Androsterone + NAD+ ⇒ 3-Oxoandrosterone + NADH + H+
The appearance of NADH is measured at 340nm by spectrophotometry.
One unit (U) is defined as the amount of enzyme which produces 1 μmol of NADH per min under the conditions described below.
Reagent I: 0.1 M sodium pyrophosphate (adjust pH 8.9 by HCl).
Reagent II: Dissolve 319 mg of NAD+ in 25 mL distilled water, adjusting to pH 7.0 – 7.5 by solid NaHCO3 and adding distilled water to 30 mL.
Reagent III: Dissolve 30 mg androsterone in 100mL methanol.
Enzyme diluent: 10 mM Tris-HCl, pH 9.0.
Sample: dilute the enzyme to 0.2-0.8 U/ml with enzyme diluent.
- Add Reagent I, Reagent II and Reagent III to the 3 mL cuvette (d=1.0 cm) in order.
- Preincubate the reaction mixture at 25 ℃ for 5 min.
Add 0.1 mL of the enzyme solution to the reaction mixture and mix to start the reaction, record the ΔAs at 340 nm in 1 minute in a spectrophotometer thermostated at 25 ℃.
At the same time, measure the blank rate
ΔAb by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution.
∆A = ∆As – ∆Ab
3.00: Total volume (mL)
0.10: Enzyme volume (mL)
1.0: Light path length (cm)
df: dilution factor
C: Enzyme concentration (mg/mL)
6.22: Millimolar extinction coefficient of
NADH under 340 nm (cm2/μmol)
- Suzuki, K. and Tamaoki, B. (1974) J. Steroid
Biochem., 5, 249–256.
- Inano, H., Hayashi, S. and Tamaoki, B. (1977)
- Steroid Biochem., 8, 41–46.
- Shikita, M. and Talalay, P. (1979) Anal.
Biochem., 92, 286–292.
- Uwajima, T., Takayama, K. and Terada, O.
(1978) Agric. Biol. Chem., 42, 1577–1583
This enzyme is useful for enzymatic cycling determination of bile acid when coupled with thio–NAD and NADH.