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√] [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 200-fold higher reactivity under conditions of [NO√] [GSH]. To help resolve these differences, the rate of loss of NO√ ( 9 μM) in aqueous solutions of GSH (2.5–20 mM) was measured by chemiluminescence. An apparent second-order rate constant of 0.080 ± 0.008 M−1 s−1 at pH 7.4, 37°C, 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 30 min for low concentrations of NO√ with 5 mM GSH, 37°C, 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 18 μM) in alidded six well plate in an anaerobic chamber in vitro; intracellular GSH levels decreased by half in 60 min. Aerobic exposure depletes GSH in cells in vitro much faster because of autoxidation of NO√ to NO2√, >108 times more reactive toward GSH.