ANALYTICAL

BIOCHEMISTRY

193,

1-5 (1991)

Analysis of Glutathione S-Transferase-Catalyzed S-Alkylglutathione Formation by High-Performance Liquid Chromatography James W. Tracy1

and Kathleen

A. O’Leary’

Departments of Comparative Biosciences and Pharmacology and Environmental University of Wisconsin-Madison, Madison, Wisconsin 53706

Received

August

Academic

Center,

6, 1990

Metabolism of alkyl halides and some organophosphorous compounds by glutathione S-transferases (EC 2.5.1.18) leads to formation of an S-alkylglutathione as a common product. We have developed an HPLC assay for formation of S-methylglutathione and S-ethylglutathione that is applicable to measuring enzyme activity toward a variety of xenobiotic substrates. The conjugates are derivatized with l-fluoro-2,4-dinitrobenzene to form the corresponding N-2,4-dinitrophenyl derivatives, which are then separated by reverse-phase HPLC with gradient elution. The utility of the method is illustrated by the use of partially purified preparations of rat liver glutathione S-transferases and several prototypic substrates including iodomethane, iodoethane, dichlorvos, and methyl parathion. The limit of detection is about 50 pmol of N-(2,4-dinitrophenyl)-S-alkylglutathione. Advantages of the method over other assays of S-alkyl transferase activity are discussed. 0 1991

Toxicology

Press,

Inc.

GSH S-transferases (EC 2.5.1.18) participate in cellular protection by catalyzing the reaction of electrophiles with GSH to form thioether conjugates (1). SAlkylglutathione derivatives constitute one class of conjugates that are formed from alkyl halides (2) and from some organophosphorous insecticides (3). Of the several methods used to determine GSH S-transferase activity toward such substrates, most are specific for a single compound and include the disappearance of sub1 To whom correspondence should be addressed at Department Comparative Biosciences, University of Wisconsin-Madison, Linden Drive West, Madison, WI 53706-1102. ’ Current address: McArdle Laboratory for Cancer Research, versity of Wisconsin, Madison, WI 53706. 0003-2697191 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

of 2015 Uni-

strate (4,5) and the appearance of dealkylated product (4-7). Production of protons is common to such reactions, but use of a titrimetric assay has been limited to the conjugation of certain alkyl halides (2). Assays for the S-alkylglutathione product generally have been based on formation of water-soluble radioactivity from radiolabeled xenobiotic substrate (4,8). Although such methods are sensitive, each substrate to be tested must first be synthesized with a radionuclide in the appropriate position. In addition, some means of quantitatively extracting unreacted radiolabeled substrate must be available. In an effort to facilitate comparisons of substrate specificity using limited amounts of enzyme, we have developed a sensitive and straightforward method of quantifying S-alkylglutathione derivative formation without using radiolabeled substrates. We report here that nanomolar amounts of GSMe3 and GSEt can be accurately determined by reverse-phase HPLC, after conversion to the corresponding N-DNP derivatives and that the method can be used to assay GSH S-transferase activity toward several prototypic alkyl donor substrates. MATERIALS

AND

METHODS

Reagents. CySMe, CySEt, -r-Glu-Gly, N-DNP-Glu, N-DNP-Gly, GSH, GSMe, GSEt, EPNP, and GSHagarose (G-4510, linked via sulfur) were purchased from Sigma (St. Louis, MO). MeI, EtI, and CDNB were ob-

3 Abbreviations used: CDNB, l-chloro-2,4dinitrobenzene; EPNP, 1,2-epoxy-3-(p-nitrophenoxy)propane; FDNB, 1-fluoro-2,4-dinitrobenzene; DNP, 2,4-dinitrophenyl; MeI, iodomethane; Etl, iodoethane; DDVP, dichlorvos; MeP, methyl parathion; GSMe, S-methylglutathione; GSEt, S-ethylglutathione; CySMe, S-methyl-L-cysteine; CySEt, S-ethyl-L-cysteine.

