Purification and characterization of epimeric estradiol dehydrogenases (17 alpha and 17 beta) from equine placenta.

This study successfully purified two enzymes known as estradiol 17 alpha-dehydrogenase and estradiol 17 beta-dehydrogenase from horse placenta. Initial findings indicate each enzyme has its own characteristics and is functionally specific to its respective type of estradiol (i.e., 17-alpha or 17-beta).

Procedures and Methods

• The enzymes were purified from the microsomal fraction of horse placenta. A microsomal fraction is a portion of a cell homogenate containing numerous vesicles derived from fragmented endoplasmic reticulum.
• These enzymes were then dissolved in a solution containing 1.5% sodium cholate, a type of bile salt that breaks down fats and oils.
• Purification required a series of steps, involving selective elution (separation through a liquid substance) from hydroxylapatite by varying concentrations of potassium phosphate. Hydroxylapatite, a form of calcium apatite, is being used in biochemistry to purify certain materials due to its selective adhesion traits.
• A more specific purification was then achieved using reactive blue 2-agarose and estriol hemisuccinate-Sepharose. Reactive blue 2-agarose is a reactive dye that binds to proteins and nucleic acids, and estriol hemisuccinate-Sepharose is a highly sensitive medium for detection and quantification of proteins.

Results and Findings

• Both enzymes showed a high level of specificity for their respective estradiol, meaning each one functions effectively with its corresponding hormone – estradiol 17-alpha or estradiol 17-beta.
• Measurements on their effectiveness or activity were termed ‘specific activity’. The 17 alpha-dehydrogenase has a specific activity of 10 IU/mg while the 17 beta-dehydrogenase reached 10.6 IU/mg. An increase in the specific activity of an enzyme preparation indicates an increased level of purification.
• Each enzyme showed a single band on polyacrylamide disc gel electrophoresis and sodium dodecyl sulfate gel electrophoresis showing their purity. Both techniques are standard methods for protein separation.
• Both enzymes function optimally at pH 9.0-9.5, indicating a slightly alkaline preference.
• The enzymes prefer NAD+, a coenzyme found in cells, over NADP+, but they can utilize both. NAD+ and NADP+ are important co-factors in biological reactions.

This study furthers understanding of how different enzymes interact with estradiol, an important sex hormone that has major effects on female reproductive system health.