A semiautomated system for measurement of glutathione in the assay of glutathione peroxidase

ANALYTICAL

BIOCHEMISTRY

89, 430-436 (1978)

A Semiautomated System for Measurement of Glutathione in the Assay of Glutathione Peroxidase JACK J. ZAKOWSKI AND AL L. TAPPEL Department of Food Science and Technology, University of California, Davis, California 95616 Received January 12, 1978 The most commonly used assay procedures for measurement of glutathione (GSH) peroxidase are unsuitable for kinetic studies; therefore, a semiautomated system that is based upon the 5,5’-dithio-bis-2-nitrobenzoic acid (DTNB) reaction with sulthydryls was developed for the assay of relatively purified samples of the enzyme. Sulfhydryl standards in the range of 0- 1.5 mM gave a linear response with r* > 0.998. In the enzyme assay, unreacted GSH was measured continuously. The -ln[GSH] was plotted vs the time of reaction to obtain first-order rate constants. The standard deviation over a twofold enzyme concentration range was 9%. The relative merits of the semiautomated DTNB, manual DTNB, and GSH reductase-NADPH coupled assays for measurement of GSH peroxidase are discussed. Sulthydryl utilization by GSH peroxidase in the presence of a fixed amount of GSH and increasing amounts of mercaptoethanol was measured by this semiautomated system. It was determined that the enzyme can utilize mercaptoethanol as one of the two donor substrates but that there is a requirement for GSH as the other donor substrate. This semiautomated assay should be applicable to other reaction systems that consume or produce sulfhydryl groups.

The most commonly used assay for the enzyme glutathione (GSH) peroxidase (EC 1.11.1.9) is the GSH reductase-NADPH coupled assay described by Paglia and Valentine (1) or some modification of this assay (26). GSH peroxidase activity has also been assayed by direct mesurement of GSH (6,7) via the reaction of the sulfhydryl group with Ellman’s reagent, 5,5’-dithio-bis-2-nitrobenzoic acid (DTNB) (8), according to the method of Sedlak and Lindsay (9) or of Beutler et al. (10). Neither of these two types of assays is useful for kinetic studies of GSH peroxidase, since NADPH is an inhibitor of the enzyme (5) and the DTNB reaction procedure is long and tedious. Accordingly, a semiautomated assay that utilizes the DTNB reaction, and that has neither of these problems, has been developed for measurement of GSH peroxidase reaction kinetics. The system can also be used to measure sulfhydryl substrate utilization and it has been determined that, contrary to previous reports (1 l), only one of the sulthydryl substrates need be GSH. 0003-2697/78/0892-0430$02.00/O Copyright 0 1978 by Academic Press, Inc. AU rights of reproduction in any fom reserved.

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Materials. Tris(hydroxymethyl)aminomethane (Tris), ethylenediaminetetraacetic acid tetrasodium salt (EDTA), mercaptoethanol, GSH, and oxidized glutathione (GSSG) were purchased from Sigma Chemical Co., St. Louis, MO. DTNB was purchased from Aldrich Chemical Co., Milwaukee, Wise. Cumene hydroperoxide was purchased from Polysciences, Inc., Rydal, Pa. A Technicon AutoAnalyzer proportioning pump I, Tygon and Solvaflex tubings of appropriate diameters, and glass connector fittings were purchased from Technicon Instruments Corp., Tarrytown, N. Y. A Cary 118C spectrophotometer (Varian Associates, Palo Alto, Calif.) was used for absorbancy measurements. The flow-through cuvette employed had a OS-cm light path. The GSH peroxidase used in the assays was purified from rat liver mitochondria (12) to a specific activity of 50,000 nmol of NADPH oxidized min/mg of protein, as determined by the coupled assay procedure (I). Methods. Figure 1 is a schematic of the semiautomated assay. The pump tubing used to carry the trichloroacetic acid and DTNB solutions was Solvaflex; all others were Tygon. The reagents used were 10% (w/v) trichloroacetic acid, and 20 mM DTNB in absolute ethanol, and the alkaline buffer was 0.4 M Tris-20 mM EDTA, pH 8.9. The volume used of each reagent is indicated in Fig. 1 by the proportioning pump flow rates, which are proportional to the diameter of the tubing used for each reagent. A delay coil is utilized to produce an approximately 3-min incubation of sample with DTNB prior to spectrophotometric assay. GSH standards in 50 mM Tris-0.1 mM EDTA, pH 7.6, were used to calibrate the instrumentation and were made fresh each day. There was a slight drift in the recorder readings with time, but this was corrected by calibration with the GSH standards several times during a 6-hr experiment. GSH peroxidase reactions were carried out at 32°C in 0.35 mM GSH, 0.2 mM cumene hydroperoxide, and 50 mM Tris-0.1 mM EDTA, pH 7.6,

