Analytical Assessment of Thiols in Biological Systems Henry Jay Forman University of California,...
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Transcript of Analytical Assessment of Thiols in Biological Systems Henry Jay Forman University of California,...
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)