THE ANTI-INFLAMMATORY NITRONE 4-OH-PBN TRAPS PROTEIN-CENTERED RADICALS

SANDRA E. GOMEZ-MEJIBA; REBECCA FARIS; NATALIYA SMITH; GARTEISER, PHILIPPE; RHEAL A. TOWNER; DARIO C. RAMIREZ

New York, NY

Conferencia; The 4th International Conference on Oxidative/nitrosative stress and diseases; 2009

The New York Academy of Sciences

THE ANTI-INFLAMMATORY NITRONE 4-OH-PBN TRAPS PROTEIN-CENTERED RADICALS         Sandra E. Gomez-Mejiba, M.Sc.1, Rebecca A. Faris, B.F.A. 1, Natalya Smith, Ph.D.2, Philippe Garteiser, Ph.D.2, Rheal A. Towner, Ph.D.2 and Dario C. Ramirez, Ph.D. M.Sc1. 1 Free Radical Biology and Aging Research Program and 2 AMRC, OMRF, Oklahoma City, OK 73104 Nitrone spin traps, such as phenyl-N-tert-butyl nitrone (PBN) and its main metabolite 4-OH-a-PBN (4OH-PBN), have emerged as anti-inflammatory drugs. Trapping of protein-centered radicals by a nitrone spin trap produces nitrone adducts and blocks further protein oxidation. We tested whether the anti-inflammatory properties of 4OH-PBN are due, at least in part, to the trapping of protein radicals. In order to approach our goal we have synthesized and purified a new spin trap probe, biotin-4OH-PBN (biotin-ST), and confirmed its structure by NMR. Addition of the biotin-ST during the reaction of 5 mM HSA with HOCl (molar ratios from 1 to 100) produced biotin-tagged nitrone adducts as assessed by ELISA and Western blot. Treatment of human serum or homogenate of A549 cells with HOCl in the presence of biotin-ST showed a number of nitrone adducts as assessed by Western blot. Immunoprecipitation and analysis by Western blot showed albumin and glycerhaldeyde-3-phosphate dehydrogenase, respectively, as major target proteins for oxidation in those reaction mixtures. In order to study the anti-inflammatory effect of our probe we used macrophages activated with lipopolysaccharide (LPS) to produce nitric oxide. In RAW 264.7 macrophages treated with LPS, inducible nitric oxide synthase expression was blocked by non-toxic concentrations of the biotin-ST and a number of biotin-ST-protein nitrone adducts were detected by Western blot and localized by confocal microscopy, suggesting that the probe is cell permeable and traps protein-centered radicals. Our results will help to explain the anti-oxidant mechanism of 4OH-PBN in inflammatory diseases. NIEHS 5R00ES015415-03