TIME Joumnr. cm BIOLOGICAL C~EMWTRY Vol. 248, No. 10, Issue of May 25, pp. 3702-3707, 1973

Printed in U.S.A.

Enzymatic Conjugation of Epoxides with Glutathione

(Received for publication, December 4, 1972)

THORSTEN A. FJELLSTEDT," ROBERT H. ALLEN,~BRUCE K. DUNCAN,~ AND WILLIAM B. JAKOBY

From the Section on Enzymes and Cellular Biochemistry, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014

SUMMARY

Glutathione epoxide transferase has been isolated in homogeneous form from the livers of male Sprague-Dawley rats. The enzyme is active in the conjugation of gluta- thione with a large number of compounds bearing an ethylene oxide ring in the terminal portion of an alkyl chain. The enzyme has a molecular weight of 40,000 and is composed of 2 subunits. Several proteins which catalyze the same reaction are demonstrated. Some of these forms, present in the purified enzyme, are interconvertible by freezing and by treatment with ethylenediaminetetraacetate.

1)espite current interest in the epoxides as intermediates in both aromatic oxidation (l-3) and in detoxication (4, 5), charac- terization of these processes on the level of the participating en- zymes has lagged. Indeed, only the enzyme responsible for the hydrolytic cleavage of epoxysuccinate has been obtained in a state of homogeneity (6) although several activities of far greater physiological importance have been recognized (cf. 4, 7). For example, liver extracts have been shown to catalyze the conjuga- tion of epoxides with glutathione (8, 9) leading to the formation of adducts which serve as intermediates in the production of the mercapturic acids. Similarly, the enzyme-catalyzed hydrolysis of arene oxides is being explored (3, 10).

The present work, undertaken to clarify one of the reactions utilizing glutathionc for the opening of the oxirane ring, has led to the isolation of a homogeneous enzyme, glutathione epoxidase (EC 4.4.1.7, X-(hydroxylalkyl)-glutathione alkyl-epoxidelyase), which catalyzes Reaction 1.

A OH SC, \ / (1)

RCHCHz + GSH -+ RCHCI-12

The enzyme, obtained from rat liver and also present in the liver of other vertebrates (8, 9), is characterized by its specificity for glutathione and for a compound with an oxirane ring at the end

* Present address, Section on Developmental Enzymology, Laboratory of Biomedical Sciences, National Institute of Child Health and Human Development, Bethesda, Md.

t Present address, Department of Medicine, Washington Uni- versity School of Medicine, St. Louis, MO.

$ Present address, Department of Biology, The Johns Hopkins University, Baltimore, Md.

of an alkyl chain (Equation 1). This report is concerned with the preparation of the enzyme and certain of its properties.

METHODS Ah‘D MATERIALS

Standard Enxym~e Assay-l ,2-Epoxy-3-(p-nitroplienoxy)pro- pane was chosen as a substrate because its conjugation product with GSH possesses a spectrum sufficiently different from that of the parent substances to allow development of a convenient spec- trophotometric assay. In a total volume of 1 ml, the assal mixture contained 0.2 M potassium phosphate at pH 6.5, 10 rnM GSH, 0.5 m&r 1,2-epoxy-3-(p-nitrophcnoxy)propane, and an appropriate amount of enzyme. The increase in absorbance at 360 nm upon the addition of the epoxide was measured with either a Gilford model 2000 or a Cary model 15 at a full-scale setting of 0.1 absorbance. An absorbance change of 0.51 at 360 nm is equivalent to the formation of 1.0 pmole of product; this value was determined by incubation of 0.25 m&f and 0.5 mi\z substrate, respectively, in the standard incubation mixture with sufficient enzyme to allow the reaction to be completed within 5 min. Activity was directly proportional to enzyme concentration when absorbance changes of less than 0.07 per min were meas- ured.

