Abstract
Quantitative P-31 NMR spin trapping techniques can be used as effective tools for the detection and quantification of many free radical species. Free radicals react with a nitroxide phosphorus compound, 5-diisopropoxy-phosphoryl-5-methyl-1-pyrroline-N-oxide (DIPPMPO), to form stable radical adducts, which are suitably detected and accurately quantified using P-31 NMR in the presence of phosphorus containing internal standards. Initially, the P-31 NMR signals for the radical adducts of oxygen-centered ((OH)-O-., O-2(.-)) and carbon-centered ((CH3)-C-., (CH2OH)-C-., (CH2CH2OH)-C-.) radicals were assigned. Subsequently, the quantitative reliability of the developed technique was demonstrated under a variety of experimental conditions. The P-31 NMR chemical shifts for the hydroxyl and superoxide reaction adducts with DIPPMPO were found to be 25.3, 16.9, and 17.1 ppm (in phosphate buffer), respectively. The P-31 NMR chemical shifts for (CH3)-C-., (CH2OH)-C-., (CH)-C-.(OH)CH3, and C-.(O)CH3 spin adducts were 23.1, 22.6, 27.3, and 30.2 ppm, respectively. Overall, this effort forms the foundations for a targeted understanding of the nature, identity, and mechanisms of radical activity in a variety of biomolecular processes. (c) 2006 Elsevier Ltd. All rights reserved.