3 8 0 ASSESSING M O L E C U L A R , CELL, AND TISSUE DAMAGE [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 un- der 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 reac- tions in which the aldehyde groups of some primary Michael addition products react with proximal lysine residues to form Schiff base cross- links, which would be stabilized by reduction with NaBH 4 .~7 This possibil- ity 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

A c k n o w l e d g m e n t s

We thank Dr. H. Esterbauer (University of Graz) for the generous gift of 4-hydroxy- nonenal 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 Thio l Groups and G lu t a th ione in P l a s m a

B y M I A O - L I N H u

Introduction

Essentially all of the plasma sulfhydryl (SH) groups are protein associ- ated.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).

Copyright 1994 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 233 All rights of reproduction in any form reserved.

[41] PLASMA SH AND GSH MEASUREMENT 381

is a powerful extracellular antioxidant. ~,3,4 Plasma SH groups are suscepti- ble to oxidative damage ~'3-5 and are often low in patients suffering from diseases such as coronary artery disease 6 and rheumatoid arthritis. 7-9 In addition to protein SH (P-SH) groups, plasma contains small amounts of glutathione (GSH), 3 Decreased plasma GSH has been reported in human immunodeficiency virus (HIV) infection. '

A spectrophotometr ic assay based on 2,2-dithiobisnitrobenzoic acid (DTNB or El lman 's reagent) 11 is commonly used for thiol assay, and modifications of the method ~2-'6 and reviews ~7,~s on the subject are avail- able. However , most of the procedures have been developed for cellular thiols, and conditions for plasma SH assay have not been well defined. For example, the DTNB method is strongly affected by pH, 14,15A9 an 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 spec- trofluorometric methods.

Assay Method

P l a s m a

Although freshly prepared human or rat plasma from EDTA- or hepa- rin-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 Biology and 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). 10 D. H. Baker, Nutr. Reo. 50, 15 (1991). ~1 G. 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). 14 p. C. Jocelyn, Biochem. J. 85, 480 (1962). 15 j. Sedlak and R. H. Lindsay, Anal. Biochern. 25, 192 (1968). J6 G. Bellomo, H. Thor, and S. Orrenius, this series, Vol. 186, p. 627. t7 p. C. Jocelyn, this series, Vol. 143, p. 45. ~8 M. E. Anderson, in "Handbook of Methods for Oxygen Radical Research" (R, A. Green-

wald, ed.), p. 317. CRC, Boca Raton, Florida, 1985. 19 D. 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) -EDTA (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 superna- tant 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 -SH, 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 super- natant 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 addi- tion, 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 GSH] '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-SH level of a plasma is calculated by subtracting the GSH level from the T-SH level. There normally is little difference between T-SH and P-SH because of the low GSH levels in the plasma. 3,~ The T-SH values obtained are around 400-600/xM for human plasma 8,25 and 300-500 /zM for rat plasma. 2'2~

Remarks. The volume of plasma and reagents can be reduced propor- tionately for the measurement of T-SH (Procedure 1) and GSH if a micro- centrifuge 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).

0.5

0.4

0 , /

o r ! tO

.<

--Q

0.3

0.2

0.1

384 ASSESSING MOLECULAR, CELL, AND TISSUE DAMAGE [41]

0 . 0 I I I I I I ~ _ _ l

5 10 15 20 25 30 35 40

M i n u t e s

FIc, I. Time course of color development and stability in T -SH 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 centrifuga- tion. 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-SH groups is demon-

26 M.-L. Hu, unpublished data.

[42] PROTEIN S - T H I O L A T I O N AND D E T H I O L A T I O N 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

In ter ferences . Tremendous interferences occur in both Procedure 1 and 2 for T-SH measurement when cigarette smoke-exposed plasma sam- ples are used. 26 The 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

27 M. Butler, T. Ginnina, D. I. Cargill, F. Popick, and B. G. Steinetz, Proc. Soc. Exp. Biol. Med. 132, 484 (1969).

28 D. A. Gerber, N. Cohen, and R. Giustra, Biochem. Pharmacol. 16, 115 (1967). 29 D. 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

By 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 hu- man 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

Copyright 1994 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 233 All rights of reproduction in any form reserved.