADH and 20 mM Luc2/ in 50 mM potassium phosphate, pH 7.8. Reactions were started by adding 100 U/ml of XO. Line 1, aerobic; line 2, anaerobic.
contribution to the absorbance. Line 1 in Fig. 1 demonstrates only a slow decrease of A367 nm under aerobic conditions and most of that can be attributed to NADH oxidation, while line 2 shows a rapid and accelerating reduction of Luc2/ under anaerobic conditions. The acceleration shown by line 2 undoubtedly corresponds with the exhaustion of residual O2 . A similar result was reported earlier in which case xanthine was the reducing substrate (3) and it was noted that LucH2 did not readily autooxidize. We can conclude that XO can reduce Luc2/ univalently and that LucH•/ readily autooxidizes, in which case it must reduce O2 to O20 . That Luc2/ does mediate O20 production in the XO plus NADH reaction mixture was shown in two ways. One of these exploited the ability of O20 / vanadate to initiate a free radical chain oxidation of NADH (24– 28) and the other the ability of O20 to reduce cytochrome c (22, 29). Line 1 in Fig. 2 demonstrates that the rate of NADH oxidation by XO was increased by Luc2/ and that subsequent addition of vanadate caused a further marked increase, which was obliterated by SOD. In line 2 the Luc2/ was added after the vanadate and, as expected, vanadate speeded the oxidation of NADH by XO and Luc2/ caused a further augmentation. Luc2/
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also augmented the reduction of cytochrome c by XO plus NADH, but this reduction was only partially inhibitable by SOD. Figure 3 demonstrates that the susceptibility to inhibition by SOD increased as the con-
FIG. 3. Reduction of cytochrome c by XO in the presence of Luc2/. Reaction mixtures contained 0.15 mM NADH, 33 mM Luc2/, the indicated concentration of cytochrome c, and 8.3 U/ml of XO in 50 mM potassium phosphate buffer at pH 7.8. Line 1, the rate of reduction of cytochrome c; line 2, the percentage inhibition caused by 5 mg/ml of SOD.
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FIG. 4. The reduction of cytochrome c by glucose oxidase. Reaction mixtures contained 1.0 mM glucose and 10 mM cytochrome c in 50 mM Tris–chloride at pH 9.5. Line 1, reaction started with 0.25 mg/ ml glucose oxidase (Boehringer) and 10 mg/ml SOD added at the arrow; line 2, reaction started with 0.10 mg/ml glucose oxidase and 20 mM Luc2/ added at the arrow.
centration of cytochrome c decreased. This can be understood on the basis of a competition between cytochrome c and O2 for reaction with LucH•/. At high cytochrome c direct reduction of cytochrome c by LucH•/ predominates, while at low cytochrome c autooxidation of LucH•/ with production of O20 becomes more important and then much of the cytochrome c reduction is due to O20 . It can be estimated that the rate constant for the direct reduction of cytochrome c by LucH•/ is 50- to 60-fold greater than for the reduction by O2 by LucH•/. Lipoamide dehydrogenase has been reported to produce O20 , as deduced from the SOD-inhibitable reduction of cytochrome c (30) and by spin trapping (31). Since this enzyme, in the presence of NADH plus O2 , also caused the luminescence of Luc2/ (30), it appeared likely that it could also catalyze the reduction of Luc2/ to LucH•/. In that case Luc2/ should increase the rate of reduction of cytochrome c and this reduction should be inhibitable by SOD to an extent which became greater as the concentration of cytochrome c diminished. This was the case with lipoamide dehydrogenase (data not shown). Luminescence elicited by glucose oxidase. Given that Luc2/ can be univalently reduced and can then give rise to O20 by autooxidation, it follows that any enzyme capable of reducing Luc2/ to LucH•/ should elicit an SOD-inhibitable luminescence. This would be the case even if there were no production of O20 by the enzyme when Luc2/ was not present. Glucose oxidase has been shown not to produce O20 during the oxidation of glucose (32, 33) and it is capable of directly reducing NBT2/ (33). Figure 4 (line 1) demonstrates that glucose
