Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
권호 표지

Cu,Zn-Superoxide Dismutase Is an Intracellular Catalyst for the H2O2-dependent Oxidation of Dichlorodihydrofluorescein

  • Kim, Young-Mi (Department of Biochemistry, Kangwon National University) ;
  • Lim, Jung-Mi (Department of Biochemistry, Kangwon National University) ;
  • Kim, Byung-Chul (Department of Biochemistry, Kangwon National University) ;
  • Han, Sanghwa (Department of Biochemistry, Kangwon National University)
  • Received : 2005.09.29
  • Accepted : 2005.11.30
  • Published : 2006.02.28
⟨ Previous Next ⟩

Abstract

Dichlorodihydrofluorescein ($DCFH_2$) is a widely used probe for intracellular $H_2O_2$. However, $H_2O_2$ can oxidize $DCFH_2$ only in the presence of a catalyst, whose identity in cells has not been clearly defined. We compared the peroxidase activity of Cu,Zn-superoxide dismutase (CuZnSOD), cytochrome c, horseradish peroxidase (HRP), $Cu^{2+}$, and $Fe^{3+}$ under various conditions to identify an intracellular catalyst. Enormous increase by bicarbonate in the rate of $DCFH_2$ oxidation distinguished CuZnSOD from cytochrome c and HRP. Cyanide inhibited the reaction catalyzed by CuZnSOD but accelerated that by $Cu^{2+}$ and $Fe^{3+}$. Oxidation of $DCFH_2$ by $H_2O_2$ in the presence of a cell lysate was also enhanced by bicarbonate and inhibited by cyanide. Confocal microscopy of $H_2O_2$-treated cells showed enhanced DCF fluorescence in the presence of bicarbonate and attenuated fluorescence for the cells pre-incubated with KCN. Moreover, DCF fluorescence was intensified in CuZnSOD-transfected HaCaT and RAW 264.7 cells. We propose that CuZnSOD is a potential intracellular catalyst for the $H_2O_2$-dependent oxidation of $DCFH_2$.