LE

SFfCTAOPHOTOMETER

FIG. reaction

1. Schematic diagram products in the assay

of the semiautomated of GSH peroxidase.

system

for

measurement

of DTNB

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with a final volume of 2 ml. The addition of the cumene hydroperoxide initiated the reaction. The sampling tube from the automated apparatus was immediately inserted into the reaction mixture after the addition of the hydroperoxide. Between enzyme assays, the sampling tube pumped 0.35 mM GSH in order to maintain an appropriate baseline. The concentration of unreacted GSH remaining in the assay mixture was measured from the absorbance reading on the recorder paper and plotted as - In[GSH] vs time of the reaction. The slope of the straight line obtained from the plots is the first-order rate constant k in units of minutes-‘. The data were processed and plotted with a Hewlett-Packard 9810A calculator and plotter. In order to determine the extent of total sulfhydryl utilization in the GSH peroxidase reaction, the assay was performed in the presence of varying amounts of mercaptoethanol and the reaction was pushed to completion by a threefold excess of cumene hydroperoxide over GSH. Reactions were carried out at 32°C in the presence of 0.15 mrvr cumene hydroperoxide, 0.05 mM GSH, 0.1 ml of GSH peroxidase preparation with an activity of 0.60 AA ,,,,/min/O. 1 ml as measured by the GSH reductaseNADPH coupled assay, varying amounts of mercaptoethanol, and 50 mM Tris-0.1 mM EDTA, pH 7.6, with a final volume of 4.0 ml. The total amount of sulfhydryl utilized was calculated from the total change in absorbance at 412 nm. Reaction blanks were run with GSSG substituting for GSH to measure disulfide interchange. RESULTS Sulfhydryl determinations. Figure 2 is representative of the GSH standard curves that were obtained. All standard curves had straight-line correlation coefficients of r* > 0.999. The range of response to GSH with the flow rates shown in Fig. 1 was O-0.80 mM GSH. The initial sulthydryl concentrations of up to 1.5 mM used in the determination of total sulfhydryl utilization (below) were also linear with Y* > 0.998. GSHperoxidase assay. Figure 3 shows examples of the determination of first-order rate constants for four concentrations of enzyme. Each deter: a t ti

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5 04 ;

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*z 08 li:i.:-

4

00

02

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GSH (mM)

FIG. 2. Representative plot of absorbance response to GSH standards. Straight-line correlation coefficient r* > 0.999.

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FIG. 3. Representative determinations of first-order rate constants for four concentrations of GSH peroxidase. The - ln[GSH] is plotted against the time of reaction to obtain the slope of the line which is k: (0) relative [GSH peroxidase] = 0, k = 0 min-*; (A) relative [GSH peroxidase] = 0.25, k = 0.151 min-r; (m) relative [GSH peroxidase] = 0.5. k = 0.353 min-*; and (4) relative [GSH peroxidase] = 1.0, k = 0.635 min-‘.

mination of k was based upon a large number of data points, and values were comparable to the rates obtained by the manual DTNB assay. The high degree of linearity confirms the first-order kinetics of the GSH peroxidase reaction with respect to GSH. Figure 4 is a plot of reaction rate vs enzyme concentration in the reaction, and it illustrates the sensitivity and reproducibility of the semiautomated GSH peroxidase assay. An 18% standard deviation was observed overall for the 20-fold enzyme concentration range shown in Fig. 4. When only the faster reactions, with the relative GSH peroxidase concentration ~0.5, were considered, there was a 9% standard deviation. Total sulfhydryl utilization. Figure 5 shows that the GSH peroxidase reaction utilizes greater amounts of total sulfhydryl in the presence of a constant amount of GSH and increasing amounts of mercaptoethanol. In the reaction blanks where GSSG was substituted for GSH, there was a small but appreciable reaction occurring. This indicated the presence of the disulfide exchange reaction since there was no reaction with mercaptoethanol alone. When this background rate was subtracted, the values shown in Fig. 5 were obtained, and it was noted that with excess amounts of mercaptoethanol relative to GSH, the total sulfhydryl utilization in-

RELATIVE

[GSH

PEROXIDASE]

4. Sensitivity and reproducibility of the semiautomated assay of GSH peroxidase. The relative amount of GSH peroxidase expressed as 1.0 was 50,000 nmol of NADPH oxidized/min/mg of protein, as determined by the coupled assay system. Assays were performed as described under Methods. (0) Single assay; bars represent the mean tt one standard deviation for six assays. FIG.

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MERCAPTOETHANOL(mM)

FIG. 5. Twofold increase in total sulfhydryl utilization with increasing concentrations of mercaptoethanol. Assays were performed as described under Methods. (0) Single assay with subtracted blanks.

creased up to, but not exceeding, a level exactly twice that observed when no mercaptoethanol was present. This leads us to believe that under these conditions of high mercaptoethanol concentrations, the GSH peroxidase reaction product is not GSSG, but rather GSSR, the disulfide adduct of GSH and mercaptoethanol. DISCUSSION

There are now available three assay systems for measurement of GSH peroxidase activity: (i) the GSH reductase-NADPH coupled assay; (ii) the manual DTNB reaction; and (iii) the semiautomated DTNB reaction system described herein. Each type of assay has its advantages and disadvantages. This coupled assay is the best for use during purification of the enzyme, since it is fast and simple and utilizes only small amounts of sample. However, this method is inappropriate for kinetic or inhibition studies because of the inhibition of GSH peroxidase by NADPH reported by Little et al. (5) and also observed in our studies (unpublished observations). The manual DTNB assay does not have this drawback, but it is a rather long and tedious procedure when it is used to assay a large number of samples. Assay of GSH peroxidase by the semiautomated DTNB technique can be performed faster than by the manual assay (ca. lo-Whr by the semiautomated assay vs 3-Yhr manually). The semiautomated method gives less reproducibility than does the coupled assay; for example, the semiautomated GSH peroxidase assay had a 9% standard deviation for values of GSH peroxidase relative concentration 20.5 and an 18% standard deviation over the whole 20-fold concentration range, while the coupled assay had a 7% standard deviation for the same 20-fold concentration range of GSH peroxidase (unpublished observations). The large standard deviation resulting when less active samples are assayed is due to the fact that the assay measures disappearance of GSH. This must be a difference measurement and is, therefore, difficult to perform accurately for small quantities. Thus, the semiautomated assay system is best restricted to samples of higher activity but is not limited to such. The semiautomated

