Stabilization and assay of a 17beta-hydroxysteroid dehydrogenase in the serum of pregnant women.
Up to the present the activity of the 17beta-hydroxysteroid dehydrogenase (17beta-HSD) in the blood of pregnant women has not been accurately measured because of its great instability. The enzyme was stabilized by mixing fresh serum with an equal volume of potassium phosphate buffer (100 mM, PH 7.4), containing 40% (v/v) glycerol, 40 muM oestradiol-17beta (Oe2) and 0.4% (v/v) ethanol. Under these conditions the 17beta-HSD was stable for several days at -20, +4 and +20 degrees C and resistant to heat denaturation at 60 degrees C. The Km-value for Oe2 with NAD or NADP as cosubstrate was 5 X 10(-6) or 2 X 10(-6) M, respectively. In the presence of NAD, Oe2 was oxidized twice as rapidly as with NADP. The Km-value for NAD was 10(-5) M. Under the conditions of assay [3H]oestrone formation was linear with time (3 h) and with protein concentration. Serum enzyme activities in normal pregnancies (between 20 and 60 muU/ml serum at the end of gestation) were nearly twice as high as those reported by other investigators. In normal single and twin births highest activities were obtained at the expulsion of the placenta which in all cases was followed by a rapid decrease in activity.
Stabilization and assay of a 17beta-hydroxysteroid dehydrogenase in the serum of pregnant women. Up to the present the activity of the 17beta-hydroxysteroid dehydrogenase (17beta-HSD) in the blood of pregnant women has not been accurately measured because of its great instability. The enzyme was stabilized by mixing fresh serum with an equal volume of potassium phosphate buffer (100 mM, PH 7.4), containing 40% (v/v) glycerol, 40 muM oestradiol-17beta (Oe2) and 0.4% (v/v) ethanol. Under these conditions the 17beta-HSD was stable for several days at -20, +4 and +20 degrees C and resistant to heat denaturation at 60 degrees C. The Km-value for Oe2 with NAD or NADP as cosubstrate was 5 X 10(-6) or 2 X 10(-6) M, respectively. In the presence of NAD, Oe2 was oxidized twice as rapidly as with NADP. The Km-value for NAD was 10(-5) M. Under the conditions of assay [3H]oestrone formation was linear with time (3 h) and with protein concentration. Serum enzyme activities in normal pregnancies (between 20 and 60 muU/ml serum at the end of gestation) were nearly twice as high as those reported by other investigators. In normal single and twin births highest activities were obtained at the expulsion of the placenta which in all cases was followed by a rapid decrease in activity.