ANALYTICAL

BIOCHEMISTRY

Continuous PATRICK

97, 340-345 (1979)

Fluorometric

M. DANSETTE,*

*Institute of Biochemistry, National Institute

Universitk

of Arthritis,

Assay of Epoxide

GARRETT

C.DuBors,t

Hydrase

AND DONALD

Paris-Sud, 91.405 Orsay, France; tLaboratory Metabolism and Digestive Diseases, National Bethesda, Maryland 20014

Activity M. JERINA~

of Bioorganic Chemistry, Institutes of Health,

Received November 15, 1978 A rapid, continuous, and highly sensitive fluorescence assay is described for the measurement of epoxide hydrase activity. The method is based on the large differences between the fluorescence spectra of certain K-region arene oxides and their corresponding transdihydrodiols. Enzymatic hydration of K-region arene oxides of phenanthrene, pyrene, benzo[a]pyrene, and 7,12-dimethyibenzo[a]anthracene was studied. The assay was most sensitive with benzo[a]pyrene-4,Soxide as substrate. With 10 /.LM benzo[a]pyrene4,Soxide, enzymatic rates of 30 pmol of dihydrodioYmin/mg of protein are three to five times those of the blank without enzyme. The fluorometric method described has been used to study site-directed inhibitors of epoxide hydrase and the stereoselective hydration of racemic arene oxides.

Microsomal epoxide hydrase catalyzes the hydration of a variety of arene and alkene oxides, reactive intermediates formed by the action of cytochrome P-450 monooxygenases on many endogeneous and exogenous compounds. The trans-dihydrodiols formed from arene oxides are either weak or inactive as mutagens (1,2) and celltransforming agents (3) without further metabolic activation. Certain dihydrodiols of some polycyclic aromatic hydrocarbons, however, may undergo further metabolism by the cytochrome P-450 system and produce highly reactive dihydrodiol epoxides, some of which are known to be ultimate carcinogens and mutagens ((4-6), and references therein). Thus, epoxide hydrase, like cytochrome P-450, plays a pivotal role in the activation and inactivation processes of many carcinogens, mutagens, and toxic compounds. Epoxide hydrase from rat liver has recently been purified to apparent homogeneity (7,8). The assays which have been developed to measure the activity of this enzyme are all endpoint assays which re0003-2697/79/120340-06$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

340

quire either a differential extraction step or a chromatographic procedure to separate substrate and product (9-15). For kinetic studies on the mechanism of action of the soluble, homogeneous enzyme, a continuous assay procedure was sought. In this paper, we report the development of a sensitive, fluorometric assay for epoxide hydrase activity. MATERIALS

AND METHODS

Chemicals. Tris and Tris-HCl were obtained from Sigma Chemical Company. Trichloropropylene oxide (TCPO) was purchased from Aldrich Chemical Company. All other chemicals were reagent grade or better and were used without further purification. Methyl-p-nitrobenzenesulfate and methyl-p-toluenesulfonate were obtained from Eastman Kodak Company. Enzyme preparations. Purified epoxide hydrase was the generous gift of Drs. W. Levin and A. Y. H. Lu at Hoffman-La Roche. The preparation of purified epoxide hydrase used in this study hydrolyzed styrene-7,8-oxide to styrene glycol at a rate

