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 @ 25 ºC

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 Safety Data Sheet (SDS) for hazards and appropriate handling precautions.

7. Procedure

7.1 CONDITIONS:
T = 25 °C, pH = 8.9, A_340nm, 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 (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 (M1775) using Anhrosterone (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 (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 (P5379) and 5.0 mg/mL solution using Albumin, Bovine, Essentially Fatty Acid Free (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:

7.4.2. Equilibrate to 25 °C. Monitor the A_340nm for a minimum of one minute, using a suitably thermostatted spectrophotometer. Then add:

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 A_340nm for at least 2.5 minutes. Obtain the ΔA_340nm/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

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

7.6. 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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