Oxidative-stress induced protein glutathione mixed-disulfide formation in the ocular lens

Biochim Biophys Acta. 1996 Aug 21;1313(1):20-8. doi: 10.1016/0167-4889(96)00049-3.


The biochemistry of protein-glutathione mixed disulfide formation in the ocular lens was examined by 13C-NMR spectroscopic measurements of glutathione oxidative metabolism in intact rabbit lenses maintained in organ culture. Lenticular amino acid uptake and glutathione biosynthetic mechanisms were employed to facilitate the incorporation of L-[3-13C]cysteine from the incubation medium into the cysteinyl residue of glutathione. Subsequent exposure to increasing levels of oxidative stress induced by tert-butylhydroperoxide resulted in decreased levels of ([3-13C]cysteinyl)-glutathione and a loss of 13C NMR resonance intensity, a reflection of protein-glutathione mixed disulfide formation. The rate of ([3-13C]cysteinyl)-glutathione loss depended on the concentration of tert-butylhydroperoxide; 13C-labeled oxidized glutathione was observed only at the highest concentration (2 mM) of oxidant tested. Removal of the oxidative stress led to a partial recovery of ([3-13C]cysteinyl)-glutathione levels and 13C resonance intensity. Recovery was significantly enhanced by the addition of 2-mercaptoethanol. The mechanism of protein-glutathione adduct formation was further characterized by the in vitro monitoring of the reaction of oxidized glutathione with bovine lens gamma-II crystallin protein using proton NMR spectroscopy. These experiments provided insight into the role of the cellular glutathione redox-couple, [GSH]/[GSSG], in maintaining reduced protein thiol groups, and suggested that protein-glutathione adduct formation may function as a mechanism for modulating the glutathione redox buffer under conditions of oxidative stress in ocular tissue. In addition, the results demonstrate the feasibility of direct chemical reduction of protein-glutathione disulfide bonds in vivo which may reflect a mechanism for the inhibition of disulfide-linked light scattering protein aggregate formation.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Crystallins / chemistry
  • Cysteine / chemistry
  • Disulfides
  • Glutathione / chemistry*
  • Glutathione Peroxidase / metabolism
  • Lens, Crystalline / metabolism*
  • Magnetic Resonance Spectroscopy
  • Organ Culture Techniques
  • Oxidation-Reduction
  • Oxidative Stress*
  • Peroxides / chemistry
  • Rabbits
  • Stress, Physiological / metabolism
  • tert-Butylhydroperoxide


  • Crystallins
  • Disulfides
  • Peroxides
  • tert-Butylhydroperoxide
  • Glutathione Peroxidase
  • Glutathione
  • Cysteine