Alkaline hydrolysis of N-ethylmaleimide allows a rapid assay of glutathione disulfide in biological samples

ANALYTICALBIoCHEMISTRY154,205-208(1986)

Alkaline Hydrolysis of N-Ethylmaleimide Allows a Rapid Assay of Glutathione Disulfide in Biological Samples PAOLO

SACCHETTA,*

DOMENICO

Received

Dr COLA,*

August

AND

GIORGIO

FEDERlCIt

27, 1985

The estimation ofglutathione disulfide (GSSG) is based on the NADPH-dependent glutathione reductase reaction. A new method has been developed to ehminate the inactivating effect of Nethylmafeimide (NEM), added to prevent glutathione oxidation, on glutathione reductase. This method takes advantage of instability ofNEM in alkaline solutions. The product of NEM hydrolysis, N-ethylmaleamic acid. obtained under accurate pH-controlled conditions, is compatible with a good activity of glutathione reductase which allows total recovery and measurement of GSSG. The method, applied to estimation of GSSG content in human erythrocytes and rat liver, gives results in optimum agreement with values reported in literature. Because of its simple performance and rapidity. the procedure can be considered an improved method in removing NEM and is particularly advantageous when a large number of biological samples must be treated and estimated. :CI 1986

Academic

Press.

Inc.

KEY WORDS: spectrophotometry: peptides; glutathione.

pH determination:

The peptide glutathione is present in biological system:s in different chemical species. The most abundant form is made up of reduced glutathione (GSH), whereas the disulfide form is present only in minimal amounts ranging from I to 2% of the total ( 1). Several methods have been developed to determine the relatively large amount of GSH in biological materials, including chemical (2) and enzymatic assays (3-5). However, the measurement of glutathione disulfide (GSSG) is more difficult because of its low concentration and the ease with which GSH is oxidized to GSSG in biological extracts. The measurement of GSSG by NADPHdependent glutathione reductase (4) ensures high specificity and good sensitivity. However, to prevent the undesirable oxidation of GSH in the course of sample treatment, a means must be intromduced for blocking free thiol groups (6). N-Ethylmaleimide (NEM)’ has been pre’ Abbreviation

used: NEM,

tissue homogenization;

clinical

chemistry:

ferred for this purpose. However, the reagent interferes with subsequent enzymatic determination of GSSG by inhibiting glutathione reductase. Several procedures that remove excess NEM, including extractions with ethyl ether (4) or column chromatography (7-9) have been described. Such methods are laborious and require additional manipulations that may lead to loss of sample. Therefore, we have developed a rapid and simple procedure for destroying excess NEM, taking advantage of instability of the reagent in alkaline solution. MATERIALS

AND

METHODS

Materials Reduced glutathione, GSSG, NADPH, 5,5’dithiobis(Z-nitrobenzoic acid), and ghttathione reductase (5 mg/ml) were obtained from Biochemia-Boehringer (Milan, Italy). NEM was purchased from Serva (Germany). All other chemical reagents were delivered from Carlo Erba (Italy).

N-ethylmaleimide. 205

0003-2697186 Copyright All

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$3.00 1986

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206

SACCHETTA.

DI

COLA.

Rat livers were from female animals (350 g) fed ad lihiturn with Purina laboratory chow. Human red blood cells were obtained from healthy male donors. All measurements were carried out in a double-beam Beckman spectrophotometer provided with a recorder. Prepcwation qf‘lhe Sample Heparinized blood samples (10 ml) were withdrawn and centrifuged at 3000~ for IO min to collect erythrocytes. Sedimented cells were washed three times with cold saline at 4°C. The residue was hemolyzed with distilled water (5 ml) in the presence or absence of 10 mM NEM. After 15 min at room temperature, samples were deproteinized by addition of perchloric acid to a final concentration of 1 M. After I5 min at 4°C the precipitate was removed by centrifugation at 50,OOOg for 30 min. Rat liver tissue (l/5. w/v) was homogenized in 0.15 M potassium phosphate at pH 7 containing 1 mM EDTA and 12.5 mM NEM with a Potter homogenizer. A separate portion of tissue was homogenized in the same buffer lacking NEM and deproteinized with perchloric acid. After I5 min at 4°C the precipitate was removed by centrifugation at 50,000~ for 30 min. All supernatant fluids obtained from either liver or red blood cells were adjusted at pH 11 with KOH to hydrolyze NEM and. after 5 min. were neutralized with HCl. All pH adjustments were made with vigorous mixing in a pH meter with care taken to avoid local extreme alkalinization. Insoluble potassium perchlorate was removed by centrifugation after freezing and thawing. The supernatant liquids could be used for GSSG determination. .drzaiJfical Prixvdwr Specf rctphntornetric~ prowdwe f&r 4 to 20 r~~lol (Mrthod ‘4). The procedure allows a direct estimation of GSSG by a stoichiometric measurement of NADPH converted into the oxidized form. A 0.8~ml amount of sample was mixed with 0.1 ml of 1 M sodium phosphate at pH 7 containing 10 mM EDTA and 0.1 ml of 1.8 mM

