Enzymatic Assay of 3α-Hydroxysteroid Dehydrogenase with Androsterone (EC 1.1.1.50)

Document History

Replaces OP SPANDR01. See CR SOP-DEK-ENZ[77].

1. OBJECTIVE
To standardize a procedure for the enzymatic determination of 3α-Hydroxysteroid dehydrogenase.

2. SCOPE
This procedure applies to products that have a specification for 3α-Hydroxysteroid dehydrogenase by enzymatic determination.

3. DEFINITIONS

3.1. Purified Water - water from a deionizing system, resistivity > or = 18MΩ•cm @ 25ºC

3.2. Unit Definition - One unit will oxidize 1.0 μmole of androsterone to 5α-Anhydrosterone-3,17-dione per minute in the presence of β-NAD at pH 8.9 at 25°C.

3.3. 3α-HSDH = 3α-Hydroxysteroid Dehydrogenase

3.4. β-NAD+ - β-Nicotinamide Adenine Dinucleotide, Oxidized Form

3.5. β-NADH - β-Nicotinamide Adenine Dinucleotide , Reduced Form

4. DISCUSSION
Androsterone + β-NAD+    3alpha-HSDH   > 5α-Anhydrosterone-3,17-dione + β-NADH

5. RESPONSIBILITIES
It is the responsibility of all Analytical Services laboratory personnel to follow this protocol as written.

6. SAFETY
Refer to the Material Safety Data Sheet (MSDS) for hazards and appropriate handling precautions.

7. PROCEDURE

7.1 CONDITIONS:
T = 25°C, pH = 8.9, A340nm, Light path = 1 cm

7.2 METHOD:
Spectrophotometric rate

7.3 REAGENTS:

7.3.1.    100 mM Sodium Pyrophosphate, pH 8.9 at 25°C (Buffer)
Prepare a 44.6 mg/ml solution in purified water using Sodium Pyrophosphate, Aldrich Product Number 221368 . Adjust to pH 8.9 at 25°C with 1 M HCl.

7.3.2.    0.015%(w/v) Androsterone (ANSD)
Prepare a 0.15 mg/ml solution in absolute Methanol, Sigma-Aldrich Product Number M1775 using Anhrosterone, Sigma-Aldrich Product Number A9755. Prepare fresh.

7.3.3.    6.0 mM β-Nicotinamide Adenine Dinucleotide, Oxidized Solution (β-NAD+)

7.3.3.1    Prepare a in purified water using β-Nicotinamide Adenine Dinucleotide sodium salt, Sigma-Aldrich Product Number N7004.

7.3.3.2    Correct concentration for percent water, percent solvent, and percent purity by high pressure liquid chromatography.

7.3.4.    10 mM Potassium Phosphate / 0.5%(w/v) Albumin, Bovine, pH 7.2 at 25°C (Enzyme Diluent)
Prepare a 1.36 mg/ml solution in purified water using Potassium Phosphate, Monobasic, Sigma-Aldrich Product Number P5379 and 5.0 mg/ml solution using Albumin, Bovine, Essentially Fatty Acid Free, Sigma-Aldrich Product Number A6003. Adjust to pH 7.2 at 25°C with 1 M KOH.

7.3.5.    3α-Hydroxysteroid dehydrogenase Dehydrogenase Enzyme Solution (Enzyme)

Immediately before use, prepare a solution at 2.0 mg/ml in cold Reagent 7.3.4 (Enzyme Diluent). Then dilute to 0.15 to 0.30 units / ml with cold Reagent 7.3.4 (Enzyme Diluent).

7.4. PROCEDURE

7.4.1    Pipette (in milliliters) the following reagents into suitable cuvettes in the following sequence:

  Test1 Test2 Test3 Blank
Purified Water 2.00 2.00 2.00 2.00
Reagent 7.3.1(Buffer) 0.60 0.60 0.60 0.60
Reagent 7.3.3 (β-NAD+) 0.20 0.20 0.20 0.20
Reagent 7.3.2 (ANSD) 0.05 0.03 ----- 0.10
Reagent 7.3.4 (Enzyme Diluent) 0.10 0.10 0.10 0.10

7.4.2    Equilibrate to 25°C. Monitor the A340nm for a minimum of one minute, using a suitably thermostatted spectrophotometer. Then add:

Reagent 7.3.5 (Enzyme) 0.05 0.07 0.10 -----

7.4.2.1    Run one aliquot level at a time. Prepare a fresh 0.15 to 0.30 units / ml solution from the concentrated enzyme solution for each aliquot level.

7.4.3.    Immediately mix by inversion and record the increase in A340nm for at least 2.5 minutes. Obtain the ΔA340nm/minute using the maximum linear rate for all Test and Blank reaction mixtures using a minimum of 5 points over a one-half minute time interval.

7.5 CALCULATIONS

7.5.1 Units/mL enzyme = ΔA340nm / min Test - ΔA340nm /min Blank)*(3)*(df)
(6.22)*(0.1)

where:
     3 = Total volume (in milliliters) of assay
     df = Dilution factor
     6.22 = Millimolar extinction coefficient of β-NADH at 340 nm
    0.1 = Volume (in milliliters) of enzyme used

7.5.1 Units/mg solid = Units/mL enzyme
mg solid/mL enzyme

7.5.3 Units/mg protein = Units/mL enzyme
mg protein/mL enzyme

7.6.    FINAL ASSAY CONCENTRATION
In a 3.00 ml reaction mix, the final concentrations are 20 mM sodium pyrophosphate,0.4 mM β-nicotinamide adenine dinucleotide, oxidized, 0.0005%(w/v) Androsterone, 0.33 mM potassium phosphate, 0.017% albumin,bovine, 3.3% methanol, and 0.0075 - 0.03 units 3α-hydroxysteroid dehydrogenase.

8. REFERENCES & ATTACHMENTS
Talalay, P. A. (1962) Methods in Enzymology 5, 512-526.

9. APPROVAL
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Materials

     
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