Purification and identification of an epoxide hydrolase from equine liver.

In the horse, drug metabolism has been shown to be complex, but little is known about the proteins involved in these processes [l]. As a result of work on the purification of equine Cytochrome P450 enzymes, an epoxide hydrolase has been purified [2]. Epoxide hydrolases catalyse the hydration of biologically active epoxides formed during the metabolism of many drugs and mutagenic compounds [3]. Fresh horse liver was obtained from non-induced animals and homogenised in 4 volumes of lOOmM potassium phosphate, 1mM EDTA, 1mM PMSF buffer pH 7.4. After centrifugation at lOOOg (1Omin; 4°C) the resulting Supernatant was spun at lO,OOOg, (10 min; 4°C). Centrifugation at lOO,OOOg,, (6Omin; 4°C) of the resulting supernatant, produced microsomes which were resuspended in 5OmM sodium phosphate buffer pH 7.25. The microsomes were then solubilised with polyoxyethylene ether W1 (0.2%wlv) and centrifuged at lOO,OOOg, (6Omin; 4°C) to obtain the solubilised supernatant [4]. A lauric acid a f f i t y column was prepared by coupling lauric acid to preswollen EAH sepharose 4B [5]. A 1.6 x 6cm column was packed with gel and solubilised microsomes (1Oml) applied to the column at a rate of OSmllmin. The column was initially eluted with a 5OmM sodium phosphate buffer containing EDTA, DTT and sodium cholate. Protein fractions were eluted by gradually modifying the buffer with varying levels of sodium chloride and polyoxyethylene ether W 1. The column effluent was monitored at a wavelength of 28Onm and fractions (5ml) were collected throughout the experiment [4]. Fractions eluted with phosphate buffer containing polyoxyethylene ether W1 (0.7% wlv) from five lauric acid column runs were combined and dialysed into 5mM potassium phosphate pH 7.4. The equilibrated fractions were then applied to a Econo-Pac HTP cartridge (BioRad) for further fractionation. Unretained protein fractions were collected and dialysed into 5mM sodium phosphate pH 6.5 and applied to an Econo-Pac CM cartridge (BioRad). After washing with phosphate buffer the column was eluted with 0.2M NaOH and fractions were again collected and analysed by SDSPAGE. The eluate was concentrated on a Centriprep 30 concentrator (Amicon) and electroblotted onto Problott membrane (Applied Biosystems) prior to sequencing. N-terminal protein sequencing was performed using an Applied Biosystems 470a Gas Phase protein sequencer equipped with a 120A on-line phenylthiohydantoin analyser [6]. Abbreviations used: EDTA, ethylenediaminetetraacetic acid; PMSF, phenylmethylsulphonyl fluoride; DTT, dithiothreitol; SDSPAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; EAH, epoxy-activated amino group (linked to sepharose 4B); HTP, hydroxyapatite; CM, carboxymethyl purified protein. MWM = molecular weight markers (kDa), X = purified protein.