3α-HSD (3α-hydroxysteroid dehydrogenase)
This enzyme is useful for enzymatic cycling determination of bile acid when coupled with thio–NAD and NADH.
Preparation and specification
|
Appearance |
White amorphous powder, lyophilized |
|
Purity(SDS-PAGE) |
≥90.0% |
|
Enzyme powder specific activity |
≥55.0 U/mg |
|
Glucose-6-phosphate dehydrogenase |
≤0.01% |
|
Lactate dehydrogenase |
≤0.005% |
|
Glutamate dehydrogenase |
≤0.001% |
|
Malate dehydrogenase |
≤0.01% |
|
Hexokinase |
≤0.01% |
Properties
|
EC number |
1.1.1.50(Recombinantfrom microorganism) |
|
Molecular weight |
30 kDa (SDS-PAGE) |
|
Isoelectric point |
5.2 |
|
Michaelis constants |
3.6×10-5M (Androsterone), 4.7×10-5 (NAD+) |
|
Inhibitors Optimum pH |
Fe3+, Co2+, Cu2+, Mn2+, Zn2+ 8.5-9.5 Fig. 1 |
|
Optimum temperature |
55 ℃ Fig. 2 |
|
pH stability |
pH 6.0 – 9.0 (25 ℃, 16 h) Fig. 3 |
|
Thermal stability |
Below 37 ℃ (pH 9.0, 30 min) Fig. 4 |
|
Storage stability |
At least one year at -25~ -15 ℃ Fig. 5 |
|
Stabilizers |
BSA |
Storage condition
Store at -25~-15°C, sealed, dry and protected from light. Valid for 2 year.
Figures
Assay principle
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 100 mL 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 2.6mL of Reagent I, 0.2mL of Reagent II and 0.1mL of Reagent III to the 3 mL cuvette (d=1.0 cm) in order.
2. Preincubate the reaction mixture at 25 ℃ for 5 min.
3. Add 0.1 mL the enzyme solution in 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
Calculation
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) J. Steroid Biochem., 8, 41–46.
3. 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.
