Download Analytical Assessment of Thiols in Biological Systems

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