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)

Where and what should we measure?

• Intracellular compartments• Extracellular

– Blood and plasma– Lung lining fluid– Other