AND

FEDERICI

NADPH. After 3 min at 25°C. the reaction was started by addition of 2 ~1 of glutathione reductase (1.2 units). The reaction was monitored by recorder for 5 min after the addition of enzyme. The absorbance decrease was used to calculate GSSG content by using the extinction coefficient of 6220 for NADPH. .~l,ec.trc,I)}roto,llc,fric.procedwe,tiw 30 lo 500 (VW)/ (izlctllc~i B). This procedure, essentially derived from that of Tietze (4), is useful for estimating small amounts of GSSG. The following reagents were added in order to a spectrophotometer cell maintained at 25°C: 0.5 ml of 0.2 M sodium phosphate at pH 7.0; 10 ~1 ofO.l M EDTA: 0.1 ml of 1.8 mM NADPH: 0.1 ml glutathione reductase solution containing 0.6 units: 20 ~1 5,5’-dithiobis(2-nitrobenzoic acid) (0.4 mg/ml): and distilled water to give a total volume of 0.95 ml. After a blank reaction was measured, the sample (50 ~1) was added and the slope of the reaction rate was measured for 0.5-2.0 min. Since the reaction rate is influenced by the presence of residual perchlorate and other lowmolecular-weight compounds occurring in the sample (lo), each single estimation was also performed in the presence of a known amount of GSSG (20 ~1, final concentration of 45 nM) added as an internal standard and the final reaction rate was measured. A separate assay was carried out to measure the reaction rate produced by the GSSG standard in the absence of sample (external standard). The sample rate was corrected taking into account the efficiency ofthe system calculated as follows. The reaction rate of the sample was subtracted from the final rate obtained after the addition of internal standard. This value was used to calculate the ratio between the internal and external standard slopes. The ratio was used as a correcting factor to calculate the efficiency of the system, which ranged from 85 to 70% according to the amount of sample added (0.0 l-0.05 PI). RESULTS Since the characteristic absorbance of NEM at 305 nm is lost by hydrolysis, the presence

GLUTATHIONE

FIG. I (0) Re:jidual absorbance (a. of initial value) at 305 nm of NEM after 5 min incubation at different pH values. NEM at linal concentration of IO mM was dissolved in 0.1 M of Tris/HCl (pH 7-9) or 0.1 M of potassium phosphate buffer (pH 10-13.7). After 5 min each sample was diluted IO-fcld in 0.2 M potassium phosphate buffer at pH 7 and the optical density was measured. (0) Residual amount of GSSG (“/ of initial value) after 20 min incubation at differen-t pH values. GSSG at final concentration of 0.25 IIIM was dissolved in the same buffers as indicated above. After 20 min each sample was diluted IO-fold in 0.2 M potassium phosphate buffer at pH 7 and utilized for GSSG determination as reported under Materials and Methods (method A).

of the reagen-t can be followed by measuring the residual optical density after incubation at different pH values. As shown in Fig. 1 the compound is completely hydrolyzed after 5 min at pH 1 I. In contrast, GSSG (Fig. 1) is not modified by incubation for 20 min in the

DISULFIDE

207

ASSAY

same medium up to pH 12. It is only at higher pH values that GSSG is decreased. To exclude the possibility that the hydrolysis product of NEM (N-ethylmaleamic acid) ( 1 1) can interfere in the enzymatic assay, comparative measurements were performed in the presence or absence of previously hydrolyzed NEM at pH 11. As seen in Fig. 3, the amount of titratable GSSG is not significantly influenced by NEM treatment. Good linearity is obtained with respect to GSSG in the ranges of 4-20 PM and SO-500 nM with a recovery greater then 9596. The method has been applied for determination of GSSG content in biological samples: human red blood cells and rat liver. Table I presents the results obtained by our method compared with values reported by others ( 12.13). Glutathione disulfide content of red blood cells and rat liver was also estimated after prolonged incubation of extracts at 4°C for 24 h in the presence and absence of NEM. DISCUSSION

The major advantage of method described here is the rapidity with which GSSG can be evaluated. The determination of GSSG in biological samples containing NEM by NADPH-dependent glutathione reductase is reliable but depends on the total removal of the excess thiol

FIG. 2. DIetermination of GSSG concentration in presence or absence of NEM. GSSG was dissolved in distilled water at Z mM concentration. Aliquots of GSSG solution (1 ml) were added to 9 ml of 0.2 M potassium phosphate buffer at pH 6.2 containing I2 mM NEM (m) or without NEM (U). All solutions were adjusted to pH I I with KOH under pH meter control. After 5 min incubation the mixtures were neutralized by HCI addition. and suitable aliquots were withdrawn and diluted in 0. I M phosphate buffer at pH 7 to obtain the rinal concentrations indicated in figure. GSSG was assayed as described under Materials and Methods by method A (A) or method B (B).