2

TRACY

AND

tained from Aldrich (Milwaukee, WI). FDNB was a product of Research Organ& (Cleveland, OH). DDVP (99%) and MeP (99%) were purchased from Chem Service (West Chester, PA). HPLC grade solvents and other reagent grade chemicals were products of J. T. Baker (Phillipsburg, NJ). Water was purified by passage through a Mini-Q system (Millipore, Bedford, MA). Synthesis of N-DNP standards. The N-DNP derivatives of GSMe, GSEt, y-Glu-Gly, CySMe, and CySEt were prepared by reaction with FDNB essentially as described by Vinson and Pepper (9), except that reactions contained excess solid Na,CO,. N,S-(DNP),-GSH was synthesized according to the method of Sokolovsky et al. (lo), while N,N-(DNP),-GSSG was synthesized using the procedure of Reed et al. (11). Crude products were washed several times with anhydrous diethyl ether and crystallized (11). Samples of the various derivatives were purified by isocratic HPLC on a PBondapak C,, column (30 X 0.46 cm i.d.; Waters Associates, Milford, MA) equilibrated with 35% (v/v) acetonitrile in 0.3 M acetic acid. Solvent was removed in a Speed Vat concentrator (Savant Instruments, Farmingdale, NY). Purified derivatives were stored at -30°C. When analyzed using the standard gradient HPLC system described below, each derivative chromatographed as a single component at both 365 and 254 nm. Molar absorption coefficients (M-’ * Cm-‘), measured at 365 nm in 35% (v/v) acetonitrile in 0.3 M acetic acid, were as follows: NDNP-GSMe, 1.7 X 104; N-DNP-GSEt, 1.4 X 104; NDNP-r-Glu-Gly, 1.5 x 104. Enzyme preparation. All operations were carried out at 0-4°C. Five-gram samples of adult male rat liver (Sprague-Dawley, Charles River, Wilmington, MA) were homogenized in 20 ml of 22 mM potassium phosphate buffer, 1 mM EDTA, pH 7.0. The homogenate was subjected to differential centrifugation, and the cytosolic fraction (supernatant after centrifugation for 90 min at 105,OOOg) was applied to a 15-ml GSH-agarose column (12) equilibrated with the same buffer containing 0.15 M NaCl. The column was washed with 75 ml of equilibration buffer to elute unbound proteins. These were precipitated between 35 and 70% saturation at 0°C with powdered ammonium sulfate (13) to give Fraction I. The precipitate was dissolved in a minimal volume of 25 mM potassium phosphate pH 7.0, containing 1 mM EDTA, and 5 mM GSH, and was dialyzed overnight against 1000 vol of the same buffer. Fraction I was stored at 4°C and was used within 3 days. Proteins that bound to the affinity column were eluted with GSHcontaining buffer and concentrated (14) to yield Fraction II. GSH S-transferase activity was routinely monitored with both CDNB and EPNP (see below). Protein concentrations were estimated by the Bradford dyebinding method (15), using crystalline bovine serum al-

O’LEARY

bumin (Miles ence protein.

Laboratories,

Elkhart,

IN)

as the refer-

Enzyme assays. GSH S-transferase activity toward CDNB and EPNP was determined spectrophotometritally at 340 and 360 nm, respectively (2). Conjugation of alkyl donor substrates with GSH was carried out in 1.5ml polypropylene centrifuge tubes containing 125 pmol 3-N-morpholinopropanesulfonate (Research Organics) buffer, pH 7.5, 1.25 pmol GSH, xenobiotic substrate (0.12 pmol MeP, 1.25 pmol DDVP, 1.25 pmol MeI, or 1.25 pmol EtI) and enzyme in a total volume of 250 ~1. Reactions were initiated by adding xenobiotic substrate (5 ~1 of a stock solution in ethanol) and were terminated after incubation at 30°C by adding 25 ~1 of ice-cold 60% HClO,. The volume of ethanol used had no effect on enzyme activity. Control reactions contained enzyme that had been boiled for 10 min. Assays were run in duplicate and three time points were used to determine linear reaction velocities. Acidified reactions could be held on ice for up to 2 h and processed together as described below, without affecting the values obtained. Assay deriuatization. The standard FDNB derivatization procedure was patterned after that of Fariss and Reed (16). Five microliters of 12 mM r-Glu-Gly internal standard was added to acidified reaction mixtures or comparable solutions of synthetic N-DNP derivatives. Next, 125 ~1 of an ice-cold solution of 2.0 M KOH and 2.4 M KHCO, was added. When gas evolution slowed (about 15 s), 200 ~1 of a freshly prepared 1.5% (v/v) solution of FDNB in ethanol was added with mixing. The reaction was allowed to proceed in the dark for at least 8 h at 22°C although it was usually more convenient to incubate samples overnight. The bright yellow mixtures were filtered by centrifugation through 0.45-pm Nylon 66 filters, using centrifugal filter holders (Rainin, Woburn, MA). The filtrates were stored in tightly capped vials in the dark at 22°C. Under those conditions, samples were stable for at least 7 days. Prolonged storage of the filtrates for up to 2 months at -30°C did not diminish the values obtained. The yields for FDNB derivatization of y-Glu-Gly, GSMe, and GSEt established by comparison to synthetic N-DNP derivatives were 97 f 4, 95 f 3, and 96 f 5%, respectively. Typically, 5 to 50 ~1 of the filtrate were analyzed. HPLC analysis. HPLC was carried out by using a gradient HPLC system (Gilson Medical Electronics, Middleton, WI) equipped with a Model 757 dual-beam uv detector (Kratos Instruments, Ramsey, NJ). Separation was carried out on a 3-pm Zorbax C,, column (0.62 cm i.d. X 8.0 cm; DuPont, Wilmington, DE) fitted with a 3.2 mm i.d. Brownlee ODS guard column cartridge (Applied Biosystems, Foster City, CA), using an acetonitrile concentration gradient. Solvent A was 0.3 M acetic acid. Solvent B was 50% (v/v) acetonitrile in 0.3 M acetic acid. The column was equilibrated with 50% of Solvent B at