A unit of activity is defined as that amount of enzyme resulting in the formation of 1 pmole of product per min at 25”. Specific activity is expressed as units of activity per mg of protein. Pro- tein was estimated calorimetrically (11) with cryst,alline bovine plasma albumin as standard.

Alternative #‘nxynLe Rssav-Since the standard assay is limited to a single epoxide substrate, a second method was developed in order to investigate enzyme specificity. Advantage was taken of the decrease in concentration of the sulfhydryl groups of GSH as determined by reaction with 5,5’-dithiobis(2-nitrobenzoate). The assay mixture was identical with that of the standard system except in that a different epoxide at a concentration of 10 rnA1 was used. Samples of 10 ~1 were removed after 1, 5, 10, and 15 min and were added to 2 ml of a solution of a reagent mixture at pH 7.8 containing 50 mM Tris chloride, 7.5 rnM potassium phosphate, and 0.8 rnM d%,hiobis(nitrobenzoate).

Ultracentrifugation-A Beckman model E analytical centrifuge wit.h optical scanning accessories was used for sedimentation equilibrium studies. In each case, centrifugation was initially conducted for 3 hours at 60,000 rpm. For the native protein, a rotor speed of 28,000 rpm at 15” was maintained for the nest 72 hours. Absorbance was measured at 280 nm at 12.hour iliter- vals. In order to obtain a final absorbance base line, rotor speed was again increased to 60,000 rpm for 150 min. The data for

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molecular weight of the enzyme were obtained with 0.15 mg of protein per ml in a buffer containing 20 rnbf ethylenediaminetetra- acetate, 30% glycerol, and 50 mM potassium phosphate at pH 6.5.

Enzyme subunits, prepared by dialysis for 3 days in 6 &T guani- dine hydrochloride (Mann) containing 0.1 RZ mercaptoethanol, were centrifuged at 25” at 32,000 rpm and at 40,000 rpm. A protein concentration of 0.3 mg per ml was used.

Gel Electrophoretic Xystem-Disc electrophoresis was per- formed by the method of Ornstein (12) and Davis (13) although a sample gel was omitted. Samples were applied directly to the stacking gel in a 30v0 glycerol solution. Approximately 2 hours were required to move the tracking dye, bromphenol blue,

kg, was pulverized with a hammer while frozen and allowed to thaw partially in 2.5 ml of water per g of liver. The mixture was homogenized in a Waring Blendor in portions of 600 ml for 75 s and the combined homogenates were centrifuged at 14,000 x g for 30 min. The supernatant fluid was decanted and adjusted from about pH 6.7 to 8.3 with 2 M Tris base. Conductivity was decreased to below 1.3 mho by addition of water to yield ap- proximately 5 liters of solution for each batch.

2. DEAE-cellulose Column Chromatograph,y--The 5 liters of extract were applied to a DEAE-cellulose column (14.5 x 30 cm) (Whatman Microgranular DE-52)) previously equilibrated with 10 mM Tris-chloride at p1-I 8.25. After application of the pro-

to within 1 cm of the end of a 6-cm gel. Gels were stained in tein solution, 4 liters of the same buffer were used to wash the 0.1% Coomassie blue-20% trichloroacetic acid for 4 hours and column. To the total effluent from this column, approsimately destained in 70/$ acetic acid. 10 liters of fluid, was added 1 M sodium phosphate at p1-l 6.50 to

