Replaces OP SPANDR01. See CR SOP-DEK-ENZ.
To standardize a procedure for the enzymatic determination of 3α-Hydroxysteroid dehydrogenase.
This procedure applies to products that have a specification for 3α-Hydroxysteroid dehydrogenase by enzymatic determination.
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
Androsterone + β-NAD+ 3alpha-HSDH > 5α-Anhydrosterone-3,17-dione + β-NADH
It is the responsibility of all Analytical Services laboratory personnel to follow this protocol as written.
Refer to the Safety Data Sheet (SDS) for hazards and appropriate handling precautions.
T = 25 °C, pH = 8.9, A340nm, Light path = 1 cm
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+)
184.108.40.206. Prepare a in purified water using β-Nicotinamide Adenine Dinucleotide sodium salt (N7004).
220.127.116.11. 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).
7.4.1. Pipette (in milliliters) the following reagents into suitable cuvettes in the following sequence:
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||-----|
18.104.22.168. 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.
|7.5.1||Units/mL enzyme =||ΔA340nm / min Test - ΔA340nm /min Blank)*(3)*(df)|
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.
Talalay, P. A. (1962) Methods in Enzymology 5, 512-526.
Review, approvals and signatures for this document will be generated electronically using DocCompliance (QUMAS). Print a “For Use” copy if hardcopy with signature verification is required.