CHROMATOGRAPHIC

ANALYSIS

OF

1.0 ml/min. The gradient was started at the time of sample injection and was increased linearly from 50% Solvent B to 100% Solvent B over a lo-min period. The solvent was maintained at 100% Solvent B for an additional 2 min, and was then returned to initial conditions over 5 min. Allowing time for column equilibration, samples could be injected at 25min intervals. Hydrolysis and analysis of N-DNP-S-alkylglutathione deriuatiues. A sample of authentic N-DNP-GSMe was hydrolyzed in 6 N HCl(8). Acid was removed in a Speed Vat concentrator and the residue was dissolved in water. One portion of the hydrolysate was analyzed by HPLC using the standard gradient elution conditions. Another portion was reacted with FDNB, before being subjected to HPLC analysis. A sample of synthetic NDNP-GSEt was similarly treated. RESULTS

AND

S-ALKYLGLUTATHIONE

DERIVATIVES

b

E 5: is CI 4

DISCUSSION

Initial attempts to analyze GSMe in the presence of GSH using the anion-exchange HPLC method of Reed et al. (11) were unsuccessful, because N-DNP-GSMe was unresolved from derivatization reagent peaks. NDNP-GSMe was resolved from reagent peaks using a reverse-phase HPLC column, but S-carboxymethyl-NDNP-GSH eluted before the S-alkyl derivative. Under conditions of substrate excess, the N-DNP-GSMe product peak could not be readily discerned because of tailing of the S-carboxymethyl-N-DNP-GSH peak. Resolution was achieved, however, by omitting the carboxymethylation step and instead, converting GSH to the N,S-(DNP), derivative. The key to the separation proved to be that N,S-(DNP),-GSH is sufficiently nonpolar to elute well after N-DNP-GSMe. Figure 1 shows the separation of authentic N-DNP-y-Glu-Gly, NDNP-GSMe, N-DNP-GSEt, andN,S-(DNP),-GSH. Capacity factors, k’, under these conditions were 1.2, 1.8, 2.3, and 3.7, respectively. By comparison with purified synthetic derivatives, the yield from the derivatization reaction exceeded 95% for all compounds, except GSH, which was about 90%. Oxidation of GSH to the disulfide did not occur to a measurable extent, because N,N(DNP),-GSSG was not detected. We found that optimal derivatization depended on careful titration with the KOH-KHCO, solution. If the pH was greater than 10, losses of up to 25% of GSMe and GSEt were noted. No loss occurred, if the pH was maintained between pH 8 and 9. Therefore, each batch of KOH-KHCO, solution was tested to ensure that the volume added gave the appropriate final pH. Various gradient elution conditions were investigated and the stated conditions represent a balance between speed of analysis and resolution of N-DNP-GSMe, N-DNP-GSEt, and the internal standard, N-DNP-r-Glu-Gly. No attempt was made to resolve longer chain length S-alkyl conjugates, although the N-DNP derivative of S-propylglutathione could be

0

8 TIME (mid

6

FIG. 1. Reverse-phase HPLC separation of a standard mixture containing 2 nmol each of (a) N-DNP-y-Glu-Gly, (b) N-DNP-GSMe, (c) N-DNP-GSEt, and 1 nmol of (d) N,S-(DNP),-GSH. Gradient elution was carried out as described under Materials and Methods. Vertitle bar, 0.01 A.