For gel electrofocusing, the method of Wrigley (14) was used yield 50 mM phosphate. Reduced GSH was added to a final with pI-I 6 to 8 Ampholine (LKB Products). Prior to applica- concentration of 5 mM and the pH was adjusted, when necessary, tion of the protein, gels were subjected to a maximum of 500 to 6.5. volts for 30 min. Thereafter samples were applied in 10% su- 3. Ammonium Suljate Fraction-Solid ammonium sulfate, 245 crose and 1% Ampholine solution by introducing them below a g per liter, was added to yield a concentration of approximately layer of 5% sucrose containing 1% Ampholine. A curreut of 357; of saturation. After stirring for 15 min, the precipitate between 1.5 to 2.0 ma was applied for 3 hours. The gels were was removed at 14,000 x g for 20 min and the supernatant fluid fixed in 5% trichloroacetic acid and stained as noted above, or was treated with an additional 245 g of ammonium sulfate per were mechanically sliced into 2-mm sections and crushed with liter of the original volume from Step 2. The resultant precipi- a Gilson gel fractionator. Each gel section was eluted with tate, suspended in 350 ml of 0.1 M sodium phosphate, pH 6.5, approximately 0.5 ml of a solution of 5 m&c GSII and 30y0 glyc- containing 5 mM GSH, was dialyzed against the same buffer erol in 50 mM sodium phosphate at pH 6.5. overnight. After centrifugation to remove a precipitate, the

Sodium dodecyl sulfate gel electrophoresis was performed with solution was concentrated to 125 ml by ultrafiltration with an the discontinuous buffer method of Neville (15) as well as by Amicon filtration apparatus and a UM-2 membrane. the method of Osborn and Weber (16). Protein solutions were 4. Gel Filtration-The concentrated protein solution from each introduced under the upper D we1 buffer and a currrnt of 1.5 ma batch of 125 livers was divided into four equal portions and ap- per gel was applied until the tracking dye had reached 1 cm from plied to Sephadex G-100 in columns (5 X 97 cm) equilibrated the end of the gel. Gels were fixed and stained as dcscribrcl with 0.1 AI sodium phosphate at pH 6.5 containing 5 mbr GSH. above. Eluate was collected after treatment with the same buffer in

Product IdentiJication-1)escending paper chromatography fractions of 10 ml and the active material, Fractions 96 through was carried out on Whatman No. 1 paper whereas thin layer 120, was pooled and concentrated by ultrafiltration to a volume chromatography utilized Eastman cellulose Chromogram plates of about 15 ml. with or without a fluorescent indicator. After chromatography, 5. C111-cellulose Chromatography--The preparations from four glycidaldehyde was identified by spraying with silver nitrate gel filtration columns were combined and the concentration of (17), whereas 1,2-epoxy-3-(p-11itrophenosy)propane was visible phosphate was reduced from 0.1 M to 0.025 M by dilution with in ultraviolet light; the latter compound and its addition product 5 mM GSH. The diluted protein was adjusted to pH 6.0 with could also be identified after elution from the rhromatographic 1 s acetic acid and concentrated by ultrafiltration to about 50 matrix by their absorbance maximum at 315 IHII. Glutathione ml. The preparation was charged onto a CM-cellulose column and its derivatives were obscrred after reaction with ninhydrin; (2.5 x 50 cm) previously equilibrated with 25 mM sodium phos- rcducrd glutathione reacted with 5,5’-dithiobis@nitrobenzoate) phate at pH 6.0 containing 5 rntif GSH. After sample applica-

(18). tion, the column was washed with 200 ml of the starting buffei followed by a linear gradient consisting of 550 ml of starting

ENZYMIS PREPARATION buffer and 550 ml of the same buffer supplemented with 0.1 M Livers were obtained from male Sprague-Dawley rats (Gibco sodium chloride. Fractions of 7 ml were collected. Three

Microbial Laboratories), 175 to 200 g in weight, that were fed major peaks of enzyme activity were observed routinely with with Purina Lab Chow for a minimum of 5 days prior to decapi- this procedure (Fig. 1). The first appeared at the end of the tation. Upon removal, livers were quickly frozen in Dry Ice and void volume, the second and main act.ivity peak at the beginning stored at -90”; under these conditions there was no loss of of the gradient, and the third near the end of the gradient. All detectable enzyme activity after 18 months of storage. All enzyme activity peaks were pooled and concentrated separately subsequent steps were carried out between 0 and 4”. by ultrafiltration. The center peak of enzyme, generally ac-