Keywords

References

  1. Aam, B. B., Myhre, O., and Fonnum, F. (2003) Transcellular signalling pathways and TNF-alpha release involved in formation of reactive oxygen species in rat alveolar macrophages exposed to tert-butylcyclohexane. Arch. Toxicol. 77, 678-684 https://doi.org/10.1007/s00204-003-0507-2
  2. Andrekopoulos, C., Zhang, H., Joseph, J., Kalivendi, S., and Kalyanaraman, B. Bicarbonate enhances alpha-synuclein oligomerization and nitration: intermediacy of carbonate radical anion and nitrogen dioxide radical. Biochem. J. 378, 435-447 https://doi.org/10.1042/BJ20031466
  3. Bonini, M. G., Fernandes, D. C., and Augusto, O. (2004) Albumin oxidation to diverse radicals by the peroxidase activity of Cu,Zn-superoxide dismutase in the presence of bicarbonate or nitrite: diffusible radicals produce cysteinyl and solvent- exposed and -unexposed tyrosyl radicals. Biochemistry 43, 344-351 https://doi.org/10.1021/bi035606p
  4. Burkitt, M. J. and Wardman, P. (2001) Cytochrome c is a potent catalyst of dichlorofluorescin oxidation: implications for the role of reactive oxygen species in apoptosis. Biochem. Biophys. Res. Commun. 282, 329−333 https://doi.org/10.1006/bbrc.2001.4578
  5. Crow, J. P. (1997) Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro: implications for intracellular measurement of reactive nitrogen and oxygen species. Nitric Oxide 1, 145−157 https://doi.org/10.1006/niox.1996.0113
  6. Elam, J. S., Malek, K., Rodriguez, J. A., Doucette, P. A., Taylor, A. B., et al. (2003) An alternative mechanism of bicarbonatemediated peroxidation by copper-zinc superoxide dismutase: rates enhanced via proposed enzyme-associated peroxycarbonate intermediate. J. Biol. Chem. 278, 21032−21039 https://doi.org/10.1074/jbc.M300484200
  7. Glebska, J. and Koppenol, W. H. (2003) Peroxynitrite-mediated oxidation of dichlorodihydrofluorescein and dihydrorhodamine. Free Radic. Biol. Med. 35, 676-682 https://doi.org/10.1016/S0891-5849(03)00389-7
  8. Goss, S. P., Singh, R. J., and Kalyanaraman, B. (1999) Bicarbonate enhances the peroxidase activity of Cu,Zn-superoxide dismutase. Role of carbonate anion radical. J. Biol. Chem. 274, 28233-28239 https://doi.org/10.1074/jbc.274.40.28233
  9. Graham, F. L. and van der Eb, A. J. (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456−467
  10. Hong, J. E., Santucci, L. A., Tian, X., and Silverman, D. J. (1998) Superoxide dismutase-dependent, catalase-sensitive peroxides in human endothelial cells infected by Rickettsia rickettsii. Infect. Immun. 66, 1293-1298
  11. Kang, J. H. and Kim, K. S. (2003) Enhanced oligomerization of the ${\alpha}$-synuclein mutant by the Cu,Zn-superoxide dismutase and hydrogen peroxide. Mol. Cells 15, 87-93
  12. Karunakaran, C., Zhang, H., Crow, J. P., Antholine, W. E., and Kalyanaraman, B. (2004) Direct probing of copper active site and free radical formed during bicarbonate-dependent peroxidase activity of bovine and human copper, zinc-superoxide dismutases. Low-temperature electron paramagnetic resonance and electron nuclear double resonance studies. J. Biol. Chem. 279, 32534−32540 https://doi.org/10.1074/jbc.M314272200
  13. Kim, Y. S. and Han, S. (2000) Nitric oxide protects Cu,Znsuperoxide dismutase from hydrogen peroxide-induced inactivation. FEBS Lett. 479, 25-28 https://doi.org/10.1016/S0014-5793(00)01874-3
  14. Kooy, N. W., Royall, J. A., and Ischiropoulos, H. (1997) Oxidation of 2′,7′-dichlorofluorescin by peroxynitrite. Free Radic. Res. 27, 245-254 https://doi.org/10.3109/10715769709065763
  15. Lawrence, A., Jones, C. M., Wardman, P., and Burkitt, M. J. (2003) Evidence for the role of a peroxidase compound Itype intermediate in the oxidation of glutathione, NADH, ascorbate, and dichlorofluorescin by cytochrome c/$H_2O_2$. Implications for oxidative stress during apoptosis. J. Biol. Chem. 278, 29410−29419 https://doi.org/10.1074/jbc.M300054200
  16. Liochev, S. I. and Fridovich, I. (2001) Copper,zinc-superoxide dismutase as a univalent NO− oxidoreductase and as a dichlorofluorescin peroxidase. J. Biol. Chem. 276, 35253-35257 https://doi.org/10.1074/jbc.M104237200
  17. Liochev, S. I. and Fridovich, I. (2004a) Bicarbonate-enhanced peroxidase activity of Cu,Zn SOD: is the distal oxidant bound or diffusible? Arch. Biochem. Biophys. 421, 255−259 https://doi.org/10.1016/j.abb.2003.11.015
  18. Liochev, S. I. and Fridovich, I. (2004b) $CO_2$, not $HCO_3$-, facilitates oxidations by Cu,Zn superoxide dismutase plus $H_2O_2$. Proc. Natl. Acad. Sci. USA 101, 743−744
  19. Mankhetkorn, S., Abedinzadeh, Z., and Houee-Levin, C. (1994) Antioxidant action of sodium diethyldithiocarbamate: reaction with hydrogen peroxide and superoxide radical. Free Radic. Biol. Med. 17, 517-527 https://doi.org/10.1016/0891-5849(94)90091-4