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system, like the coupled assay and unlike the manual DTNB assay, is continuous and produces a large number of data points, with initial points produced less than 30 set after the start of the enzyme reaction (Fig. 3). This rapid initial response, relative to that obtained by the manual DTNB assay, is due to the almost immediate mixing of the reaction components with the 10% trichloroacetic acid after the addition of the hydroperoxide substrate. Assignment of the zero time point varies k5 set, but this is not crucial as it does not affect the value obtained for the first-order rate constant k. The semiautomated system allows the investigator to alter experimental conditions more easily than does the coupled assay because there is no concern for effecters of GSH reductase. Also, the range of response to GSH can be altered easily in the semiautomated system by changing the ratio of flow rates of sample and buffer, thus producing a desired dilution or by calibration of the Cary spectrophotometer to produce adequate zero suppression. This procedure is most applicable to the assay of GSH peroxidase that has been purified sufficiently so that no protein precipitates when the sample is mixed with the trichloroacetic acid. Use of the semiautomated system in measuring total sulfhydryl utilization has led us to the conclusion that GSH peroxidase does not have a strict substrate specificity for both of its sulfhydryl substrates to be GSH, but rather that one of the sulfhydryl substrates must be GSH while the other may be some alternative sulfhydryl. The disulfide reaction product in this situation would thus be GSSR instead of GSSG. This explains why the GSH reductase-NADPH coupled assay for GSH peroxidase shows mercaptoethanol and other sulfhydryls such as cysteine and dithiothreitol to be inhibitors of GSH peroxidase (unpublished observations). These sulfhydryl compounds were participating in the GSH peroxidase reaction and yielding GSSR as products which could not then be assayed by the GSH reductase-NADPH because of the high specificity of GSH reductase for GSSG. The effect of interfering substances on the semiautomated assay was not determined since the DTNB reaction used is well-known and has been studied previously (8). Since the basis of this assay is sulfhydryl determination, the system is not specific for GSH peroxidase but should be directly applicable for measurement of reaction rates, kinetics, or sulfhydryl utilization for any enzymatic reaction that consumes or produces free sulfhydryl groups. Clinically, the system could be adapted for the determination of nutritional selenium status by measurement of the GSH peroxidase, since this is a selenium-containing enzyme (13), or for the determination of nutritional flavin status by measurement of GSH reductase, which is a flavin-containing enzyme (14). ACKNOWLEDGMENT This research was supported by NIH Research Grant AM 06424 from the National Institute of Arthritis, Metabolism, and Digestive Diseases.

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REFERENCES 1. 2. 3. 4.

Paglia, D. E., and Valentine, W. N. (1967) J. Lab. Clin. Med. 70, 158. Hopkins, J., and Tudhope, G. R. (1973) Brir. J. Haetnatol. 25, 563. Nakamura, W., Hosoda, S., and Hayashi, K. (1974) Biochim. Biophys. Acta 358, 251. Giinzler, W. A., Kremers, H., and Flohe, L. (1974) Z. Klin. Chem. Klin. Biochem. 12, 444.

5. Little, C., Olinescu, R., Reid, K. G., and O’Brien, P. J. (1970) J. Biol. Chem. 245,3632. 6. Emerson, P. M., Mason, D. Y., and Cuthbert, J. E. (1972) Brit. J. Haematol. 22, 667. 7. Necheles, T. F., Boles, T. A., and Allen, D. M. (1968) J. Pediat. 72, 319. 8. Ellman, G. L. (1958) Arch. Biochem. Biophys. 74, 443. 9. Sedlak, J., and Lindsay, R. H. (1968) Anat. Biochem. 25, 192. 10. Beutler, E., Duron, O., and Kelly, B. M. (1963) J. Lab. C/in. Med. 61, 882. 11. FlohC, L., Gtinzler, W. A., Jung, G., Schaich, E., and Schneider, F. (1971) HoppeSeyler’s

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12. Stults, F. H., Forstrom, J. W., Chiu, D., and Tappel, A. (1977)Arch.

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13. Rotruck, J. T., Pope, A. L., Ganther, H. E., Swanson, A. B., Hafeman, D. G., and Hoekstra, W. G. (1973) Science 179, 588. 14. Beutler, E. (1973) in Glutathione (FIohC, L., Benohr, Ch., Sies, H., Wailer, H. D., and Wendel, A., eds.), pp. 109- 114, Thieme, Stuttgart.