The larger preparations, one of which is reported in detail here, counting for 50% or more of the total activity, was used in all involved a total of 250 rat livers which were processed in two subsequent steps. At this stage, the preparation could be stored separate batches through the first four steps of purification, i.e. at -90” while the remaining batch was brought to the same passage through a DEAE-cellulose column, ammonium sulfate point of purification. fraction, Sephadex G-100 chromatography, and CM-cellulose 6. Hydroxylapatite Chromatography-The product of two chromatography. The product was combined for subsequent batches of Step 5, representing 250 livers, was diluted by addition operations. of 5 mM GSH to a phosphate concentration of 10 mM. The

1. Extraction-Each batch of 125 rat livers, approximately 1 diluted protein was adjusted to pH 6.8 with 1 N sodium hydroxide

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FRACTIONS

Fro. 1. Elution pattern of enzyme activity and protein from CM-cellulose. l , activity; 0, protein as measured by absorbance at 280 rim. The salt gradient is indicated by an uninterrupted solid line.

and concentrated by ultrafiltration with an Amicon UM-2 mem- brane to about 20 ml. After concentration, the supernatant fluid was applied to a column of hydroxylapatite (2.5 X 30 cm) (19) equilibrated with 10 mM sodium phosphate, pH 6.8, con- taining 5 mM GSH. The column was washed with 50 ml of the starting buffer and followed by a linear gradient consisting of 450 ml of the same buffer and 450 ml of 0.3 M potassium phos- phate, pI-I 7.0, containing 5 mM GSH. Fractions of 5 ml were collected, and those containing enzyme Fractions 102 through 115 were pooled and concentrated to 12 ml.

7. Preparative Electrofocusing-An LKS electrofocusing col- umn of 110.ml capacity was assembled and operated according to the manufacturer’s recommendations. Cooling was carried out by circulating ice wat.er throughout the apparatus which was housed in a cold room at 4”. A 1% Ampholine solution of the indicated pH range was used with a 0 to 60 o/0 (v/v) discontinuous glycerol gradient supplemented to 1 mM with mercaptoethanol. The protein solution, consisting of half of the material from Step 6, was added to five of the center gradient fractions. Operation was at 500 volts and at an initial current of 1.6 ma. After 40 hours, the contents of the column were pumped out with an LKl3 l’erpex peristaltic pump at a rate of 1.4 ml per min and fractions of 2 ml were collected. The pH of the undiluted frac- tions was determined at 4” with a Radiometer model 26 pH meter. The fractions between pI-I 7 and 7.5 were retained.

8. second Gel Filtration-Active fractions from the previous step were made 5 mM with respect to GSH and 50 mM with respect to phosphate at a final pH of 6.5. The enzyme solution, concentrated to 3 ml, was applied to a Sephadex G-100 column (1.5 X 90 cm) equilibrated with 50 mM sodium phosphate at p1-I 6.5, 5 m&r GSH, and 30% glycerol. Fractions of 1.4 ml were collected and those selected on the basis of enzyme activity, Fractions 58 through 72, were pooled and concentrated to 2 ml.

9. Second Hydroxylapatite Chromatography-Since the prepa-

TABLE I

Summary of purijkations of glutathione-S-transferuse from rat liver

Procedure

Cell-free extract (250 livers)

