Behavior of 3α- and 7α-hydroxysteroid dehydrogenases on chenodeoxycholate substituted sepharose

Chenodeoxycholate (3α-, 7α-dihydroxy-5β-cholanoate) was linked to Sepharose 4B by an ethylenediamine bridge. When 3α-hydroxysteroid dehydrogenase and 7α-hydroxysteroid dehydrogenase preparations were applied to a column of covalently linked Chenodeoxycholate, both enzymes were retarded at pH 6.7; the 7α-OH oriented enzyme more than the 3α-OH enzyme. Approximately forty-fold purification of 7a-hydroxysteroid dehydrogenase was achieved in one step. Although no significant purification of 3α-hydroxysteroid dehydrogenase occurred, the background value in the fluorometric enzymatic estimation of bile acids by eluted 3α-hydroxysteroid dehydrogenase was markedly reduced. Molecular weight estimation by Sephadex G-200 gave the values of 47,000 for 3α-hydroxysteroid dehydrogenase and 105,000 for 7α-hydroxy-steroid dehydrogenase.