Kinetics of the reaction between nitric oxide and glutathione: Implications for thiol depletion in cells

Nitric oxide in the absence of oxygen was suggested to react with 5-50 mM glutathione (GSH) over many minutes when [NO.] much less than [GSH] (N. Hogg et al., FEBS Lett. 382:223-228; 1996). However, Aravindakumar et al. (J. Chem. Soc. Perkin Trans. 2:663 - 669; 2002) provided data suggesting similar to 200-fold higher reactivity under conditions of [NO.] much greater than [GSH]. To help resolve these differences, the rate of loss of NO. (similar to9 muM) in aqueous solutions of GSH (2.520 mM) was measured by chemiluminescence. An apparent second-order rate constant of 0.080 +/- 0.008 M-1 s(-1) at pH 7.4, 37degreesC, was calculated based on the total [GSH] and "pseudo-first-order" kinetics; thiolate anion was much more reactive than undissociated thiol. These data imply a half-life of similar to30 min for low concentrations of NO. with 5 mM GSH, 37degreesC, pH 7.4, in the absence of oxygen. Possible kinetic schemes that can partially explain the divergent literature reports are discussed, notably an equilibrium in the reaction between NO. and GSH. Human breast carcinoma MCF-7 cells were exposed to NO. (initially similar to18 muM) in alidded six well plate in an anaerobic chamber in vitro; intracellular GSH levels decreased by half in similar to60 min. Aerobic exposure depletes GSH in cells in vitro much faster because of autoxidation of NO. to NO2. > 10(8) times more reactive toward GSH. (C) 2004 Elsevier Inc. All rights reserved.