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oxidase plus glucose is able to reduce cytochrome c and that this inhibition was not inhibitable by SOD, while line 2 shows that Luc2/ speeds the reduction of cytochrome c by this system. This indicates that this enzyme too can reduce Luc2/ to LucH•/ which then reduces cytochrome c. The reduction of Luc2/ to LucH2 by glucose oxidase could also be followed at 367 nm when O2 was excluded (data not shown). That glucose oxidase plus glucose can elicit the luminescence of Luc2/ is shown by line 1 in Fig. 5, while line 2 shows that this luminescence was largely inhibitable by SOD. It should be noted that SOD did not inhibit completely and moreover that luminescence in the presence of SOD increased with time. H2O2 would be expected to accumulate in these reaction mixtures and it can act as both an oxidant and a reductant, the latter action being more pronounced at elevated pH. Indeed, we observed that H2O2 per se elicited the chemiluminescence of lucigenin at pH 9.5 but not at pH 7 (data not shown). A similar effect has been previously reported (34). It should be noted that commercial sources of glucose oxidase may be contaminated with SOD. Thus, we noted that a sample purchased from Sigma contained 6.5 units (22) of SOD per milligram of protein and since
FIG. 5. Luc2/ luminescence elicited by the glucose oxidase reaction. Reaction mixtures contained 0.75 mM glucose, 0.25 mg/ml glucose oxidase (Boehringer), and 0.1 mM Luc2/ in 50 mM Tris–chloride at pH 9.5. Line 1, no SOD; line 2, 10 mg/ml SOD present.
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reduced by XO. However, the relative reactivities of PQ•/ and LucH•/ with O2 and cytochrome c differ. Thus, PQ•/ reduces O2 much more rapidly than cytochrome c, with the consequence that the reduction of cytochrome c by the XO reaction remains inhibitable by SOD in the presence of PQ2/. In contrast, LucH•/ reduces cytochrome c more rapidly than O2 , with the consequence that cytochrome c reduction by the XO reaction is made relatively insensitive to SOD by the presence of Luc2/. Since Luc2/ and PQ2/ must be in competition for reduction by xanthine oxidase, we can expect that PQ2/ will make cytochrome c reduction in the presence of Luc2/ more sensitive to inhibition by SOD. Figure 7 demonstrates this effect. Thus, line 1 shows that Luc2/ speeds cytochrome c reduction by xanthine oxidase plus NADH and that the inhibitory effect of SOD was increased by subsequent additions of PQ2/. In line 2 the order of addition is different and PQ2/ added after Luc2/ is seen to further enhance the rate of cytochrome c reduction, which is then sensitive to inhibition by SOD. DISCUSSION FIG. 6. Luc2/ luminescence elicited by KO2 . 0.2 ml of 0.5 mM KO2 , 1.0 mM 18-Crown-6 in dry DMSO was injected into 0.2 ml of 0.2 mM Luc2/ in 50 mM Tris–Cl. Line 1, no SOD; line 2, 10 mg/ml SOD present in the Tris–Cl.
most of this was inhibitable by 1.0 mM CN0 it was probably due to a Cu,ZnSOD. Boehringer glucose oxidase was much purer, having a higher glucose oxidase activity and only Ç0.5 U/mg of SOD. Can O20 reduce Luc2/? The foregoing demonstrates that several enzymes, and even H2O2 at pH 9.5, can reduce Luc2/ to LucH•/; that LucH•/ can autooxidize with the formation of O20 ; and that SOD-inhibitable luminescence will then be observed. It remained to be seen whether O20 per se can reduce Luc2/. This was tested by rapidly mixing 0.25 mM KO2 , solubilized in dry DMSO, with 0.5 mM 18-Crown-6 (35), with Luc2/ buffered at pH 7.8. Line 1 in Fig. 6 demonstrates that this admixture was followed by an intense and rapidly declining luminescence, while line 2 shows the profound inhibition caused by SOD. This indicates that O20 can reduce Luc2/, in agreement with the results of earlier electrochemical studies (5). There was a relatively small luminescence that decayed slowly and that was due to H2O2 produced by the spontaneous dismutation of O20 . This H2O2-dependent luminescence at pH 7.8 was inhibited by catalase and was largely dependent on the simultaneous presence of DMSO. Luc2/ and paraquat: Similarities and differences. Both paraquat (PQ2/) and Luc2/ can be univalently
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Although the luminescence of Luc2/ depends upon O and thus is inhibitable by SOD, it cannot be considered a measure of O20 . This the case because Luc2/ can be reduced univalently by diverse enzymes including XO, glucose oxidase, dihydrolipoamide dehydrogenase, liver aldehyde oxidase (3), and bacterial dehydroorotate dehydrogenase (3). The LucH•/ so produced then 0 2
FIG. 7. Reduction of cytochrome c by XO: competition between Luc2/ and PQ2/. Reaction mixtures contained 10 mM cytochrome c, 0.1 mM NADH, and 15 U/ml of XO. Where indicated additional components were added as follows: Luc2/ to 40 mM, SOD to 10 mg/ml, and PQ2/ as indicated.
