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

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, Howard J. Halpern

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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.

Original languageEnglish (US)
Pages (from-to)297-300
Number of pages4
JournalJournal of the Chemical Society. Perkin Transactions 2
Issue number2
DOIs
StatePublished - Feb 1999

ASJC Scopus subject areas

  • Chemistry(all)

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    Rosen, G. M., Pou, S., Cohen, M. S., Hassett, D. J., Britigan, B. E., Barber, M. J., Cao, G. L., Cosby, K., Sturgeon, B. E., & Halpern, H. J. (1999). 14N-Spin trapping of free radicals in the presence of 15N-spin labeled Neisseria gonorrhoeae. Journal of the Chemical Society. Perkin Transactions 2, (2), 297-300. https://doi.org/10.1039/a806100i