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Analytical Assessment of Thiols in Biological Systems

Analytical Assessment of Thiols in Biological Systems. Henry Jay Forman University of California, Merced. What needs to be measured?. Glutathione (GSH, GSSG) Nitrosoglutathione (GSNO) Thioredoxin (Trx) Cysteine, Cystine Protein thiols (PSH, PSSP, PSSG)

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Analytical Assessment of Thiols in Biological Systems

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  1. Analytical Assessment of Thiols in Biological Systems Henry Jay Forman University of California, Merced

  2. What needs to be measured? • Glutathione (GSH, GSSG) • Nitrosoglutathione (GSNO) • Thioredoxin (Trx) • Cysteine, Cystine • Protein thiols (PSH, PSSP, PSSG) • Other modification of protein thiols, -SNO, -SOH, -SO2H, -SO3H Oxidative stress compared with signaling

  3. amino acid -glu-amino acid GSX GSSG cys cys-gly GSH GGT GSX transporter gly dipeptidase GSH transporter Amino acid transporters acivicin BSO GSSG NADPH ROH -glu-amino acid GR GPx amino acid 5-oxoproline ROOH ATP NADP+ ADP gly cys glu -glu-cys GSH GST GCL GSH synthase GSX ADP ATP ADP ATP X Glutathione metabolism extracellular intracellular GSNO

  4. Protein mixed disulfide (PSSG) • Thiol (SH) exchange with disulfide (SS) • versus thiolate (S-) oxidation PDI but remember the ratio of GSH/GSSG What about cys/cys2 exchange?

  5. Dithionitrobenzoic acid Glutathione methods • Total • GSH and GSSG • Recycling method To measure GSSG first modify GSH N-ethylmaleimide (NEM) vinylpyridine To measure PSSG - release GSH with NaBH4

  6. A365 5 min HPLC method Others: cys cys2 cysteic acid -glu-cys cys-gly homocys

  7. Nitrosoglutathione (GSNO) First tie up GSH with NEM GSNO treated with mercaptoethanol to release GSH GSH reacted with orthophthaldehyde (OPT) Separated by HPLC Tsikas, D., et al. 1999. Determination of S-nitrosoglutathione in human and rat plasma by high-performance liquid chromatography with fluorescence and ultraviolet absorbance detection after precolumn derivatization with o-phthalaldehyde. Anal Biochem. 273: 32-40. Neuschwander-Tetri, B. A. & F. J. Roll. 1989. Glutathione measurement by high-performance liquid chromatography separation and fluorometric detection of the glutathione- orthophthalaldehyde adduct. Anal Biochem. 179: 236-41. A sensitive quantitative method was also developed that involves 15N labeling of GSNO, HgCl2 oxidation to nitrite, conversion to the pentafluorobenzyl derivatives, separation by HPLC and measurement by GC-MS. Tsikas, D., et al. 1999. Gas chromatographic-mass spectrometric detection of S-nitroso-cysteine and S-nitroso-glutathione. Anal Biochem. 272: 117-22.

  8. S- ASK1 inactive Trx SH H2O2 H+ ASK1 active Trx Reductase S S- H2O Trx Trx SH S NADP+ NADPH Thioredoxin Saitoh, M., et al. 1998. Mammalian thioredoxin is a direct inhibitor of apoptosis signal- regulating kinase (ASK) 1. Embo J 17: 2596-2606.

  9. Thioredoxin Holmgren, A. & B. M. Sjöberg. 1972. Immunochemistry of thioredoxin. I. Preparation and cross-reactivity of antibodies against thioredoxin from Escherichia coli and bacteriophage T4. J Biol Chem. 247: 4160-4. Antibody Liu, H., et al. 2006. The ADP-stimulated NADPH oxidase activates the ASK-1/MKK4/JNK pathway in alveolar macrophages. Free Radic Res. 40: 865-874.

  10. Protein Thiol Oxidation • Protein mixed disulfides (see above) • Protein disulfides • Other modification of protein thiols, -SNO, -SOH, -SO2H, -SO3H NBD-Cl Ellis, H. R. & L. B. Poole. 1997. Novel application of 7-chloro-4-nitrobenzo-2-oxa- 1,3-diazole to identify cysteine sulfenic acid in the AhpC component of alkyl hydroperoxide reductase. Biochemistry 36: 15013-15018.

  11. Biotin switch Methyl methanethiosulfonate (MMTS) N-[6-(biotinamido)hexyl]-3-(2-pyridyldithio)propionamide (Biotin-HPDP) Jaffrey, S. R. & S. H. Snyder. 2001. The biotin switch method for the detection of S-nitrosylated proteins. Sci STKE. 2001: PL1. Saurin, A. T., et al. 2004. Widespread sulfenic acid formation in tissues in response to hydrogen peroxide. Proc Natl Acad Sci U S A. 101: 17982-7. Huang, B. & C. Chen. 2006. An ascorbate-dependent artifact that interferes with the interpretation of the biotin switch assay. Free Radic Biol Med. 41: 562-7. Gladwin, M. T., X. Wang & N. Hogg. 2006. Methodological vexation about thiol oxidation versus S-nitrosation -- a commentary on "An ascorbate-dependent artifact that interferes with the interpretation of the biotin-switch assay". Free Radic Biol Med. 41: 557-61.

  12. Where and what should we measure? • Intracellular compartments • Extracellular • Blood and plasma • Lung lining fluid • Other

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