3α–HYDROXYSTEROID DEHYDROGENASE [3α–HSDⅡ]
from Pseudomonas sp.
(3 α –Hydroxysteroid: NAD+ oxidoreductase, EC 1.1.1.50)
Preparation and Specification
- Appearance
- : White amorphous powder, lyophilized
- Specific activity
- : More than 30 U/mg solid
Properties
- Substrate specificity
- : See Table 1
- Molecular weight
- : 41 kDa(gel filtration)
- Isoelectric point
- : pH 4.8±0.2
- Michaelis constants
- : Androsterone 2.1 × l0-4M
- Optimum pH
- : 8.0–10.0Figure 1
- pH stability
- : 6.0–10.0(37℃, 10 min)Figure 2
- Optimum temperature
- : 50℃(Phosphate buffer) Figure 3
- Thermal stability
- : Stable at 50℃ and below(pH 8.0, 10 min)Figure 4
- Effect of metal ions
- : See Table 2
- Effect of detergents
- : See Table 3
- Inhibitor
- : MnCl2
Applications for Diagnostic Test
This enzyme is useful for enzymatic cycling determination of bile acid when coupled with thio–NAD and NADH.
Table 1. Substrate specificity
Substrate (0.95mM) | Relative activity (%) |
---|---|
Cholic acid | 100 |
Androsterone | 131 |
Deoxycholic acid | 115 |
Chenodeoxycholic acid | 89.0 |
Glycocholic acid | 103 |
Taurocholic acid | 99.0 |
Taurodeoxycholic acid | 128 |
Table 2. Effect of metal ions on 3α–HSDⅡ activity
Metal ion | Relative activity (%) |
---|---|
None | 100 |
NaCl | 105 |
KCl | 102 |
LiCl | 101 |
MgCl2 | 106 |
MnCl2 | 16.0 |
CaCl2 | 105 |
Table 3. Effect of detergents on 3α–HSDⅡ activity
Detergent | Relative activity (%) |
---|---|
None | 100 |
Triton X–100 | 71.0 |
Triton X–305 | 71.0 |
Triton X–114 | 71.0 |
Adekanol SO–120 | 104 |
Adekanol NP–720 | 75.0 |
Adekanol B–795 | 78.0 |
Emulgen B–66 | 75.0 |
Emulgen 911 | 76.0 |
Emulgen 709 | 84.0 |
Emulgen 810 | 50.0 |
Emulgen 109P | 112 |
Rheodol 460 | 71.0 |
Rheodol TWL–103 | 72.0 |
Fig.1 pH Optimum

buffer
●: Phosphate buffer
□: Tris-HCI buffer
■: Glycine-NaOH buffer
Fig.2 pH Stability

〇: Acetate buffer
●: Phosphate buffer
□: Tris-HCI buffer
■: Glycine-NaOH buffer
Fig.3 Optimum Temperature

