Origin
Pseudomonas testosteroni
Formula
Appearance
White amorphous powder.
Activity
More than 25 units/mg protein.
Unit definition
One unit oxidize 1 μmol of testosterone as a substrate per minute in the presence of NAD at pH8.9 at 25 ºC.
Storage
Stable for one year when stored below 5 ºC and also stable at room temperature for at least one week. For prolonged storage, keep at -20 ºC.
Contaminants
Malate dehydrogenase | < 0.01% |
Lactate dehydrogenase | < 0.01% |
Alcohol dehydrogenase | < 0.01% |
3α-Hydroxysteroid dehydrogenase | 約 0.1 % |
Properties
Molecular weight | Approx. 80,000 Da |
---|---|
Optimum pH | 10.1~10.3 |
Optimum temperature | 34℃ |
pH stability | 7.0~8.7 |
Thermal stability | below 42 ºC (pH 7.2, 10 min) |
Michaelis constant | 2.5×10-6M(Testosterone) 1.8×10-4M(NAD) |
Assay method
(Spectrophotometric method)
Reagents
- Sodium pyrophosphate buffer , pH 8.9 : 100 mM
- NAD solution : 15 mM (adjust pH to 7.0–7.5 with solid NaHCO3)
- Substrate solution : 3 mM Testosterone in methanol
- Enzyme solution : Prepare approximately 10 units/ml solution of β-Hydroxysteroid dehydrogenase in 30 mM potassium phosphate buffer, pH 7.2. This solution, stored at 5 ºC, is stable for at least one week. For assay of activity dilute this enzyme solution to approximately 0.4 unit/ml with enzyme diluent.
- Enzyme diluent : 30 mM Potassium phosphate buffer (pH 7.2) containing 0.1% bovine serum albumin.
Procedure
- Prepare the following reaction mixture in a cuvette (d =1.0cm) and equilibrate at 25 ºC for about 10 minutes.
1.6 ml H2O
1.0 ml Sodium pyrophosphate buffer (A)
0.2 ml NAD solution (B) - Add 0.1 ml enzyme solution (approx. 0.04 unit/cuvette) (D) and mix.
- Add 0.1 ml substrate solution (C) and mix.
- Read the increase of optical density at 340 nm against water as reference for several minutes at timed intervals in a spectrophotometer thermostated at 25 ºC, and calculate the DA340 per minute from the initial linear portion of the increase of optical density (usually, DA340 per minute is calculated from the increase between 30 and 90 seconds after the addition of substrate).
Calculation
unit / cuvette | = DA340 x 3 ÷ 6.22 |
where 3 | = volume (ml) of solution in a cuvette |
6.22 | = millimolar extinction coefficient of NADH |
Package
10 units, 50 units