Radiometric assay for cytochrome P-450-catalyzed progesterone 16 alpha-hydroxylation and determination of an apparent isotope effect
In the course of studies on the oxygenation of steroids by purified P-450 cytochromes, particularly rabbit liver microsomal cytochrome P-450 form 3b, a rapid and reliable radiometric assay has been devised for progesterone 16 alpha-hydroxylation. In view of the lack of a commercially available, suitably tritiated substrate, (1,2,6,7,16,17-3H)progesterone was treated with alkali to remove the label from potential hydroxylation sites other than the 16 alpha position. The resulting (1,7,16-3H)progesterone was added to a reconstituted enzyme system containing cytochrome P-450 form 3b, NADPH-cytochrome P-450 reductase, and NADPH, and the rate of 16 alpha-hydroxylation was measured by the formation of /sup 3/H/sub 2/O. This reaction was shown to be linear with respect to time and to the cytochrome P-450 concentration. An apparent tritium isotope effect of 2.1 was observed by comparison of the rates of formation of tritium oxide and 16 alpha-hydroxyprogesterone, and the magnitude of the isotope effect was confirmed by an isotope competition assay in which a mixture of (1,7,16-/sup 3/H)progesterone and (4-14C)progesterone was employed.
- Research Organization:
- Univ. of Michigan, Ann Arbor, MI (United States)
- OSTI ID:
- 5842132
- Journal Information:
- Anal. Biochem.; (United States), Vol. 164:2
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
PROGESTERONE
HYDROXYLATION
RADIOASSAY
TRITIUM COMPOUNDS
ISOTOPE EFFECTS
CARBON 14 COMPOUNDS
CYTOCHROMES
IN VITRO
LIVER
MICROSOMES
OXIDOREDUCTASES
RABBITS
TRACER TECHNIQUES
ANIMALS
BODY
CELL CONSTITUENTS
DIGESTIVE SYSTEM
ENZYMES
GLANDS
HORMONES
ISOTOPE APPLICATIONS
KETONES
LABELLED COMPOUNDS
MAMMALS
ORGANIC COMPOUNDS
ORGANOIDS
ORGANS
PIGMENTS
PREGNANES
PROTEINS
STEROID HORMONES
STEROIDS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques