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

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.