20 mM Phosphate buffer
Fig.4 Thermal Stability

20 mM Phosphate buffer
Assay
Principle
The assay is based on the increase in absorbance at 550 nm as formazan dye is formed in the following reactions:
3α–HSD Ⅱ | ||
Cholic acid+NAD+ | → | 3–Oxocholic acid+NADH+H+ |
DI | ||
NADH+H++NTB | → | NAD++NTBH2 |
NAD : Nicotineamido adenine dinucleotide,
NTB : Nitrotetrazolium blue
DI : Diaphorase
Unit definition
One unit is defined as the amount of enzyme which oxidizes 1 μmole of cholic acid to 3–oxocholic acid per minute at 37℃ under the conditions specified in the assay procedure.
Reagents
- Reaction mixture
10 mM NAD solution 0.05 ml 0.25%(W/V)NTB solution 0.05 ml 100 U/ml DI solution 0.025 ml 2%(W/V)Triton X–100 solution 0.10 ml 0.2 M Tris–HCl buffer pH 8.0 0.10 ml Distilled water 0.175 ml 1): 100 U/ml DI solution
Dissolve 100 U of DI with 1 ml of 10 mM Tris–HCl buffer pH 8.0. - Substrate solution(20 mM Androsterone)
Dissolve 23 mg of androsterone with 4 ml of methanol. - Reaction stopper
0.5%(W/V)Sodiumdodecyl sulfate(SDS)solution - Enzyme dilution buffer
10 mM Tris–HCl buffer pH 8.0 - Reagents
NAD: NACALAI TESQUE, INC. #24334–84
- NTB: Dojindo Laboratories #344–02033
DI: Nagase Diagnostics Co., Ltd. #T–06
Triton X–100: The Dow Chemical Company
Androsterone: Sigma Chemical Co. #A–9755
SDS(Sodium Dodecyl Sulfate):NACALAI TESQUE, INC. Extra pure #31606–75
Enzyme solution
Accurately weigh about 20 mg of the sample and add enzyme dilution buffer to make a total of 20 ml. Dilute it with enzyme dilution buffer to adjust the concentration as required.
Procedure
- Pipette accurately 0.5 ml of reaction mixture into a
small test tube, then add 20 μl of enzyme solution into the same test tube and preincubate at 37℃.※ In the case of a test blank, add 20 μl of enzyme dilution buffer in place of enzyme solution. - After 5 min, add exactly 25 μl of substrate solution and mix to start the reaction at 37℃.
- At 5 min after starting the reaction, add 2.5 ml of the reaction stopper to stop the reaction.
- Measure the absorbance at 550 nm.
△A =(As−Ab)≦ 0.20 AbsAbsorbance sample : As blank : Ab
Calculation
- Activity(U/mg of powder)= {(△A/5)/(16.7)} × 3.045/0.02 × 1/x
16.7 : millimolar extinction coefficient of NTBH2 at 550 nm(cm2/ μmole) 5 : reaction time(min) 3.045 : final volume(ml) 0.02 : volume of enzyme solution(ml) X : concentration of the sample in enzyme solution ( mg/ml)
Storage
Storage at −20℃ in the presence of a desiccant is recommended.
References
- Suzuki, K. and Tamaoki, B.(1974)J. Steroid Biochem., 5,
249–256. - Inano, H., Hayashi, S. and Tamaoki, B.(1977)J. Steroid Biochem., 8, 41–46.
- Shikita, M. and Talalay, P.(1979)Anal. Biochem., 92, 286–292.
- Uwajima, T., Takayama, K. and Terada, O.(1978)Agric.
Biol. Chem., 42, 1577–1583. - Talalay, P.(1962)Methods Enzymol., 5, 512.
- Mowszowicz, I. and Bardin, C. W.(1974)Steroids, 23, 793–807.
- Nimrod, A., Lamprecht, S. A. and Lindner, R. H.(1975)J. Steroid Biochem., 6, 1205–1209.
- Nozu, K., Inano, H. and Tamaoki, B.(1974)Proteins Nucleic Acids and Enzymes(Japan), 19, 397–410.
3α–HSD Ⅱ活性測定法(Japanese)
試薬液
- 反応試薬混合液
10mM NAD 溶液 0.05 ml 0.25%(W/V)NTB 溶液 0.05 ml 100U/ml DI 溶液1) 0.025 ml 2%(W/V)トリトンX–100 溶液 0.10 ml 0.2M トリス−HCl 緩衝液pH8.0 0.10 ml 精製水 0.175 ml 1): 100U/ml DI 溶液
DI 100 単位(U)を10mM トリス−HCl 緩衝液 pH8.0 1ml で溶解する。 - 基質溶液(20mM アンドロステロン溶液)
アンドロステロン23mg をMeOH4ml で溶解する。 - 反応停止液
0.5%(W/V)SDS 溶液 - 酵素溶解希釈用液
10mM トリス−HCl 緩衝液pH8.0 - 試薬
NAD(ニコチンアミドアデニンジヌクレオチド):ナカライテスク製 #24334–84NTB(ニトロテトラゾリウムブルー):同仁化学製 #344–02033DI(ジアフォラーゼ):ナガセダイアグノスティックス製 #T–06
トリトンX–100:Dow Chemical 製
アンドロステロン:シグマ製 #A–9755
SDS(ドデシル硫酸ナトリウム):ナカライテスク製 一級 #31606–75
酵素試料液
- 検品約20mg を精密に量り、酵素溶解希釈用液で溶解して全容20ml とする。
その液を酵素溶解希釈用液で適宜希釈する。
測定操作法
- 小試験管に反応試薬混合液0.5ml を正確に分注し、後に酵素試料液20 μl を正確に分注して37℃で予備加温する。
※ 盲検は酵素試料液の代わりに酵素溶解希釈用液20μl を加える。 - 5 分経過後、基質溶液25 μl を正確に加えて混和し、37℃で反応を開始する。
- 5 分経過後、反応停止液2.50ml を正確に加えて混和し、反応を停止する。
- 550nm における吸光度を測定する。
求められた吸光度を試料液はAs、盲検液はAb とする。
ΔA =( As−Ab) ≦ 0.20 Abs
計算
活性(U/mg)= {(ΔA/5)/(16.7)} × 3.045/0.02 × 1/x16.7 : | NTBH2 の550nm におけるミリモル分子吸光係数 (cm2 / μmole) |
5 : | 反応時間(min) |
3.045 : | 反応総液量(ml) |
0.02 : | 反応に供した酵素試料液量(ml) |
X : | 酵素試料液中の検品濃度(mg/ml) |