STUDIES ON THE ISOLATION AND PAPER ELECTROPHORESIS OF ASPERGILLUS NIGER 12Y PROGESTERONE HYDROXYLASES

Abstract

Ammonium sulphate was unsuitable for salting out active fractions of progesterone hydroxylases from the cell-free preparation of Aspergillus niger 12Y while ethanol provided precipitates possessing weak activities. Acetone afforded precipitates possessing moderate activities and the precipitate obtained by treatment with 6 volumes of acetone showed only 11β-hydroxylase activity. Centrifugation of a buffered cell-free preparation at different velocities provided fractions rich in 11α-hydroxylase and others rich in 11β-hydroxylase. The supernatant fluid remaining after centrifugation at 18, 000rpm and the sediment obtained at 7, 000rpm showed maximal activities of 11α-hydroxylase and 11β-hydroxylase, respectively. Paper electrophoresis of the cell-free preparation of progesterone hydroxylases was studied using 26 different buffer solutions. Successful separation of 11α-hydroxylase and 11β-hydroxylase was achieved with acetate buffer of pH 3.42 (0.02M and 0.2M) and pH 4.05 (0.02M), as well as with phosphate buffer of pH 5.29 (0.0006M) and pH 8.0 (0.0001M). Acetate buffer of low pH had an inhibitory effect on the electrophoresed 11β-hydroxylase. However, both 11α-hydroxylase and 11β-hydroxylase became inactive when electrophoresed with acetate buffer of pH 5.89. In no case 21-hydroxylase appeared on the electropherograms, probably due to its presence as a minor component of the enzyme sample.