DEAE-cellulose Ammonium sulfate Gel filtration CM-cellulose :main

peak Early peak Late peak

Hydroxylapatite Electrofocusing Second gel filtration Second hydroxylapa-

tite

VOlUIlW

ml

7,500

10,400 250

90 37

144 30 12.5 11.7 26.6

3.8

Total Total protein xtivity

Specific activity

w 321,000

65,000 44,000

9,400 830

101

12 9

units 4,035

3,543 5,150 4,140 1,200

475 210

1,113 461 256 262

units/mg 0.013

0.054 0.12 0.44 1.4

11.0

21.1 29.2

ration from Step 8 was always contaminated by a faster moving band of catalytically inactive protein, as observed on disc gel electrophoresis, the pooled and concentrated enzyme from that step was again applied to a column (1.5 X 30 cm) of hydroxyl- apatite equilibrated with 10 mM sodium phosphate at pH 6.5 containing 5 rnA[ GSH and 30% glycerol. After washing with 50 ml of this buffer, a linear gradient of 200 ml of the same buffer and 200 ml of 0.3 M potassium phosphate of pH 6.5 containing 5 mM GSH and 30% glycerol was used for elution. The enzyme which appeared between 250 and 265 ml was concentrated by ultrafiltration.

Instead of using hydroxylapatite in Step 9, the contaminating protein band can also be removed with Sephadex G-100 by repeating Step 8; a similar yield and an identical specific activity are achieved.

The results of purification of one lot of 250 rat livers are sum- marized in Table I.

PROPERTIES OF ENZYME

Homogeneity and Molecular Weight-Disc gel electrophoresis of the purified enzyme containing 100 pg of protein revealed a single band of protein which stained with Coomassie blue and was catalytically active. Sedimentation equilibrium centrifuga- tion in 20 mM GSH, 20 mM ethylenediaminetetraacetate, 50 mM potassium phosphate at pH 6.5, and 307, glycerol resulted in a linear plot of the log of protein concentration versus the square of the radius, i.e. consistent with the presence of a single protein species. Preliminary data suggest a molecular weight of 40,000 at protein concentrations of both 0.12 and 0.15 mg per ml at a measured density and an assumed partial specific volume of 0.73. Estimation of molecular weight with a (1.5 X 100 cm) column of Sephadex G-100 resulted in a value of 39,500 as based on calibration with bovine carbonic anhydrase, horseradish per- oxidase, and equine skeletal muscle myoglobin.

In 6 M guanidine hydrochloride-O.1 M mercaptoethanol, a molecular weight of 24,700 was calculated from the data ob- tained by equilibrium sedimentation on the basis of an assumed partial specific volume of 0.73. Neither the results at a rotor speed of 32,000 nor those at 40,000 were indicative of the presence of more than one protein species. Electrophoresis on SDS gels resulted in a protein band at a molecular weight of 27,000 and a

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TABIJ: II

Epoxide subslrates Standard assay conditions were employed with measurement of

the decrease in the concentration of sulfhydryl groups. Except where indicated, each epoxide was tested at a concentration of 10 mM. Rates are expressed relative to the epoxide used for the spectrophotometric assay.

PH

FIG. 2. The effect of pH on the conjugation of GSH with the assay substrate in 0.2 M potassium phosphate ( l ) and 0.2 M Tris chloride (0 1. The dashed li,ne represents the enzyme catalyzed rate and is calculated from the experimental rates of reaction with and without enzyme.

second band bearing less stain at 25,000. Estimation of molecu- lar weights with this procedure is based on comparison with the following proteins used as standards: equine myoglobin, bovine oc-chymotrypsin, and porcine heart malate dehydrogenase.

Effect of pH-Under otherwise standard assay conditions, the enzyme activity increased with increasing pH after correction for the spontaneous, base-catalyzed reaction (Fig. 2). Because the nonenzymatic reaction rate was negligible below pH 6.8, enzyme assays were carried out in this range rather than at the more effective pH.

Specificity and Kinetics-With the standard assay system, GSH was the only mercaptan which served as substrate; P-mercapto- ethanol, cysteine, and dithiothreitol, each tested in the range of 0.1 to 10 mM, were inactive. The K, for GSH was a function of pH in a manner consistent with ionization of a thiol group; K, values of 8.1 mM, 2.3 mM, and 1.6 rnM were obtained at pH 6.5, 7.0, and 7.5, respectively, after correcting for the nonenzy- matic reaction. In the presence of 30% glycerol in the assay solution at pH 6.5, the K, for GSH was 44 rnB[ (cj. 20).

