Kinetics of the reactions of nitrogen dioxide with glutathione, cysteine, and uric acid at physiological pH

Nitrogen dioxide (NO2.) is a key biological oxidant. It can be derived from peroxynitrite via the interaction of nitric oxide with superoxide, from nitrite with peroxidases, or from autoxidation of nitric oxide. In this study, submicromolar concentrations of NO2. were generated in < 1 mus using pulse radiolysis, and the kinetics of scavenging NO2. by glutathione, cysteine, or uric acid were monitored by spectrophotometry. The formation of the urate radical was observed directly, while the production of the oxidizing radical obtained on reaction of NO2. with the thiols (the thiyl radical) was monitored via oxidation of 2.2'-azino-bis-(3-ethylthiazoline-6-sulfonic acid). At pH 7.4, rate constants for reaction of NO2. with glutathione, cysteine, and urate were estimated as similar to2 x 10(7), 5 x 10(7), and 2 x 10(7) M-1 s(-1). respectively. The variation of these rate constants with pH indicated that thiolate reacted Much faster than undissociated thiol. The dissociation of urate also accelerated reaction with NO2. at pH > 8. The thiyl radical from GSH reacted with urate with a rate constant of similar to3 x 10(7) M-1 s(-1). The implications of these values are: (i) the lifetime of NO2. in cytosol is < 10 mus; (ii) thiols are the dominant 'sink' for NO2. in cells/tissue, whereas urate is also a major scavenger in plasma; (iii) the diffusion distance of NO2. is similar to0.2 mum in the cytoplasm and < 0.8 mum in plasma; (iv) urate protects GSH against depletion oil oxidative challenge from NO2.; and (v) reactions between NO2. and thiols/urate severely limit the likelihood of reaction of NO2. with NO2(.) to form N2O3 in the cytoplasm. (C) 2002 Elsevier Science Inc.