  20. Moreno, S. N., Stolze, K., Janzen, E. G., and Mason, R. P. (1988) Oxidation of cyanide to the cyanyl radical by peroxidase/ $H_2O_2$ systems as determined by spin trapping. Arch. Biochem. Biophys. 265, 267−271 https://doi.org/10.1016/0003-9861(88)90127-0
  21. Myhre, O. Vestad, T. A., Sagstuen, E., Aarnes, H., and Fonnum, F. (2000) The effects of aliphatic (n-nonane), naphtenic (1,2,4-trimethylcyclohexane), and aromatic (1,2,4-trimethylbenzene) hydrocarbons on respiratory burst in human neutrophil granulocytes. Toxicol. Appl. Pharmacol. 167, 222-230 https://doi.org/10.1006/taap.2000.9008
  22. Myhre, O., Andersen, J. M., Aarnes, H., and Fonnum, F. (2003) Evaluation of the probes 2′,7′-dichlorofluorescin diacetate, luminol, and lucigenin as indicators of reactive species formation. Biochem. Pharmacol. 65, 1575−1582 https://doi.org/10.1016/S0006-2952(03)00083-2
  23. Oyama, Y., Hayashi, A., Ueha, T., and Maekawa, K. (1994) Characterization of 2′,7′-dichlorofluorescin fluorescence in dissociated mammalian brain neurons: estimation on intracellular content of hydrogen peroxide. Brain Res. 635, 113-117 https://doi.org/10.1016/0006-8993(94)91429-X
  24. Paz-Miguel, J. E., Flores, R., Sanchez-Velasco, P., Ocejo- Vinyals, G., Escribano de Diego, J., et al. (1999) Reactive oxygen intermediates during programmed cell death induced in the thymus of the Ts(1716)65Dn mouse, a murine model for human Down's syndrome. J. Immunol. 163, 5399-5410
  25. Peled-Kamar, M., Lotem, J., Wirguin, I., Weiner, L., Hermalin, A., et al. (1997) Oxidative stress mediates impairment of muscle function in transgenic mice with elevated level of wild-type Cu/Zn superoxide dismutase. Proc. Natl. Acad. Sci. USA 94, 3883−3887
  26. Pias, E. K., Ekshyyan, O. Y., Rhoads, C. A., Fuseler, J., Harrison, L., et al. (2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells. J. Biol. Chem. 278, 13294−13301 https://doi.org/10.1074/jbc.M208670200
  27. Possel, H., Noack, H., Augustin, W., Keilhoff, G., and Wolf, G (1997) 2,7-Dihydrodichlorofluorescein diacetate as a fluorescent marker for peroxynitrite formation. FEBS Lett. 416, 175-178 https://doi.org/10.1016/S0014-5793(97)01197-6
  28. Rota, C., Fann, Y. C., and Mason, R. P. (1999) Phenoxyl free radical formation during the oxidation of the fluorescent dye 2',7'-dichlorofluorescein by horseradish peroxidase. Possible consequences for oxidative stress measurements. J. Biol. Chem. 274, 28161-28168 https://doi.org/10.1074/jbc.274.40.28161
  29. Sankarapandi, S. and Zweier, J. L. (1999) Bicarbonate is required for the peroxidase function of Cu, Zn-superoxide dismutase at physiological pH. J. Biol. Chem. 274, 1226-1232 https://doi.org/10.1074/jbc.274.3.1226
  30. Sato, K., Akaike, T., Kohno, M., Ando, M., and Maeda, H. (1992) Hydroxyl radical production by $H_2O_2$ plus Cu,Zn-superoxide dismutase reflects the activity of free copper released from the oxidatively damaged enzyme. J. Biol. Chem. 267, 25371-25377
  31. Tampo, Y., Kotamraju, S., Chitambar, C. R., Kalivendi, S. V., Keszler, A., et al. (2003) Oxidative stress-induced iron signaling is responsible for peroxide-dependent oxidation of dichlorodihydrofluorescein in endothelial cells: role of transferrin receptor-dependent iron uptake in apoptosis. Circ. Res. 92, 56−63 https://doi.org/10.1161/01.RES.0000048195.15637.AC
  32. Wrona, M., Patel, K., and Wardman, P. (2005) Reactivity of 2',7'-dichlorodihydrofluorescein and dihydrorhodamine 123 and their oxidized forms toward carbonate, nitrogen dioxide, and hydroxyl radicals. Free Radic. Biol. Med. 38, 262−270 https://doi.org/10.1016/j.freeradbiomed.2004.10.022
  33. Zhang, H., Andrekopoulos, C., Joseph, J., Chandran, K., Karoui, H., et al. (2003) Bicarbonate-dependent peroxidase activity of human Cu,Zn-superoxide dismutase induces covalent aggregation of protein: intermediacy of tryptophan-derived oxidation products. J. Biol. Chem. 278, 24078−24089 https://doi.org/10.1074/jbc.M302051200
  34. Zhang, H., Joseph, J., Felix, C., and Kalyanaraman, B. (2000) Bicarbonate enhances the hydroxylation, nitration, and peroxidation reactions catalyzed by copper, zinc superoxide dismutase. Intermediacy of carbonate anion radical. J. Biol. Chem. 275, 14038−14045 https://doi.org/10.1074/jbc.275.19.14038
  35. Zhang, H., Joseph, J., Gurney, M., Becker, D., and Kalyanaraman, B. (2002) Bicarbonate enhances peroxidase activity of Cu,Zn-superoxide dismutase. Role of carbonate anion radical and scavenging of carbonate anion radical by metalloporphyrin antioxidant enzyme mimetics. J. Biol. Chem. 277, 1013-1020 https://doi.org/10.1074/jbc.M108585200