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reduces O2 to O20 which can react with LucH•/ to give the dioxetane, whose decomposition is responsible for luminescence (4). H2O2 can weakly elicit Luc2/ luminescence at pH 9.5 and this probably depends upon HO20 acting as a reductant for Luc2/. When that happens the immediate products are LucH•/ and O20 , the prerequisites for luminescence. At pH 7.8 H2O2 did not elicit Luc2/ luminescence unless DMSO was present and we can propose that the Luc2/ reductants generated in that case were derived from the oxidation of DMSO by H2O2 . It should be noted that dilution of the Luc2/ in phosphate buffer with an equal volume of DMSO caused an increase in pH from 7.8 to 8.3 due to the effect of diminished dielectric constant on the pKa of the phosphate buffer. Raising the pH by this amount in the absence of DMSO did not cause significant luminescence from the H2O2 plus Luc2/ mixture. We have seen that KO2 per se causes the luminescence of Luc2/, which implies that O20 can reduce Luc2/. This is, in effect, the reverse of the autooxidation of LucH•/. However, in enzymatic systems, this does not happen to a detectable degree. Thus, the reduction of Luc2/ to LucH2 by XO plus xanthine (3), or by XO plus NADH (this study), could be seen anaerobically but not aerobically. This implies that the autooxidation of LucH•0 was effectively irreversible. The following scheme of reactions serves to clarify the reactions of Luc2/. Luc2/ / EnzH2 r EnzH• / LucH•/ LucH•/ / EnzH• r LucH2 / Enz LucH•/ / LucH•/ r LucH2 / Luc2/ LucH•/ / Cyt c3/ r Luc2/ / H/ / Cyt c2/ LucH•/ / O2 r Luc2/ / O20 / H/ O uw f. LucH•/ / O20 r Luc{O r light / acridone g. O20 / O20 / 2H/ r H2O2 / O2 h. O20 / Cyt c3/ r O2 / Cyt c2/ a. b. c. d. e.
When O2 was present, reactions e together with f would outcompete reactions b and c and would prevent net reduction of Luc2/ to LucH2 . The inhibition of Luc2/ luminescence is a more sensitive assay of SOD activity than is the inhibition of cytochrome c reduction (36). This indicates that the reaction of O20 with cytochrome c (h) must be faster than its reaction with LucH•/ (f), under the specified conditions of the two assays. In contrast, the reduction of cytochrome c by LucH•/ (d) is faster than the autooxidation of LucH•/ (e), which explains why cytochrome c reduction in the XO / NADH / Luc2/ mixture was relatively insensitive to inhibition by SOD. Luc2/, like PQ2/, and NBT2/ (33, 37–39) can undergo
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a cycle of univalent reduction followed by autooxidation. These compounds thus share the capacity to mediate the production of O20 , even when that radical would not have been made in their absence. They can thus serve as the basis of assays for SOD, but cannot be used to measure, or even to reliably detect, O20 . REFERENCES 1. Totter, J R., Medina, V. J., and Scoseria, J. L. (1960) J. Biol. Chem. 235, 238–241. 2. Totter, J. R., DeDugros, E. C., and Riveiro, C. (1960) J. Biol. Chem. 235, 1839–1842. 3. Greenlee, L., Fridovich, I., and Handler, P. (1962) Biochemistry 1, 779–783. 4. Faulkner, K., and Fridovich, I. (1993) Free Radical Biol. Med. 15, 447–451. 