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
extracellular
intracellular
cys glu-glu-cys
ADP ATPGCL
GSHgly
ATPADP GSHsynthase
-glu-amino acid
amino acid
ATP
ADP
5-oxoproline
cys
gly
Amino acid transporters
dipeptidase
-glu-amino acid
cys-gly
amino acid
GGTGSH
GSHtransporter
GSX
XGSX GST
NADPH
NADP+ ROOH
ROH
GSSG
GPxGR
GSSG
GSXtransporter
GSNO
Glutathione metabolismGlutathione metabolism
acivicin
BSO
Thiol (SH) exchange with disulfide (SS)
versus thiolate (S-) oxidation
2GSH H2O2GSHPx GSSG 2H2O
Protein mixed disulfide (PSSG)
PS H2O2 PSO H2O
PSO GSH PSSG OH
PDI
but remember the ratio of GSH/GSSGPSH GSSG PSSG GSH
What about cys/cys2 exchange?
Glutathione methods• Total• GSH and GSSG
– Recycling method
DTNB GSH GS TNB TNB
GS TNB GSH TNB GSSG
NADPH GSSG GSSG Reductase NADP H 2GSH
Akerboom, T. P. M. & H. Sies. 1981. Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples. Methods in Enzymology 77: 373-382
To measure GSSG first modify GSHN-ethylmaleimide (NEM)vinylpyridine
To measure PSSG - release GSH with NaBH4
-O
N+
OO OH
S
N+
O
OH
O-
O
S
NH
HN
NH2
OHS
O
O
HO
O
OH
+
NH
HN
NH2
O
S
O
O
HO
O
OH
S
N+
O
OH
O-
O
-O
N+
O
O OH
S-
+
Dithionitrobenzoic acid
HPLC method
Fariss, M. & D. J. Reed. 1987. High-performance liquid chromatography of thiols and disulfides: dinitrophenol derivatives. Methods in Enzymology 143: 101-109.
IOH
O
+ RSHRS
OH
O
+ I- + H+
F
N+
O-
O
N+
-O O
iodoacetic acid
1-fluoro-2,4-dinitrobenzene
+
+ F- + H+
N
N+
O-
O
N+
-O O
H
-O O
R
N
H
-O O
R
H
IOH
O
+ RSHRS
OH
O
+ I- + H+
F
N+
O-
O
N+
-O O
iodoacetic acid
1-fluoro-2,4-dinitrobenzene
+
+ F- + H+
N
N+
O-
O
N+
-O O
H
-O O
R
N
H
-O O
R
H
Others:cyscys2
cysteic acid-glu-cyscys-glyhomocys
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
5 min
A365
Nitrosoglutathione (GSNO)
O
O
NH
HN
NH2
OHS
O
O
HO
O
OH
+
N
NH
HN
O
S
O
O
HO
O
OH
First tie up GSH with NEMGSNO treated with mercaptoethanol to release GSHGSH 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.
Thioredoxin
Trx(SH )2 H2O2Prx 1-5 TrxS2 2H2O
TrxS2 NADPH Trx Reductase Trx(SH )2 NADP H
ASK1inactive
Trx S-
SH
NADPH
Trx S-SHNADP+
TrxReductase
ASK1active
H2OSS
H2O2
Trx
H+
Saitoh, M., et al. 1998. Mammalian thioredoxin is a direct inhibitor of apoptosis signal- regulating kinase (ASK) 1. Embo J 17: 2596-2606.
Thioredoxin
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.
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
Antibody
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.
ADP - + - +
TrxASK1
Time, min 0 1 0 1
IP: ASK1
Fol
d co
ntro
l
0
1.0
2.0
*
TrxASK1
Time, min 0 1 0 1
IP: ASK1
Fol
d co
ntro
l
0
1.0
2.0
*
Catalase - - + + ADP - + - +
TrxASK1
Time, min 0 1 0 1
IP: ASK1
Fol
d co
ntro
l
1.0
2.0
*
TrxASK1
Time, min 0 1 0 1
IP: ASK1
Fol
d co
ntro
l
1.0
2.0
*
Catalase - - + +
Trx(SH)2
Trx(SS)
1 2 3 4 5 6 7 8 9 10
Time, min 0 1 5 1 5 1 5 1 5
H2O2
ADPCatalaseDPI
- + + - - - - - -- - - + + + + + +- - - - - - - + +- - - - - + + - -
LA
DD
ER
Trx(SH)2
Trx(SS)
1 2 3 4 5 6 7 8 9 10
Time, min 0 1 5 1 5 1 5 1 5
H2O2
ADPCatalaseDPI
- + + - - - - - -- - - + + + + + +- - - - - - - + +- - - - - + + - -
LA
DD
ER
Protein Thiol Oxidation• Protein mixed disulfides (see above)• Protein disulfides • Other modification of protein thiols, -SNO, -SOH, -
SO2H, -SO3H
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.
RSOH +
Cl
NO2
N
O
N
S
NO2
N
O
N
O
NO2
N
O
N
O
+ H+ + Cl-
+ H+ + Cl-
SR
R
RSH +
Cl
NO2
N
O
N
S
NO2
N
O
N
+ H+ + Cl-
R
NBD-Cl
Biotin switch
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.
SH
S
S
SNO
H3C S SCH3
O
O
S
S
S
SNO
SCH3
Mild reduction
S
S
S
SH
SCH3 S
S
S
S-Biotin
SCH3
N S S Biotin
Methyl methanethiosulfonate (MMTS)
N-[6-(biotinamido)hexyl]-3-(2-pyridyldithio)propionamide(Biotin-HPDP)
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