Analytical Assessment of Thiols in Biological Systems

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Analytical Assessment of Thiols in Biological
Systems

• Henry Jay Forman
• University of California, Merced

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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
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Glutathione metabolism
extracellular
intracellular
GSNO
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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?
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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
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HPLC method
Others cys cys2 cysteic acid ?-glu-cys cys-gly ho
mocys
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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.
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Thioredoxin
Saitoh, M., et al. 1998. Mammalian thioredoxin is
a direct inhibitor of apoptosis signal-
regulating kinase (ASK) 1. Embo J 17 2596-2606.
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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.
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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-di
azole to identify cysteine sulfenic acid in the
AhpC component of alkyl hydroperoxide reductase.
Biochemistry 36 15013-15018.
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Biotin switch
Methyl methanethiosulfonate (MMTS)
N-6-(biotinamido)hexyl-3-(2-pyridyldithio)propio
namide (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.
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Where and what should we measure?

• Intracellular compartments
• Extracellular
• Blood and plasma
• Lung lining fluid
• Other