3α-Hydroxysteroid Dehydrogenase

Assay principle

Androsterone + NAD+      ⇒     3-Oxoandrosterone + NADH + H+

The appearance of NADH is measured at 340nm by spectrophotometry.

Unit definition

One unit (U) is defined as the amount of enzyme which produces 1 μmol of NADH per min under the conditions described below.

Reagents preparation

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.

Procedure

1. Add Reagent I, Reagent II and Reagent III to the 3 mL cuvette (d=1.0 cm) in order.

2. 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)

References

1. Suzuki, K. and Tamaoki, B. (1974) J. Steroid

Biochem., 5, 249–256.

2. Inano, H., Hayashi, S. and Tamaoki, B. (1977)
3. Steroid Biochem., 8, 41–46.
4. Shikita, M. and Talalay, P. (1979) Anal.

Biochem., 92, 286–292.

4. 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.