3-α-hydroxysteroid dehydrogenase (3α-HSD), CAS No.9028-56-2

CAS: 9028-56-2 Cat. No.: rp216186 EC Number: 232-839-5

GRADE & PURITY Suitable for molecular biology, BioReagent, EnzymoPure™, ≥90%(SDS-PAGE), ≥ 55 U/mg

Overview

Application
It is used for the development and mass preparation of total bile acid (TBA) reagents.
Enzymatic properties (note that 2+, +, 3+ are superscripts)
Source: Microorganism
Enzymology Committee Number: EC1.1.1.50
Molecular weight: 30 kDa (SDS-PAGE)
Isoelectric point: 5.2
Km value: 3.6×10-5M (androsterone), 4.7×10-5M (NAD+)
Inhibitors:  Cu²⁺，Ag⁺，Hg²⁺，Zn²⁺，Fe³⁺
Optimum pH: 8.5-9.5                                                 Figure 1
Optimum temperature: 55℃                                      Figure 2
pH stability: 6.0-9.0 (25℃,16h)                                  Figure 3
Thermal stability: Stable below 37℃ (pH9.0, 30min) Figure 4
Stability: -25 ~ -15℃ standing store for 12 months
More than 90% activity                                               Figure 5
Protective agent: BSA


 Assay method for activity
1. Principle

The NADH produced by the reaction can be detected with a spectrophotometer at 340nm.
2. Definition of enzyme activity
Unit enzyme activity is defined as the amount of enzyme required to catalyze the production of 1μmolNADH per minute under the following conditions.
3. Reagent preparation
Reagent I: 0.1M sodium pyrophosphate (adjust pH to 8.9 with HCl).
Reagent II: Dissolve 319mg NAD+ into 25mL double steaming water, adjust the pH to 7.0-7.5 with solid NaHCO3, and adjust the volume to 30mL with double steaming water.
Reagent III: Dissolve 30mg into 100mL methanol.
Enzyme diluent: 10mMTris-HCl, pH 9.0.
4. Operation procedure
1. Add 2.6mL reagent I, 0.2mL reagent II and 0.1mL reagent III into a 3mL colorimetric dish and mix well.
2. Preheat the reaction mixture at 25°C for 5min.
3. Add 0.1mL enzyme liquid to the reaction mixture, mix it well, react at 25°C, and record the absorbance change within 1min at 340nm with a spectrophotometer (∆As).
* Replace enzyme liquid with enzyme diluent, other steps are the same, the absorbance of the resulting solution is blank absorbance (∆Ab)
∆A=∆As-∆Ab
5. Vitality computing

3.00: total volume of reaction liquid (mL);
0.10: enzyme liquid volume (mL);
1.0: optical path length (cm);
df: dilution ratio;
C: Enzyme concentration (mg/mL);
6.22: Nanomolar absorption coefficient of NADH at 340nm (cm2/μmol).

Specifications

Product Name

3-α-hydroxysteroid dehydrogenase (3α-HSD), CAS No.9028-56-2

Grade

BioReagent, Suitable for molecular biology, EnzymoPure™

Specifications & Purity

BioReagent, Suitable for molecular biology, EnzymoPure™, ≥90%(SDS-PAGE), ≥ 55 U/mg

CAS

9028-56-2

Molecule Type

Enzyme

Storage and Shipping

Concentration

≥ 55 U/mg

Storage

Store at -20°C,Avoid repeated freezing and thawing

Shipped In

Ice chest + Ice pads

Stability And Storage

Store at -20℃ long term (12 months). Upon receipt, it is recommended to aliquot. Avoid freeze/thaw cycle.