380

ASSESSING M O L E C U L A R , C E L L , A N D T I S S U E D A M A G E

[41]

tively, is often considerably lower than the number of these residues that
disappeared on treatment with 4-hydroxynonenal. For example, reaction
of 4-hydroxynonenal with glyceraldehyde-3-phosphate dehydrogenase under the conditions described here led to the modification of 5 histidine,
3.5 lysine, and 2.5 cysteine residuesJ 7 By means of the Raney nickel
procedure, only 17% of the modified cysteine could be attributed to a
simple Michael addition reaction, whereas 90 and 28%, respectively, of
the histidine and lysine residues were present as simple Michael addition
products, as determined by HPLC of the o-phthalaldehyde derivatives of
NaBH4-treated acid-hydrolyzed samples. It was proposed that the poor
recovery of lysine and cysteine residues might be due to secondary reactions in which the aldehyde groups of some primary Michael addition
products react with proximal lysine residues to form Schiff base crosslinks, which would be stabilized by reduction with NaBH 4.~7This possibility is supported by (1) the observation that the number of cysteine plus
histidine residues that could not be accounted for as Michael addition
products is equal to the number of lysine residues that could not be
accounted for and (2) by the appearance of protein conjugates which
sodium dodecyl sulfate (SDS) gel electrophoresis exhibited molecular
weights about two times that of the native subunit.~7
Acknowledgments
We thank Dr. H. Esterbauer (University of Graz) for the generous gift of 4-hydroxynonenal diethylacetal. We also thank Dr. R. L. Levine and B. S. Berlett (National Institutes
of Health) for advice on amino acid analysis.

[41] M e a s u r e m e n t of P r o t e i n T h i o l G r o u p s a n d G l u t a t h i o n e
in P l a s m a
By MIAO-LIN Hu

Introduction
Essentially all of the plasma sulfhydryl (SH) groups are protein associated.t,2 Albumin is the most abundant plasma protein (40-60 mg/ml) and
l D. D. M. Wayner, G. W. Burton, K. U. Ingold, L. R. C. Barclay, and S. J. Locke,
Biochim. Biophys. Acta 924, 408 (1987).
2 A. Hamvas, R. Palazzo, L. Kaiser, Jo Cooper, T. Shuman, M. Valazquez, B. Freeman,
and D. P. Schuster, J. Appl. Physiol. 72, 621 (1992).

METHODS IN ENZYMOLOGY, VOL. 233

Copyright 1994 by Academic Press, Inc.

All rights of reproduction in any form reserved.

[41]

PLASMA SH AND GSH MEASUREMENT

381

is a powerful extracellular antioxidant. ~,3,4Plasma SH groups are susceptible to oxidative damage ~'3-5 and are often low in patients suffering from
diseases such as c o r o n a r y artery disease 6 and rheumatoid arthritis. 7-9 In
addition to protein S H ( P - S H ) groups, plasma contains small amounts o f
glutathione (GSH), 3 Decreased plasma G S H has been reported in human
immunodeficiency virus (HIV) infection. '
A spectrophotometric assay based on 2,2-dithiobisnitrobenzoic acid
( D T N B or E l l m a n ' s reagent) 11 is c o m m o n l y used for thiol assay, and
modifications o f the method ~2-'6 and reviews ~7,~s on the subject are available. H o w e v e r , most of the procedures have been developed for cellular
thiols, and conditions for plasma S H assay have not been well defined.
F o r example, the D T N B method is strongly affected by pH, 14,15A9an effect
often unappreciated by researchers. This chapter describes convenient
assays for P - S H a n d G S H in plasma using spectrophotometric and spectrofluorometric methods.

Assay Method
Plasma

Although freshly prepared h u m a n or rat plasma from E D T A - or heparin-treated blood is preferred, samples stored at 4 for up to 2 days or
frozen at - 7 0 are also satisfactory.
3 B. Halliwell and J. M. C. Gutteridge, "Free Radicals in Biologyand Medicine." Clarendon,
Oxford, 1989.
4 B. Halliwell and J. M. C. Gutteridge, Arch. Biochem. Biophys. 280, 1 (1990).
5 B. Frei, R. Stocker, and B. V. Ames, Proc. Natl. Acad. Sci. U.S.A. 85, 9448 (1988).
6 K. Kadota, Y. Tui, R. Hattori, Y. Murohara, and C. Kawai, lpn. Circ. J. 55, 937 (1991).
7 A. Lorber, C. M. Pearson, W. L. Meredith, and L. E. Gantz-Mandall, Ann. Int. Med.
61, 423 (1964).
s M. Haataja, Scand. J. Rheurnatol. 4 (Suppl), 1 (1975).
9 N. D, Hall and A. H. Gillan, J. Pharm. Pharmacol. 31, 676 (1979).
10D. H. Baker, Nutr. Reo. 50, 15 (1991).
~1G. L. Ellman, Arch. Biochern. Biophys. 82, 70 (1959).
1l A. F. Boyne and G. L. EUman, Anal. Biochem. 46, 639 (1972).
t3 p. H. W. Butterworth, H. Baum, and J. W. Porter, Arch. Biochem. Biophys. 118, 716
(1967).
14p. C. Jocelyn, Biochem. J. 85, 480 (1962).
15j. Sedlak and R. H. Lindsay, Anal. Biochern. 25, 192 (1968).
J6G. Bellomo, H. Thor, and S. Orrenius, this series, Vol. 186, p. 627.
t7 p. C. Jocelyn, this series, Vol. 143, p. 45.
~8M. E. Anderson, in "Handbook of Methods for Oxygen Radical Research" (R, A. Greenwald, ed.), p. 317. CRC, Boca Raton, Florida, 1985.
19D. R. Grassetti and J. R. Murray, Arch. Biochem. Biophys. 119, 41 (1967).