5. Legg, K. D., and Hercules, D. M. (1969) J. Am. Chem. Soc. 91, 1902–1907. 6. Minkenberg, I., and Ferber, E. (1984) J. Immunol. Methods 71, 61–67. 7. Stevens, P., and Hong, D. (1984) Microchem. J. 30, 135–146. 8. Afanas’ev, I. B., Dorozhko, A. I., Polozova, N. I., Kuprianova, N. S., Brodskii, A. V., Ostrachovitch, E. A., and Korkina, L. G. (1993) Arch. Biochem. Biophys. 320, 200–205. 9. Allen, R. C. (1986) Methods Enzymol. 133, 449–493. 10. Mer’etey, K., Antal, M., Rozsnyay, Z., Bo¨hm, U., Elekes, E., and Genti, G. (1987) Inflammation 11, 417–425. 11. Gyllenhammar, H. (1987) J. Immunol. Methods 97, 209–213. 12. Storch, J., and Ferber, E. (1988) Anal. Biochem. 169, 262–276. 13. Grunfeld, S., Hamilton, C. A., Mesaros, S., McClain, S. W., Dominiczak, A. F., Bohr, D. F., and Malinski, T. (1995) Hypertension 26, 854–857. 14. Okuda, M., Lee, H. C., and Chance, B. (1992) Circ. Shock 38, 228–237. 15. Samuni, A., Krishna, C. M., Cook, J., Black, C. D. V., and Russo, A. (1991) Free Radical Biol. Med. 10, 305–313. 16. Okuda, M., Lee, H.-C., Chance, B., and Kumar, C. (1992) Free Radical Biol. Med. 12, 271–279. 17. Tosi, M. F., and Hamedaru, A. (1992) Am. J. Clin. Pathol. 97, 566–573. 18. Pagano, P. T., Ito, Y., Tomheim, K., Callop, P. M., Tauber, A. I., and Cohen, R. A. (1995) Am. J. Physiol. 268, H2274–H2280. 19. Carceni, P., Ryu, H. S., van Thiel, D. H., and Borle, A. B. (1995) Biochim. Biophys. Acta 1268, 249–254. 20. Scott, M. D., and Eaton, J. W. (1996) Redox Reports 2, 113–119. 21. Waud, W. R., Brady, F. O., Wiley, R. D., and Rajagopalan, K. V. (1975) Arch. Biochem. Biophys. 169, 699-701. 22. McCord, J. M., and Fridovich, I. (1969) J. Biol. Chem. 244, 6049– 6055. 23. Hodgson, E. K., McCord, J. M., and Fridovich, I. (1973) Anal. Biochem. 51, 470–473. 24. Darr, D., and Fridovich, I. (1984) Arch. Biochem Biophys. 232, 562–565. 25. Liochev, S., and Fridovich, I. (1986) Free Radical Biol. Med. 1, 287–292. 26. Liochev, S., and Fridovich, I. (1986) Arch. Biochem. Biophys. 250, 139–145.
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27. Liochev, I., and Fridovich, I. (1988) Arch. Biochem. Biophys. 263, 299–304. 28. Liochev, S. I., and Fridovich, I. (1990) Arch. Biochem. Biophys. 279, 1–7. 29. McCord, J. M., and Fridovich, I. (1968) J. Biol. Chem. 243, 5713– 5760. 30. Grinblat, L., Sreider, C. M., and Stoppani, A. O. (1991) Biochem. Int. 23, 83–92. 31. Bando, Y., and Aki, K. (1991) J. Biochem. (Tokyo) 109, 450–454. 32. Massey, V., Strickland, S., Mayhew, S. G., Howell, L. G., Engel, P. C., Mathews, R. G., Schuman, M., and Sullivan, P. A. (1969) Biochem. Biophys. Res. Commun. 36, 891–897.
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33. Liochev, S. I., and Fridovich, I. (1995) Arch. Biochem. Biophys. 318, 408–410. 34. Gleu, K., and Petsch, W. (1935) Angew. Chem. 48, 57–59. 35. Valentine, J. S., and Cortis, A. B. (1975) J. Am. Chem. Soc. 97, 224–226. 36. Corbisier, P., Houbion, A., and Remacle, J. (1987) Anal. Biochem. 164, 240–247. 37. Epel, B. L., and Neuman, J. (1973) Biochim. Biophys. Acta 325, 520–529. 38. Auclair, C., Torres, M., and Hakim, J. (1978) FEBS Lett. 89, 26– 28. 39. Picker, S. D., and Fridovich, I. (1984) Arch. Biochem. Biophys. 228, 155–158.
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