The specificity of the enzyme appears to be confined to com- pounds containing an oxirane ring in the terminal portion of an alkyl chain. Table II presents a list of compounds which were found to be active and documents the rate of activity of the enzyme relative to 1,2-epoxy-3-(p-nitrophcnoxy)propane, the epoxide used in the standard assay. In the presence of 10 mM GSH at pH 6.5, 1,2-epoxy-3-(p-nitrophenoxy)propane had a K, of 0.2 mM. 1,2-Epoxyethylbenzene and allylglycidyl ether, tested as competitive inhibitors of the standard assay substrate, had Ki values of 0.8 mM and 2 mM, respectively. The presence of 30y0 glycerol in the standard assay did not affect the K, for I ,2-epoxy+(p-nitrophenoxy)propane (cf. 20).

All of the arene oxides tested, i.e. benzene oxide, 1,4-dimcthyl- benzene oxide, naphthalene-l,2-oxide, and phenanthrene-9, lo- oxide, each prepared and provided by Donald M. Jerina, were entirely inactive as substrates when tested over a broad range of concentration. The following compounds were inactive as substrates when tested at a concentration of 10 mM: cyclohexene oxide, cis-epoxysuccinate, trans.epoxysuccinate, ethyl-2,3-epoxy- butyrate, glycidol, glycidyl-lauryl ether, scopolamine, trans- stilbene oxide.

Product and Stoichiometry-The structure of the product of the enzymatic reaction of GSH and any of the epoxides has not been established by rigorous criteria. However, paper chromato- graphic studies (Table III) suggest that the products are, indeed,

Substrate Relative rate

1,2-Epoxy-3-(p-nitrophenoxy)propane

1,2-Epoxy-3-phenoxypropane

2,3-Epoxypropyl methacrylate

2,3-Epoxypropyl acrylate

Allylglycidyl ether

3,3,3-Trichloro-1,2-epoxypropane

1,2-Epoxybutane

1,2-Epoxyethylbenzene

1,2-Epoxy-3-(p-chlorophenoxy)propane

Epichlorohydrin

Glycidaldehyde

1,2-Epoxypropane

Methyl-lO,ll-epoxyundecanoate

Butadiene monoxide

0 H2d&-CH2-O-NO2 lo@

H21dLli-CH2-0Q 145=

0 0 P3 Ji2scJ+cH2-o-E-c=cH, 114

0 9 H2c%H-CH2-O-C-C”=CH2 110

0 H2acH-CH2-0-cH2-CH=cH2 106

0 H2&H-CC13 97

0 Jr2 CGH-CH2 -CH3 66

H,l&HO 612

0 H2CCJl-CH2-OOC1 3@

0 H2ca”-CH2C1 26

0 H2CGH-CHO 26

0 H2&+CH3 25

0 H2ca”-(cH2)*-coocH3 85

0 Hz&H-CH=CH2 8

a Present as a saturated solution of the epoxide because of low solubility.

unsymmetrical thioethers between glutathione and one of the former oxirane carbon atoms. Attempts mere made to prepare thioethers by allowing GSH and the cpoxide to react for 3 hours at room temperature in aqueous solution at pH 9. The reaction product of the base-catalyzed reaction of GSH with 1,2-epoxy-3- (p-nitrophenoxy)propane and with glycidaldehyde was, in each case, a compound which co-chromatographed in four solvent systems with the enzymatic reaction product for the respective cpoxide (Table III). In the case of the aromatic substrate, the product was found to react with ninhydrin but not with dithio- bis(nitrobenzoate). When elutcd from cellulose plates following thin layer chromatography (Table III), the product had the same absorbance maximum, 315 nm, as observed by following the reaction spectrophotometrically. The molar extinction coefficient at 315 nm for the product was 1.24 x 104, whereas that of the aromatic substrate was 1.17 X 104.