FLUOROMETRIC

ASSAY

OF EPOXIDE

HYDRASE

341

of 640 to 660 nmoYmin/mg of protein in the Inhibition experiments. Purified epoxide standard radiometric assay (15,16). Liver hydrase (7.7 pg/lOO ~1 of incubation mixmicrosomes were prepared from adult, male ture) was incubated at room temperature Wistar rats (200 g) which had received with 2.0 mM reagent in 0.125 M phosphate phenobarbital in their drinking water (1 g/ buffer at the desired pH. Aliquots (18 ~1) liter) for 10 days. Preparations of nuclei were were removed at appropriate time intervals obtained from lung tissue of adult male and assayed fluorometrically by the above Wistar rats. procedure with benzohzlpyrene-4,5-oxide Substrates. The phenanthrene-9, lo-oxide, as substrate. pyrene-4,5-oxide, benzo[u]pyrene4,5oxide, RESULTS and 7,1Zdimethylbenzo[u]anthracene used as substrates in this study were prepared Assay Conditions from the corresponding K-region cis-dihyThe fluorescence spectra of four polydrodiols as previously described (17). Prepcyclic aromatic hydrocarbon arene oxides aration of the (+)- and (-)-enantiomers of and their corresponding dihydrodiols formed benzo[a]pyrene-4,Soxide has also been by hydration were examined to establish described (18). conditions under which the reactions could Assay of epoxide hydrase. In a typical be followed. At optimal conditions of assay of microsomal activity, microsomes excitation and emission (Table l), the (2-800 pg/ml) in l-2 ml of Tris-HCl buffer fluorescence spectra of the dihydrodiols (0.015 M, pH 8.7 at 37°C) were thermostated at 37°C in a stirred spectrofluorimeter cell. were markedly different from those of the the Reaction was initiated by the addition of starting arene oxides. In addition, quantum yields for fluorescence from the substrate in 5-20 ~1 of acetonitrile. With dihydrodiols were much higher than those the purified enzyme, protein concentrations of the corresponding arene oxide subin the range of 0.2 to 0.5 pg/ml were used strates. A representative set of spectra for in the absence of added detergent or lipid. Relative fluorescence was recorded as a the conversion of benzo[a]pyrene-4,5-oxide is shown function of time, and standardization of to benzo[u]pyrene-4,5-dihydrodiol in Fig. 1. Similar results were obtained for product formation was achieved by addition the other arene oxides listed in Table 1. of known amounts of product dihydrodiol. Low substrate concentrations were used Kinetic Data (0.1-20 PM) so that the absorbance of the solution (~0.1) was sufficiently low at both Initial reaction rates were proportional to excitation and emission wavelengths to the protein concentrations. Since the assay avoid quenching. Protein concentrations is continuous, true initial rates can be eswere determined by the method of Lowry timated from a single experiment (Table 1). et al. (19). Product formation was quantitated by caliAlthough several different instruments bration of the instrument with known were used to record increases in fluo- amounts of dihydrodiol. Maximum rates rescence with time, most of the studies were (V,,,) for phenanthrene-9, IO-oxide and conducted with a Perkin-Elmer fluores- benzo[u]pyrene-4,5-oxide were within 5% cence spectrophotometer, model MPF-3L. of those obtained by a radiometric assay Excitation slits of 2 to 5 nm and emission (15,16) when parallel determinations were slits of 5- 10 nm were used. Due to the strong made with microsomes or homogeneous fluorescence of the reaction products, the epoxide hydrase from immature Longleast sensitive instrument settings were gen- Evans rats. erally adequate. The most linear product formation with

342

DANSETTE,

DUBOIS, AND JERINA TABLE

ASSAM

OF MICROSOMAL WITH

EPOXIDE

SEVERAL

I

HYDRASE

K-REGION

ARENE

A Excitation

BY FLUORESCENCE OXIDES

CHANGES

AS SUBSTRATES”

Substrate

(nm)

A Emission (nm)

Rate* (nmol/min/mg)

K, (PM)

Phenanthrene-9, IO-oxide Pyrene-4,5-oxide Benzo[u]pyrene-4,5-oxide 7,12-dimethylbenzo[a]anthracene-5,6-oxide

270 310 310

320 372 385

140 (10) 94 (15) 39 (40)

1.3 0.4 0.13

310

380

37 (40)C

0.54

” Liver microsomes were prepared from adult, male Wistar rats (200 g) which had received phenobarbital in their drinking water (1 g/liter) for 10 days. Kinetic data were obtained from double reciprocal plots in which the concentration of each substrate was varied from 0.1 to 40 PM. b Rates are expressed as nmol dihydrodiol formed/min/mg and are computed values of V, (apparent) determined from initial rates. The values in the parentheses are the concentrations of microsomal protein used (&ml). r These reactions were biphasic, and only the initial rate is given (see Figs. 2 and 5).

time and the fewest problems with substrate solubility were obtained with phenanthrene9,10-oxide as substrate. Quality of the rate data was greatly improved by stirring the contents of the cell. Since arene oxides are photolabile, reactions can only be followed to completion successfully when exposure

I

I

1

250

300

I

I

350 WAVELENGTH

400

I

450

fnm)

FIG. 1. Repetitively scanned fluorescence spectra as a function of time for conversion of benzo[u]pyrene-4,5-oxide to benzo[a]pyrene-4,5-dihydrodiol by microsomal epoxide hydrase. Excitation spectra (left) were recorded on an Aminco-Bowman fluorimeter with the emission wavelength fixed at 385 nm; for emission spectra (right), the excitation wavelength was fixed at 310 nm. The bottom traces were run in the presence of substrate and absence of enzyme. Microsomes (5 @ml) in Tris-HCI buffer (0.15 M, pH 8.7, 37°C) were incubated with 20 PM substrate. Spectral traces were recorded at 3-min intervals.

to the lamp is short (ca. 15-30 min). For experiments which require longer time periods, better kinetics are obtained if the cell is exposed to the lamp for only brief periods during the course of the run. Sensitivity

of the Method

The substrate for which the assay procedure was most sensitive was benzo[a]pyrene4,5-oxide, due in part to a very low apparent spontaneous hydrolysis rate (no enzyme) of

Continous fluorometric assay of epoxide hydrase activity.

ANALYTICAL BIOCHEMISTRY Continuous PATRICK 97, 340-345 (1979) Fluorometric M. DANSETTE,* *Institute of Biochemistry, National Institute Univers...
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