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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, -SO2H, -SO3H Oxidative stress compared with signaling Glutathione metabolism -glu-amino acid amino acid GSX GSSG cys-gly GSH GGT GSH transporter extracellular cys gly dipeptidase Amino acid transporters acivicin intracellular GSSG NADPH ROH GR GPx amino acid 5-oxoproline ROOH NADP+ GSH GSX -glu-amino acid GSNO GST ADP X ATP gly GSH synthase -glu-cys ATP ADP cys glu GCL BSO ATP ADP Protein mixed disulfide (PSSG) Thiol (SH) exchange with disulfide (SS) versus thiolate (S-) oxidation 2GSH H 2O2 GSHPx GSSG 2H 2O PSH GSSG PDI PSSG GSH but remember the ratio of GSH/GSSG PS H 2O2 PSO H 2O PSO GSH PSSG OH What about cys/cys2 exchange? Glutathione methods • Total • GSH and GSSG HS O O HO N H – Recycling method DTNB GSH O H N OH O O NH2 N+ + OOH S GS TNB TNB S -O GS TNB GSH O N+ TNB GSSG Dithionitrobenzoic acid O O OH Reductase NADPH GSSG GSSG NADP H 2GSH O To measure GSSG first modify GSH N-ethylmaleimide (NEM) vinylpyridine N+ OOH S S O O O O H N HO To measure PSSG - release GSH with NaBH4 N H O S- OH NH2 + Akerboom, T. P. M. & H. Sies. 1981. Assay of glutathione, glutathione disulfi de, and glutathione mixed disulfi des in biological samples. Methods in Enzymology 77: 373-382 -O N+ O O OH HPLC method iodoacetic iodoacetic acid acid O O O O + + I I RSH RSH + + RS RS OH OH II-- + + + H H+ OH OH A365 1-fluoro-2,4-dinitrobenzene 1-fluoro-2,4-dinitrobenzene O O 5 min O O N++ N O-O -O -O O O R R N N N++ N QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. O-O F F -O -O N++ N O O + + H H -O -O R R O O N N H H -O -O N++ N O O + + - + F F- + + H H+ Others: cys cys2 cysteic acid -glu-cys cys-gly homocys H H Fariss, M. & D. J. Reed. 1987. High-performance liquid chromatography of thiols and disulfi des: dinitrophenol derivatives. Methods in Enzymology 143: 101-109. 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. O HS O O H N + HO Neuschwander-Tetri, B. A. & F. J. Roll. 1989. Glutathione measurement by high-performance liquid chromatography separation and fluorometric detection of the glutathioneorthophthalaldehyde adduct. Anal Biochem. 179: 236-41. N H O O N OH S O H N N H HO O OH NH2 O 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. O O Thioredoxin Trx(SH )2 H 2O2 TrxS2 2H 2O Prx 1-5 TrxS2 NADPH Trx(SH )2 NADP H Trx Reductase ASK1 Trx S SH inactive H+ ASK1 active Trx S- SH Trx Reductase NADP+ H2O2 Trx S S H2O NADPH Saitoh, M., et al. 1998. Mammalian thioredoxin is a direct inhibitor of apoptosis signal- regulating kinase (ASK) 1. Embo J 17: 2596-2606. Thioredoxin Antibody ADP Catalase Time, min 0 + 1 + 0 Trx ASK1 Fold control IP: ASK1 + + 1 Time, min H2O2 ADP Catalase DPI LADDER 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. Quic kTime™ and a TIFF (LZW) decompres sor are needed to see this picture. 0 - 1 + - 5 + - 1 + - 5 + - 1 + + 5 + + 1 + + - 5 + + - 2.0 Trx(SS) Trx(SH) 2 1.0 * 0 1 2 3 4 5 6 7 8 9 10 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. Protein Thiol Oxidation • Protein mixed disulfides (see above) • Protein disulfides • Other modification of protein thiols, -SNO, -SOH, SO2H, -SO3H R O S N R Cl Cl S N RSH + O N NO2 + H+ + Cl- NO2 O + H+ + Cl- NO2 O RSOH + N N + H+ + Cl- N N N O O R S O N NO2 N NBD-Cl NO2 Ellis, H. R. & L. B. Poole. 1997. Novel application of 7-chloro-4-nitrobenzo-2-oxa1,3-diazole to identify cysteine sulfenic acid in the AhpC component of alkyl hydroperoxide reductase. Biochemistry 36: 15013-15018. Biotin switch Methyl methanethiosulfonate (MMTS) O SH H3C S S O SCH3 S SCH3 S SCH3 S SCH3 S S S S S S S SNO SNO SH S-Biotin Mild reduction N S S Biotin 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 Snitrosylated 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. Where and what should we measure? • Intracellular compartments • Extracellular – Blood and plasma – Lung lining fluid – Other