382

ASSESSING MOLECULAR, CELL, AND TISSUE DAMAGE

[41]

Total Thiols in Plasma

Reagents
DTNB, 10 mM (4 mg/ml) in absolute methanol; the reagent is stable
for up to 2 weeks when stored at 4
Tris base (0.25 M ) - E D T A (20 raM) buffer, pH 8.2
Procedure 1. An aliquot of plasma (0.20 ml) is mixed in a 10-ml test
tube with 0.6 ml of the Tris-EDTA buffer followed by addition of 40/zl
of 10 mM DTNB and 3.16 ml of absolute methanol. The test tube is capped,
and the color is developed for 15-20 rain, followed by centrifugation at
3000 g for I0 rain at ambient temperature. The absorbance of the supernatant is measured at 412 nm (A) and subtracted from a DTNB blank (B)
and a blank containing the sample without DTNB. In agreement with
Sedlak and Lindsay, ~s a value of 0.03 at 412 nm for the sample blank is
consistently obtained. Consequently, individual sample blanks are not
critical and can be taken as 0.03.
Total SH groups are conveniently calculated using an absorptivity of
13,600 cm -l M -I as follows:
(A - B - 0.03) (4.0/0.2)/13.6 = (A - B - 0.03) 1.47 mM

(1)

Remarks. The assay procedure 2 is modified from that of Sedlak and

Lindsay 15 originally developed for simultaneous determination of total
thiols (T-SH), P - S H , and non-protein-bound SH groups in animal tissues
and red blood cells. The method employs a mild precipitation of proteins
with methanol (80% final concentration) during color formation. The supernatant is relatively clear and free of interferences.
Procedure 2. An aliquot of plasma (50/~1) is mixed with 1.0 ml of the
Tris-EDTA buffer, and the absorbance at 412 nm is measured (A~). To
this is added 20 t~l of 10 mM DTNB. After 15 rain at ambient temperature
the absorption is measured again (A2) together with a DTNB blank (B).
Total SH groups are calculated as follows:
(A 2 - A~ - B) (1.07/0.05)/13.6 = (Az - A~ - B) 1.57 mM

(2)

Remarks. The author has consistently found that use of GSH (reduced
glutathione) as standard (1.0 mM dissolved in deionized water) is required
to ensure the recovery and reproducibility of the measurement. In addition, normalization of T - S H for total protein may be necessary when
changes in plasma protein content may occur)

2o M.-L. Hu, C. J. Dillard, and A. L. Tappel, Agents Actions 25, 132 (1988).

['41]

PLASMA S H AND G S H MEASUREMENT

383

Plasma Glutathione Measurement

Principle. As plasma contains approximately 5 and 20/xM GSH for
humans 6 and rats, 2-22 respectively, the spectrophotometric method is
not sensitive enough for the measurement. A convenient method using a
fluorescent reagent, o-phthalaldehyde, for measuring tissue G S H 23'24 has
been modified for measurement of plasma G S H ] '21
Reagents
Trichloroacetic acid (TCA), 10%, (w/v)
Sodium phosphate 0.1 M/EDTA 5 mM, pH 8.0
o-Phthalaldehyde, 1 mg/ml in absolute methanol
GSH, 1.0 mM in deionized water
Procedure. A 0.5-ml aliquot of plasma is added to 0.5 ml of cold 10%
TCA. After 10 min in ice, the mixture is centrifuged (3000 g for 15 rain
at 4), and 0.2 ml of the supernatant is mixed with 1.7 ml of the phosphate/
EDTA buffer and 0.1 ml of o-phthalaldehyde. After 15 min the fluorescence
at 350 nm excitation and 420 nm emission is read against a blank that
contains deionized water to replace plasma. The concentration of GSH
is determined using a GSH standard to replace plasma.
Plasma Protein Thiol Groups
The P - S H level of a plasma is calculated by subtracting the GSH level
from the T - S H level. There normally is little difference between T - S H
and P - S H because of the low GSH levels in the plasma. 3,~ The T - S H
values obtained are around 400-600/xM for human plasma 8,25and 300-500
/zM for rat plasma. 2'2~
Remarks. The volume of plasma and reagents can be reduced proportionately for the measurement of T - S H (Procedure 1) and GSH if a microcentrifuge and a spectrophotometer capable of handling small volumes
are available.
Discussion