By following the course of the reaction both spectrophoto- metrically and by titration with dithiobis(nitrobenzene) under otherwise standard assay conditions over the course of 40 min, it was observed that 1 mole of -SH was removed per mole of product formed. During the first 20 min a linear rate of -SH disappearance, 0.158 pmole per min, was found to accompany the formation of 0.16 pmole of product per min.

Multiple Enzyme Forms-Enzyme isolated in the manner out-

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TABLE III Rp values of components of reaction

Chromatography was conducted on plates of Eastman cellulose Chromagram S. The RF values of the base-catalyzed reaction products are not listed since they are identical with those pre-

Component

1,2-Epoxy-3-(p-nitrophenoxy)propane Enzymatic product of GSH and (1,2-

epoxy-3-(p-nitrophenoxy)propane Glycidaldehyde Enzymatic product of GSH and glycid-

aldehyde

sented below for the enzymatic products. -

ITO

-

1 2 3

GSH GSSG

5 6

-

T Solvent systema .06 - A

-

31 49 97 62

RF X 100

11 6 11 19 8 0 95 100 100 39 73 33

31 14 82 12 90 75 32 84

0 Solvent systems: A, 3-butanol-formic acid-water (70: 15: 15) ; B, 1-butanol-acetic acid-water (4:l:l); C, 3-butanol-methylethyl ketone-formic acid-water (40:30:15:15) ; D, ethanol-water (77:23). 1

ined and stored frozen gradually decreased in catalytic activity despite the presence of 30% glycerol and 5 mM glutathione. Such preparations, when subjected to isoelectric focusing in gels, displayed catalytically active protein bands in the range of pII 6.5 to 7.3. The results with one preparation, stored for 3 weeks at 4”, are shown in Fig. 3B. Upon incubating this preparation at room temperature for 6 hours in the presence of 5 mM EDTA, 30% glycerol, and 5 mM GSH, the enzyme pattern changed and both protein and activity were found to migrate at a position corresponding to a p1 of 7.3’ (Fig. 3A). Following incubation at room temperature, electrofocusing patterns have been ob- tained wherein the sole band of protein and activity was at pH 7.3.

Associated with the increase in p1 is an increase in catalytic activity, the increase being greater for enzymes stored for longer periods and ranging to more than 300yo. The preparation used in the experiments summarized by Fig. 3 was initially obtained after purification in the absence of EDTA. This material, 1.6 units, was incubated for 6 hours at room temperature in the presence of 5 mu EDTA and of the normally present 30% glyc- erol and 5 mM GSH yielding 5.1 units of enzyme activity. After storage of this activated preparation at -90” for 3 weeks, ac- tivity was reduced to approximately 2.5 units; this is the prepa- ration shown in Fig. 3B. The product of a second cycle of reac- tivation with EDTA, 5.0 units, is shown in Fig. 3A.

It will have been noted that purification of the enzyme by chromatography on CM-cellulose reveals at least three distinct peaks of activity (Fig. l), of which only the middle peak is used for further purification. Enzyme in the early peak, that eluting soon after the salt gradient is applied, presents a pattern upon gel electrofocusing which appears as a continuum of activity along the entire pH range of pH 6.4 and 7.3. Enzyme in the last peak of activity to be eluted from CM-cellulose was itself composed of three distinct activity bands: a major one at pH 7.8 and less active bands at pH 7.3 and 8.9. This pattern re- mains unchanged after incubation at room temperature in 30%

1 On preparative electrofocusing this species migrates at pH 7.14, a difference assumed to be due to the fact that the pH was measured directly in this case rather than after the lo-fold dilution required to elute slices of polyacrylamide gel.

