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 approximately 200-fold higher reactivity under conditions of [NO*] >> [GSH]. To help resolve these differences, the rate of loss of NO* ( approximately 9 microM) 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 degrees 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 approximately 30 min for low concentrations of NO* with 5 mM GSH, 37 degrees 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 approximately 18 microM) in alidded six well plate in an anaerobic chamber in vitro; intracellular GSH levels decreased by half in approximately 60 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.
Folkes
Lisa K
LK
Gray Cancer Institute, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR, UK.
Wardman
Peter
P
D000818:Animals; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D005609:Free Radicals; D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D009569:Nitric Oxide / Q000737:chemistry* / Q000378:metabolism; D009585:Nitrogen Dioxide / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D051381:Rats; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D005609:Free Radicals; D013438:Sulfhydryl Compounds; D009569:Nitric Oxide; D005978:Glutathione; D009585:Nitrogen Dioxide; D010100:Oxygen