separated from N-DNP-GSEt, when mixtures of the pure compounds were analyzed (not shown). Resolution of longer chain length derivatives from derivatization reagent peaks probably could be achieved by appropriate adjustment of the elution solvent. Detector response (peak height at 365 nm) was proportional to the amount of N-DNP-GSMe injected at least over the range of 0.5 to 10 nmol. Five replicate injections of 2 nmol of N-DNP-GSMe gave a peak absorbance of 0.0690 +- 0.0017 A. Over a period of 3 weeks the variation in detector response was less than 4%. Using a full-scale detector sensitivity of 0.01 A, the limit of detection was about 50 pmol injected with a signal-tonoise ratio of 3.5. Similar results were obtained with N-DNP-y-Glu-Gly and N-DNP-GSEt. To confirm the identity of the S-alkyl conjugate, a sample of N-DNP-GSMe that had been purified by reverse-phase HPLC was subjected to acid hydrolysis. A single yellow product that cochromatographed with authentic N-DNP-Glu was detected, when the hydrolysate was analyzed by HPLC under the standard conditions (not shown). When a sample of the hydrolysate was derivatized with FDNB prior to analysis, three products were found in relative molar ratios of 0.93, 1.00, and

4

TRACY

AND

1.06. By comparison with authentic standards, they were identified as N-DNP-Glu, N-DNP-Gly, and NDNP-CySMe, respectively. In agreement with Hollingworth (8), we found that the thioether bond of CySMe was stable to acid hydrolysis under the conditions used. Analysis of a comparable N-DNP-GSEt hydrolysate gave the expected products (N-DNP-Glu, N-DNP-Gly, and N-DNP-CySEt) in approximately equimolar amounts. To demonstrate the applicability of the method, we partially purified rat liver GSH S-transferases by affinity chromatography on GSH-agarose. Two protein fractions were obtained. Fraction I was composed of those proteins that did not bind to the column and that precipitated between 35 and 70% saturation with ammonium sulfate. One form of GSH S-transferase, isoenzyme E,4 does not bind to the column (2). That isoenzyme shows relatively high catalytic activity toward EPNP, a model epoxide substrate, and toward MeI, but little activity toward the prototypic aryl halide substrate, CDNB (2). In contrast to transferase E, other rat liver GSH S-transferases bind to GSH-agarose and can be eluted with GSH-containing buffer (12). This affinity-purified preparation (Fraction II), shows relatively high catalytic activity toward CDNB, but much lower activity toward EPNP and Me1 (1). Because transferase E is unstable, particularly.when highly purified (13), no attempt was made to further purify Fraction I. Figure 2 shows a typical chromatogram obtained when GSH and Me1 were incubated in the presence of Fraction I (Fig. 2A) or in the presence of heat-denatured Fraction I (Fig. 2B). The enzymatic rate of GSMe formation in the case shown was about seven times greater than the nonenzymatic rate. In addition to peaks corresponding to the internal standard and N-DNP-GSMe, other large peaks were attributable to N,S-(DNP),GSH and derivatization reagents. Product peak heights from replicate injections never varied by more than l-2%. Fraction I-catalyzed formation of GSMe from Me1 was proportional both to reaction time (0 to 8 min) and to the total amount of protein added over a range of 0 to 25 pg. Similar dependence also was observed with EtI, DDVP, and MeP, although because MeP was used at a lower concentration due to limited aqueous solubility, a linear reaction rate was obtained only when less than 6-8 nmol of GSMe was formed. Fraction I and Fraction II were compared for their ability to catalyze the conjugation of the standard sub-

’ Although various nomenclatures GSH S-transferase isoenzymes (l), tem of Habig et al. (2).

have been suggested for rat liver we have retained the original sys-

O’LEARY

i

8

16

0

8

16

TIME(min)

FIG. 2. Reverse-phase HPLC separation of FDNB-derivatized reactions mixtures showing GSH-dependent metabolism of Me1 catalyzed by rat liver Fraction I. (A) Chromatogram of a complete reaction mixture containing GSH, MeI, and enzyme (25 rg protein) after a 5-min incubation at 30°C. (B) S ame as in (A) except that boiled enzyme was used. Ten-microliter injections. The enzymatic rate was about seven times the nonenzymatic rate. Verticle bar, 0.01 A.