Procedures for Total Thiols. For normal appearing plasma, Procedure

2 is simple and appropriate. However, the procedure is not satisfactory
2I M.-L. Hu, C. J. Dillard, and A. L. Tappel, Res. Commun. Chem. Pathol. Pharmacol.$9,
147 (1988).
22 M. E. Anderson and A. Meister, J. Biol. Chem. 255, 9530 (1980).
23 V. H. Cohn and J. Lyle, Anal. Biochem. 14, 434 (1966).
24 p, j. Hissin and R. Hill, Anal. Biochem. 74, 214 (1976).
25 M.-L. Hu, S. Louie, C. E. Cross, P. Motchnik, and B. HaUiwell, J. Lab. Clin. Med. 121,
257 (1993).

384

[41]

ASSESSING MOLECULAR, CELL, AND TISSUE DAMAGE

0.5

0.4

0,/

--Q

0.3

o
r!
tO

0.2
.<
0.1

0.0

10

15

20

25

30

~ _ _ l

35

40

Minutes
FIc, I. Time course of color development and stability in T - S H measurement (Procedure
2). (0) pH 8.2, (V) pH 7.4, (V) pH 7.0.

for plasma samples with turbidity that cannot be removed by centrifugation. The problem can be avoided using Procedure 1, which employs mild
precipitation of proteins and solubilization of lipids with 80% methanol.
The T - S H values obtained from the same plasma samples by the two
procedures agree within 5%. 26
Stability of Color. The formation of color (due to liberated
p-nitrothiophenol anion) is completed within 15 min for both Procedures
1 and 2. The color is stable for at least 30 min thereafter. One factor that
can affect the stability of color is the amount of plasma or protein used
in the T - S H assay (equivalent to - 3 . 5 mg plasma protein/ml assay mix
in both procedures). This dilution factor (->20) will greatly minimize the
instability of p-nitrothiophenol as affected by any oxidant that may be
present in plasma. 25
Effect ofpH. Using DTNB, Jocelyn 14 reported a value of 0.6 SH per
mole of bovine serum albumin at pH 7.6 and 0 per mole at pH 6.8. In
contrast, Sedlak and Lindsay ]5 found that color was produced at any pH
above 4.7. The effect of pH on human plasma T - S H groups is demon26 M.-L. Hu, unpublished data.

[42]

PROTEIN S-THIOLATION AND DETHIOLATION

385

strated in Fig. 1 (assayed using Procedure 2). 26 The color develops to

maximum intensity within 10 min at pH 8.2, whereas the maximum color
is not reached and the absorbance continues to increase at pH 7.4 and
7.0 even at 40 min after addition of DTNB. These findings are in agreement
with those of Sedlak and Lindsay, 15 who observed that maximum color
is only obtained for various types of samples at pH 8.0-9.0. The slow
reaction of DTNB with plasma SH groups at physiological pH (-7.4)
has been used to determine plasma SH reactivity, that is, the rate of
SH-disulfide exchange reaction, 9,2 and the effect of antiarthritic drugs
on the reactivity. 9'2,21'27-29
I n t e r f e r e n c e s . Tremendous interferences occur in both Procedure 1
and 2 for T - S H measurement when cigarette smoke-exposed plasma samples are used. 26The absorbance at 412 nm continues to rise with increased
exposure and assay time. Precipitation of proteins (50/xl of plasma) in
1 ml of 5% TCA (containing 5 mM EDTA) followed by suspension in the
Tris-EDTA buffer (Procedure 2) appears to remove such interferences. 26
27M. Butler, T. Ginnina,D. I. Cargill,F. Popick, and B. G. Steinetz,Proc. Soc. Exp. Biol.
Med. 132, 484 (1969).
28D. A. Gerber, N. Cohen, and R. Giustra, Biochem. Pharmacol. 16, 115 (1967).
29D. T. Waltz and M. J. DiMartino, Proc. Soc. Exp. Biol. Med. 1411,263 (1972).

[42] P r o t e i n S - T h i o l a t i o n a n d D e t h i o l a t i o n
B y JAMES A. THOMAS, YUH-CHERNG CHAI, and CHE-HUN JUNG

Introduction
S-Thiolated proteins (mixed disulfides of proteins and low molecular
weight thiols) are very early products of protein oxidation during oxidative
stress, occurring within seconds after the generation of oxygen radicals.
As a result, assessment of the extent and specificity of this process during
oxidative stress is one of the best measures of the primary effects of
oxygen radical generation on intact cells (see Fig. 1). The development
of methods for measuring the S-thiolation status of individual proteins,
eventually studying organs of intact animals and even humans, is essential
for a more complete understanding of the role of oxidative stress in human disease.
The list of proteins that participate in S-thiolation/dethiolation is quite
long, and in many cases S-thiolation has been correlated with an alteration
in protein function. The complexity of the process is increased by the
METHODS IN ENZYMOLOGY, VOL. 233

Copyright 1994by Academic Press, Inc.

All rights of reproduction in any form reserved.