PH 6.5 7.0 7.5

I I I -32

. A

P

i

FIG. 3. The distribution of activity obtained on electrofocusing the purified enzyme before (B) and after (A) treatment at room temperature with 5 mM EDTA. The history of each of these preparations is detailed in the text.

glycerol, 5 mM GSH, and 5 mM EDTA. Indeed, the same may be said of the middle activity peak of Fig. 1 as obtained from CM-cellulose; this material consists mainly of enzyme with a p1 of 7.3, contaminated slightly with that of p1 7.8 from the later enzyme peak, and is entirely stable when frozen.

It is only after chromatography on hydroxylapatite that activity is found to decrease upon freezing with concomitant change in p1. This effect suggested that components of the hydroxylapatite preparation could be responsible, particularly in view of the correcting effect of EDTA. However, incubation of the product of CM-cellulose chromatography from the middle enzyme peak with large quantities of hydroxylapatite or with 1 m CaC12, MgC12, ZnClz, or FeSO., for 1 hour did not decrease enzyme activity. Finally, the addition of diisopropylfluoro- phosphate, 25 mg per liter immediately after extraction, 25 mg per liter during dialysis, and 25 mg per liter at Step 3 of the puri- fication procedure, did not affect activity or the distribution of any of the catalytic species.

There is at least one additional species of enzyme. Cell-free extracts of liver prepared from certain strains of male Sprague-

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Dawley rats2 were found to contain an active protein which was adsorbed by DEAE-cellulose and could be eluted therefrom.3 The percentage of activity adsorbed varied between 0 and 100% and appeared to be related to the nutritional state of the animal; starved rats had most of their activity adsorbed by the ion ex- changer. This phenomena was not investigated further since the variety of Sprague-Dawley rats adopted for the preparation reported here was satisfactory, i.e. essentially all of the gluta- thione epoxide transferase was not adsorbed to DEAE-cellulose.

Acknowledgments-We are grateful for the help and advice of Werner Klee with the work involving ultracentrifugation. David Rogerson, Jr. and Herbert A. Sober generously provided all of the hydroxylapatite used here.

REFERENCES

1. BOYLANL), E., AND SIMS, P. (1965) Biochem. J. 95, 788 2. GIBSON, D. T. (1968) Science 161, 1093

2 This includes Sprague-Dawley rats obtained from Flow Labo- ratories, from General Biochemical Company, and from those produced at the National Institutes of Health.

3 Extracts from bovine liver were found to contain a similar pat- tern of enzyme activity, i.e. approximately half of the total activ- ity adsorbed to DEAE-cellulose and the remainder found in three

3. JERINA, D. M., DALY, J. W., WITKOP, B., ZALTZMAN-NIREN- BERG, P., AND UDENFRIEND, S. (1970) Biochemistry 19,147

4. BOYLAND, E., AND CHASSEAUD, L. F. (1969) Advan. Enzymol. 32, 173

5. BOYLAND, E. (1971) in Concepts in Biochemical Pharmacoloau (BRODI&, B. B., AND GILLETTE, I. R., eds) Vol. 28, Part -2”, v. 584. Svrineer-Verlae. Berlin

6. ALLEN, R.-H., AND JAK&Y, W. B. (1969) J. Biol. Chem. 244, 2078

7. JAKOBY, W. B., AND FJELLSTEDT, T. A. (1972) in The Enzymes (BOYER, P. D., ed) 3rd ed., Vol. 7, p. 199, Academic Press, New York

8. BOYLAND, E., AND WILLIAMS, K. (1965) Biochem. J. 94, 190 9. WIT, J. G., AND SNEL, J. (1968) Eur. J. Pharmacol. 3, 370

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peaks of activity as eluted from CM-cellulose. Sci. 69, 2373

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Thorsten A. Fjellstedt, Robert H. Allen, Bruce K. Duncan and William B. JakobyEnzymatic Conjugation of Epoxides with Glutathione

1973, 248:3702-3707.J. Biol. Chem. 

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