Purification of mouse liver benzene dihydrodiol dehydrogenases

Multiple forms of cytosolic benzene dihydrodiol dehydrogenase (designated DD1, DD2, DD3, and DD4 according to order of elution from DEAE-cellulose column) were purified to homogeneity from liver of male Swiss-Webster mice, primarily by DEAE-cellulose, affinity, gel filtration, and hydroxylapatite chromatography. The purified enzymes were shown to have specific activities of 3.7, 0.94, 0.98, and 0.76 units/mg of protein, respectively, when assayed with 1.8 mM benzene dihydrodiol and 2.3 mM NADP+. DD1 and DD2 were monomers with molecular weights of 30,000 and 34,000, respectively, while DD3 and DD4 were dimers in the native state with molecular weights of 64,000 and 65,000. The isoelectric points were 8.1, 6.2, 5.5, and 5.4, respectively. The different forms of dihydrodiol dehydrogenase were also studied with respect to their Michaelis constants and substrate specificity.