View PDF Version
 
DOI: 10.1039/A806100I (Paper) Reference Section for: J. Chem. Soc., Perkin Trans. 2, 1999, 297-300

14N-Spin trapping of free radicals in the presence of 15N-spin labeled Neisseria gonorrhoeae

(Note: The full text of this document is currently only available in the PDF Version )

Gerald M. Rosen, Sovitj Pou, Myron S. Cohen, Daniel J. Hassett, Bradley E. Britigan, Michael J. Barber, Guan-Liang Cao, Kennyatta Cosby, Bradley E. Sturgeon and Howard J. Halpern

Abstract

One of the difficult tasks confronting the study of free radicals in biology is the inability to measure “on line” injury to a biological target, while characterizing the reactive species responsible for the toxic event. This is particularly relevant in light of the fact that specific free radicals play a critical role in host immune response. An approach towards addressing this important issue draws upon the unique EPR spectral properties of 14N/15N-labeled compounds. In particular, Neisseria gonorrhoeae has been covalently labeled with 15N-deuterium17-containing 4-maleimido-2,2,6,6-tetramethylpiperidin-1-yloxyl (15N-D17-4-MAL-TEMPO). The EPR spectrum from bacteria so labeled exhibited two low-field peaks: (a) a broad, strongly immobilized species classified as “S”; (b) a more narrow, weakly immobilized component termed “W”. The W/S ratio is an indicator of changes in membrane organization. In the presence of the superoxide-generating system, hypoxanthine/xanthine oxidase, an increase in the W/S ratio from 3.3 for control to 6.4 was observed, which was only partially inhibited by superoxide dismutase (W/S ratio of 4.4). When the spin trap 5,5-dimethyl-1-pyrroline 14N-oxide (DMPO) was included in the above reaction mixture, an EPR spectrum was recorded, which was a composite of 2,2,-dimethyl-5-hydroperoxypyrrolidin-1-yl-14N-oxyl (DMPO-OOH) and 15N-D17-4-MAL-TEMPO-labeled Neisseria gonorrhoeae. With the use of computer subtraction procedures, the W/S ratio was found to be 6.4. The experiments demonstrate the utility of 14N/15N-labeled aminoxyls as a valuable tool in accessing the effects of specific free radicals on the fluidity of cell membranes.

References

  1. P. Van der Valk and C. J. Herman, Lab. Invest., 1987, 57, 127 Search PubMed.
  2. (a) G. M. Rosen, S. Pou, C. L. Ramos, M. S. Cohen and B. E. Britigan, FASEB J., 1995, 9, 200 Search PubMed; (b) B. M. Babior, R. S. Kipnes and J. T. Curnette, J. Clin. Invest., 1973, 52, 741 Search PubMed; (c) E. L. Thomas, R. I. Lehrer and R. F. Rest, Rev. Infect. Dis., 1988, 10, S450 Search PubMed.
  3. (a) D. A. Butterfield, A. D. Roses, S. H. Appel and D. B. Chesnut, Arch. Biochem. Biophys., 1976, 177, 226 CAS; (b) R. Subramaniam, F. Roediger, B. Jordan, M. P. Mattson, J. N. Keller, G. Waeg and D. A. Butterfield, J. Neurochem., 1997, 69, 1161 CAS; (c) D. A. Butterfield, B. J. Howard, S. Yatin, K. L. Allen and J. M. Carney, Proc. Natl. Acad. Sci. U.S.A., 1997, 94, 674 CrossRef CAS.
  4. G. M. Rosen, M. J. Barber and E. J. Rauckman, J. Biol. Chem., 1983, 258, 2225 CAS.
  5. B. A. Freeman, G. M. Rosen and M. J. Barber, J. Biol. Chem., 1986, 261, 6590 CAS.
  6. J. S. Beckman, R. L. Minor, Jr., C. W. White, J. E. Repine, G. M. Rosen and B. A. Freeman, J. Biol. Chem., 1988, 263, 6884 CAS.
  7. M. Iwamura and N. Inamoto, Bull Chem. Soc. Jpn., 1967, 40, 703 CAS.
  8. (a) E. G. Janzen and B. J. Blackburn, J. Am. Chem. Soc., 1968, 90, 5909 CrossRef CAS; (b) E. G. Janzen and B. J. Blackburn, J. Am. Chem. Soc., 1969, 91, 4481 CrossRef CAS.
  9. (a) E. G. Janzen, Acc. Chem. Res., 1971, 4, 31 CrossRef CAS; (b) E. G. Janzen, in Free Radicals in Biology, ed. W. A. Pryor, Academic Press, New York, 1980, vol. 4, p. 116 Search PubMed.
  10. (a) E. Finkelstein, G. M. Rosen and E. J. Rauckman, Arch. Biochem. Biophys., 1980, 200, 1 CAS; (b) G. M. Rosen and E. Finkelstein, Adv. Free Radical Biol. Med., 1985, 1, 345 Search PubMed.
  11. A. H. Beth, S. D. Venkataramu, S. D. , K. Balasubramanian, L. R. Dalton, B. H. Robinson, D. E. Pearson, C. R. Park and J. H. Park, Proc. Natl. Acad. Sci. U.S.A., 1981, 78, 967 CAS.
  12. F. Radner, A. Rassat and C.-J. Hersvall, Acta Chem. Scand., 1996, 50, 146 CAS.
  13. (a) D. A. Butterfield, F. E. Ordaz and W. R. Markesbery, J. Gerontol., 1982, 37, 535 Search PubMed; (b) M. J. Barber, G. M. Rosen and E. J. Rauckman, Biochim. Biophys. Acta, 1983, 732, 126 CrossRef CAS.
  14. J. F. W. Keana, S. Pou and G. M. Rosen, Magn. Reson. Med., 1987, 5, 525 CrossRef CAS.
  15. (a) B. Zhang, M. Centra, G. L. Cao, R. M. Taylor, R. E. Ratych and G. M. Rosen, J. Infect. Dis., 1996, 174, 1001 CAS; (b) B. Zhang, M. Centra, G. L. Cao, R. E. Ratych, J. B. Domachowske, H. L. Malech and G. M. Rosen, Immunol. Lett., 1997, 58, 113 CrossRef CAS.
  16. (a) R. J. Hamill, J. M. Vann and R. A. Proctor, Infect. Immun., 1987, 54, 833; (b) J. M. Vann and R. A. Proctor, Infect. Immun., 1987, 55, 2155 CAS; (c) J. M. Vann and R. A. Proctor, Micro. Pathogen., 1988, 4, 443 Search PubMed; (d) U. S. Ryan, in Endothelial Cells, ed. U. S. Ryan, CRC Press, Boca Raton, FL, 1988, vol. III, p. 33 Search PubMed.
  17. R. Bonnett, R. F. C. Brown, V. M. Clark, I. O. Sutherland and A. J. Todd, J. Chem. Soc., 1959, 2094 RSC.
  18. D. S. Kellogg, Jr., W. L. Peacock, Jr., W. E. Deacon, L. Brown and C. I. Prickle, J. Bacteriol., 1963, 85, 1274 CAS.
  19. H. Kuthan, V. Ullrich and R. W. Estabrook, Biochem. J., 1982, 203, 551 CAS.
Click here to see how this site uses Cookies. View our privacy policy here.