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

1. Sodium pyrophosphate buffer , pH 8.9 : 100 mM
2. NAD solution : 15 mM (adjust pH to 7.0–7.5 with solid NaHCO₃)
3. Substrate solution : 3 mM Testosterone in methanol
4. 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.
5. Enzyme diluent : 30 mM Potassium phosphate buffer (pH 7.2) containing 0.1% bovine serum albumin.

Procedure

1. 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)
2. Add 0.1 ml enzyme solution (approx. 0.04 unit/cuvette) (D) and mix.
3. Add 0.1 ml substrate solution (C) and mix.
4. 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