Rapid purification of cytosolic epoxide hydrolase from normal and clofibrate-treated animals by affinity chromatography

Epoxide hydrolase from liver cytosol (cEH) of both normal and clofibrate-treated mice can be bioselectively absorbed onto an affinity column prepared from epoxy-activated Sepharose and 7-methoxycitronellyl thiol. The free ligand is a modest inhibitor of cEH (I₅₀, ≃ 3 x 10^-4 M) and lacks the epoxide function necessary for it to be turned over as a substrate. This study demonstrates that a methoxy group can be used to mimic an oxirane in a vertebrate system. Bioselective elution of cEH can be accomplished with several chalcone oxides, which are selective potent inhibitors (I₅₀, 1-50 x 10^-7 M), and activity can be recovered by dialysis. This procedure thus enhances the purification by offering independent opportunities for selective binding and selective elution. Conservatively, a 40%-80% recovery of partially inhibited enzyme activity can be achieved in 4-48 hr with a 30- to 90-fold purification. The purified cEH from clofibrate-induced animals was essentially homogeneous by NaDodSO₄/PAGE and had an apparent subunit molecular weight of 58,000. The cEHs from normal and clofibrate-induced animals appeared identical by NaDodSO₄/PAGE. Since the cEH activity in normal and clofibrate-treated animals is due to the same enzyme, the increase in cEH activity caused by selected hypolipidemic agents appears to be true induction.