Behavior of 3alpha- and 7alpha-hydroxysteroid dehydrogenases on chenodeoxycholate substituted Sepharose

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