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) Other modification of protein thiols, - SNO, -SOH, -SO 2 H, -SO 3 H Oxidative stress compared with signaling

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

Thiol (SH) exchange with disulfide (SS) versus thiolate (S - ) oxidation Protein mixed disulfide (PSSG) PDI but remember the ratio of GSH/GSSG What about cys/cys 2 exchange?

Glutathione methods Total GSH and GSSG –Recycling method To measure GSSG first modify GSH N-ethylmaleimide (NEM) vinylpyridine To measure PSSG - release GSH with NaBH 4 Dithionitrobenzoic acid

HPLC method Others: cys cys 2 cysteic acid  -glu-cys cys-gly homocys 5 min A 365

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 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: Neuschwander-Tetri, B. A. & F. J. Roll Glutathione measurement by high-performance liquid chromatography separation and fluorometric detection of the glutathione- orthophthalaldehyde adduct. Anal Biochem. 179: A sensitive quantitative method was also developed that involves 15 N labeling of GSNO, HgCl 2 oxidation to nitrite, conversion to the pentafluorobenzyl derivatives, separation by HPLC and measurement by GC-MS. Tsikas, D., et al Gas chromatographic-mass spectrometric detection of S-nitroso-cysteine and S-nitroso-glutathione. Anal Biochem. 272:

Thioredoxin ASK1 inactive Trx S-S- SH NADPH Trx S-S- SH NADP + Trx Reductase ASK1 active H2OH2O S S H2O2H2O2 Trx H+H+ Saitoh, M., et al Mammalian thioredoxin is a direct inhibitor of apoptosis signal- regulating kinase (ASK) 1. Embo J 17:

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

Protein Thiol Oxidation Protein mixed disulfides (see above) Protein disulfides Other modification of protein thiols, -SNO, -SOH, - SO 2 H, -SO 3 H Ellis, H. R. & L. B. Poole 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: NBD-Cl

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

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