Enzymatic Assay of 3α-Hydroxysteroid Dehydrogenase with Androsterone (EC 184.108.40.206)
Replaces OP SPANDR01. See CR SOP-DEK-ENZ.
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
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, Aldrich Product Number 221368 . Adjust to pH 8.9 at 25°C with 1 M HCl.
7.3.3. 6.0 mM β-Nicotinamide Adenine Dinucleotide, Oxidized Solution (β-NAD+)
220.127.116.11 Prepare a in purified water using β-Nicotinamide Adenine Dinucleotide sodium salt, Sigma-Aldrich Product Number N7004.
18.104.22.168 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).
7.4.1 Pipette (in milliliters) the following reagents into suitable cuvettes in the following sequence:
|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||-----|
22.214.171.124 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.
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