Determination of the thiol redox state of organisms: new oxidative stress indicators
This study describes a new methodology by which the concentrations of non-protein (NP) thiols glutathione (GSH), cysteine (CSH), N-acetylcysteine (AcCSH), and protein (P) thiols (PSH), as well as the contribution of these components to symmetric and mixed disulfides (NPSSR, NPSSC, NPSSCAc, PSSR, PSSC, PSSCAc, PSSP) can reliably be measured. The methodology consists of a strict sequence of methods which are applied to every sample. Free thiols at any given state of the procedure are measured by Ellman’s assay, the CSH fraction is measured by its unique response in the ninhydrin assay, AcCSH is selectively measured with ninhydrin after enzymatic deacylation, proteins are separated from non-protein thiols/disulfides by precipitation with trichloroacetic or perchloric acid, disulfides are reduced into free thiols with borohydride, mixed disulfides between a protein and a non-protein component are measured by extracting the non-protein thiol from the protein pellet after borohydride treatment, and protein thiols/disulfides are measured after resolubilization of the protein pellet.
When this method was applied to animal and fungal tissue, new molecular indicators of the thiol redox state of living cells were identified. The findings of the present study clearly show that the new parameters are very sensitive indicators of redox state, while at the same time the traditional parameters GSH and GSSG often remain constant even upon dramatic changes in the overall redox state of biological tissue. Therefore, unbiased assessment of the redox state also requires explicit measurement of its most sensitive thiol indicators.
KeywordsN-Acetylcysteine Protein/non-protein thiols/disulfides Thiol redox state
This work was financially supported by the Greek Ministry of Education and by ‘K. Karatheodoris Program’, University of Patras, Greece. Dr D. Synetos and Dr Nikolaos Matsokis (from Biochemistry Department of School of Medicine and from Biology Department, respectively, of the University of Patras, Greece), contributed significantly to this work by providing yeast and mice, respectively.
- 2.Halliwell B, Gutteridge CMJ (1999) Free radicals in biology and medicine. Oxford University Press, OxfordGoogle Scholar
- 4.Akerboom MPT, Sies H (1981) In: Colowick SP, Kaplan ON (eds) Methods in enzymology, vol 77. Academic Press, New York, pp 373–382Google Scholar
- 7.Gilbert FH (1995) In: Packer L (ed) Methods in enzymology, vol 251. Academic Press, New York, pp 8–28Google Scholar
- 8.Ratbbun BW (1990) In: Vina J (ed) Glutathione: metabolism and physiological functions. CRC Press, Boca Raton, pp 193–206Google Scholar
- 9.Miquel J, Weber H (1990) In: Vina J (ed) Glutathione: metabolism and physiological functions. CRC Press, Boca Raton, pp 187–192Google Scholar
- 10.Levitt J (1972) Responses of plants to environmental stresses. Academic Press, New YorkGoogle Scholar
- 11.van der Vliet A, Cross EC, Halliwell B, O’Neill AC (1995) In: Packer L (ed) Methods in enzymology, vol 251. Academic Press, New York, pp 448–455Google Scholar
- 12.Cotgreave AI, Weis M, Atzori L, Moldéus P (1990) In: Vina J (ed) Glutathione: metabolism and physiological functions. CRC Press, Boca Raton, pp 155–175Google Scholar
- 18.Ellman LG (1959) Arch Biochem Biophys 82:70–77Google Scholar
- 19.Redegeld FAM, Koster AS, van Bennekom WP (1990) In: Vina J (ed) Glutathione: metabolism and physiological functions. CRC Press, Boca Raton, pp 11–20Google Scholar
- 27.Wong KB, Corcoran BG (1990) In: Vina J (ed) Glutathione: metabolism and physiological functions. CRC Press, Boca Raton, pp 255–262Google Scholar
- 29.Means EG, Feeney ER (1971) Chemical modification of proteins. Holden-Day, San FranciscoGoogle Scholar
- 31.Margenau H, Murphy MG (1976) The mathematics of physics and chemistry. Krieger, New YorkGoogle Scholar
- 34.Buege JA, Aust SD (1978) In: Fleisher S, Packer L (eds) Methods in enzymology, vol 52. Academic Press, New York, pp 302–310Google Scholar