208

SACCHETTA.

DI

COLA, TABLE

GSSG

CONTENT

IN HUMAN

AND

FEDERlCI

I

ERYTHROCY

TES AND IN RAT LIVER

TISSUES

GSSG Tissue Erythrocytes (n = 5) Rat liver (11 = 3)

NEM

(10 mM)

5.6 + 0.8” 20.0 * 1.2”

5.1 + 0.7* 18.9 k 1.2”

None

Ref.

91 rt lob 212 * 15b

20 (13)

4(12)

Note. GSSG is expressed as nmol/ml of blood and as nmol/g of wet rat liver. Erythrocytes and rat liver tissue prepared as described under Materials and Methods were treated in the presence or absence of NEM. “An aliquot was withdrawn and GSSG immediately assayed. b The remaining part of the sample was stored at 4°C for 24 h before the GSSG was assayed.

reagent. This step is the most critical one in the entire procedure. In our method. NEM is not removed, but advantage is taken of the instability of the compound in alkaline solutions, which destroy remaining excess NEM without affecting GSSG stability: GSSG remains unmodified up to pH 11, but is affected at higher pH values (Fig. 1). Therefore we require accurate control of pH, taking care to avoid local extreme alkalinization. All other operations are not critical steps. The method presented here ensures good linearity within a relatively large interval of GSSG concentration (Fig. 2) allowing the direct determination of low amounts of GSSG usually present in biological materials. Confirmation of reliability of the present procedure was attained by determinating GSSG in erythrocytes and in liver. The concentrations of GSSG found are in optimum agreement with data previously reported by Akerboom and Sies (6) for rat liver, and also fit the range of values indicated by others for human erythrocytes (4,12). It should be mentioned that the addition of NEM in biological extracts at concentrations greater than 30 mM produces an apparent partial loss of GSSG (up to 30%). Nevertheless, this drawback does not seem to be a severe limitation to our method since such a high concentration of NEM is not absolutely required to block all available sulfhydryl groups in cellular extracts.

extracts

A recent paper (14) reports that NEM must be added at a concentration of at least 100 mM to ensure total blockage of thiol groups. However, under our different extraction conditions the reactivity of NEM is significantly enhanced, thereby allowing the use of a lower concentration of reagent. REFERENCES 1. Kosower, N. S., and Kosower, E. M. (1978) Inf. Rev. C.wl. 54, IOO- 160. 2. Hissin, P. J., and Hilf, R. (I 976) .dnn/. BIOX~WII. 74, 2 14-226. 3. Racker, E. (195 I) J Biol. Chern. 190,685-696. 4. Tietze, F. ( 1969) itnal. Bioclrem. 27, 502-522. 5. Brehe, J. E., and Burch. H. B. ( 1976) Anal. Biochem. 74, 189-197. 6. Akerboom, T. P. M.. and Sies. H. (1982) rn Methods in Enzymology (Jacoby. W. B.. ed.). Vol. 77. pp. 373-382. Academic Press. New York. 7. Guthenberg, H.. and Rapaport, S. (1968) .tc!r( Viol. A4ed. Ger. 20, 559-563. 8. Wendell, P. L. (1970) Eiochem. J. 117, 66 l-665. 9. Akerboom, T. P. M., Bilzer, M., and Sies. H. (1982) .I. Biol. Churn. 257, 4248-4252. IO. Meister, A.. and Anderson, M. E. ( 1983) .tnn~ Rev. Biochwz. 52, 7 I I-760. I I. Riordan, J. F., and Vallee. B. L. (1967) in Methods in Enzymology (Colowick. S. P.. and Kaplan, N. 0.. eds.). Vol. 11. pp. 541-548, Academic Press. New York. 12. Srivastava. S. K.. and Beutler, E. (1968) .dnal. Biochern. 25, 70-76. 13. Brigelius, R.. Muckel. C.. Akerboom, T. P. M., and Sies, H. (1983) &o&m. Pharmacol. 32, 25292534. 14. Alpert, A. J.. and Gilbert, H. F. (1985) Arm/. Biochern. 144, 553-562.