strates, CDNB and EPNP (Table 1). As expected, Fraction I displayed very little catalytic activity toward CDNB, but substantial activity toward EPNP. The converse was true with Fraction II. When four prototypic alkyl donor compounds were compared as substrates for Fraction I, Me1 showed the highest specific activity. The activity observed with Me1 was about 3-fold greater than with EtI. That value is in close agreement with the ratio obtained by Habig et al. (2) using purified transferase E and a titrimetric assay. Fraction I was about lofold more active with Me1 than with DDVP, whereas activity with the latter was only slightly greater than with MeP. Motoyama and Dauterman (17) prepared two fractions of rat liver cytosol by anion-exchange chromatography. They further fractionated the preparations to give multiple forms of GSH S-transferase that they compared for activity with several substrates. Although direct comparisons are not possible because different methods of protein fractionation were used, some similarities are apparent. One of their fractions, which was quite active with MeI, showed almost no activity toward CDNB, whereas the fraction that was most active toward CDNB, showed much lower activity toward the alkyl halide (17). The individual forms of GSH S-trans-

CHROMATOGRAPHIC

ANALYSIS

OF

S-ALKYLGLUTATHIONE

TABLE Expt. IlO.

Protein fraction

CDNB

EPNP

5

DERIVATIVES

1

Me1

Et1

DDVP

MeP

1

I II

20 22,500

341 f 26 16i 3

389 -c 20 17* 4

127 f 6 15 + 2

32 f 4 7i3

26 AZ 3 2+5

2

I II

16 25,700

297 + 31 9f 4

364 f 18 19f 7

116 f 8 9+3

41 f 2 12 f 8

24 2 2 l&4

Note. Specific activity expressed as nmol * min-’ * (mg protein))‘. three determinations for other substrates, and have been corrected

Average of two determinations for nonenzymatic product

ferase in their Fraction I preparation were 6- to Z&fold more active toward Me1 than MeP. Those values are in agreement with the relative activity were observed with our Fraction I. Motoyama and Dauterman also found that the fraction that was most active toward Me1 did not catalyze aryl transfer of MeP (17). We detected no N-DNP-S-(p-nitrophenyl)-GSH in our reaction mixtures (data not shown), indicating that aryl transfer of MeP was not taking place. The method described here for quantitation of GSMe and GSEt by HPLC is both sensitive and straightforward and can be used to assay GSH S-transferase activity using limited amounts of protein. The major advantage of our approach over other methods is twofold. First, unlike assays that monitor disappearance of substrates (43) or appearance of the dealkylated product (4-7), this method allows direct determination of the relevant GSH S-conjugate which helps establish a role for GSH S-transferase in the reaction. Second, this method is particularly useful for making comparisons of different xenobiotic substrates that produce a common S-alkylglutathione conjugate, because each compound to be tested need not be synthesized with a radiolabel in the alkyl moiety.

ACKNOWLEDGMENTS This work was supported States-Japan Cooperative supported by NIH Training 230 of the Environmental sin-Madison.

by NIH Grant AI22520 from the United Medical Sciences Program. K.A.O. was Grant T32 ES07015. This is Contribution Toxicology Center, University of Wiscon-

with formation.

CDNB.

Values

are the mean

f SD of

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J. Agric. Food Chem. 17, 987-996. L. D. (1972) Anal. Chim. Acta 58,

T., and

Patchornik,

A. (1964)

J. Amer.

11. Reed, D. J., Babson, J. R., Beatty, P. W., Brodie, A. E., Ellis, W. W., and Potter, D. W. (1980) Anal. Biochem. 106.55-62. 12. Simons, P. C., and vander Jagt, D. L. (1981) in Methods in Enzymology (Jakoby, W. B., Ed.), pp. 235-237, Academic Press, New York. 13. Meyer, D. J., Christodoulides, L. G., Tan, K. H., and Ketterer, B. (1984) FEBS Lett. 173, 327-330. 14. O’Leary, K. A., and Tracy, J. W. (1988) Arch. Biochem. Biophys. 264, 1-12. 15. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. 16. Fariss, M. W., and Reed, D. J. (1983) in Isolation, Characterization, and Use of Hepatocytes (Harris, R. A., and Cornell, N. W., Eds.), pp. 349-355, Elsevier, New York. 17. Motoyama, N., and Dauterman, W. C. (1978) J. Agric. Food Chem. 26, 1296-1301.