Worthington hydroxysteroid dehydrogenase technical description and determination scheme

Hydroxysteroid dehydrogenase catalyzes the conversion of hydroxyl and carboxyl groups of steroids .  There are two types of testosterone derived hydroxysteroid dehydrogenase ：3-α- Hydroxysteroid dehydrogenase （α enzyme ） and 3-β- Hydroxysteroid dehydrogenase （β enzyme ）.α The molecular weight of the enzyme is 47,000 Dalton .α Enzymes only oxidize  C19、C21  and  C24  Series of  3-α- Hydroxysteroid . It is inhibited by heavy metals and sulfhydryl binders .β Enzymatic catalysis C19 and C21 Series of 3-β- Hydroxysteroid 、C18、C19 and C21 Series of 17-β- Hydroxy steroids and some 16-β- Oxidation of hydroxysteroid . It is inhibited by heavy metals and reducing agents . The oxidation of testosterone is  3,17-α- Estradiol and others  1,3,5- Estradiene derivatives inhibit .

AI Meijie Worthington  Two preparations are provided ： One from the conventional P. testosteroni(ATCC 11966)  Culture production α and β enzyme , The other comes from almost complete production α Mutant strain of enzyme . By using both , Can be determined by differences β- Hydroxysteroid .

 

AI Meijie Worthington Hydroxysteroid dehydrogenase Technical specification ：STDHP  and  STDH  Also include α and β activity . However ,STDHMP  Contains only α activity . 

Unit definition ： A unit in  25°C、pH 9.0  Decrease per minute  1  Micromore  NAD, Use androgen or testosterone as a substrate .

 

AI Meijie Worthington Hydroxysteroid dehydrogenase Determination ：

Method :  The determination is  Marcus  and  Talalay (1956)  Determination of , The reaction rate is measured as  NAD  The decrease in  340 nm  Increase in absorbance at . A unit in  25°C  and  pH 9.0  Reduce per minute under  1  Micromore  NAD, Use androgen or testosterone as substrate under specific conditions .

Reagents  :

0.03 M Tris⋅HCl  Buffer ,pH 7.2, contain  0.001 M EDTA

0.166 M  Sodium pyrophosphate buffer ,pH 9.0

0.0043 M NAD  In reagent grade water . Be careful ：NAD  The salt form and degree of hydration may vary . Analytical grade and correct molecular weight should be used with care .

0.015%  Androgen . By way of  15 mg  Androgen dissolved in  100 ml  From anhydrous methanol .

0.015%  testosterone . By way of  15 mg  Testosterone dissolves in  100 ml  From anhydrous methanol .

enzyme  :

Purify the enzyme with  1 mg/ml  The concentration of dissolved in contains  0.001 M EDTA  Of  0.03 M Tris⋅HCl pH 7.2  In buffer . This buffer is also used for further dilution .

Crude enzyme ： Enzymes can be extracted from cells by  0.001 M EDTA  Yes  0.03 M Tris·HCl  Buffer （pH 7.2） Medium  50 mg/ml  The cell suspension is ultrasonically treated . Clarify by centrifugation and determine the supernatant .

For determination , Dilute the enzyme to obtain  0.02-0.04 ΔA/min  Rate .

Program  :

Adjust the spectrophotometer to  340 nm  and  25°C.

Move the pipette into each cuvette , As shown below ：

0.166 M Sodium pyrophosphate  0.6  ml

0.0043 M NAD 0.2  ml

Reagent grade water  2.0 ml

enzyme  0.1  ml

Incubate in a spectrophotometer  3-4  Minutes to reach temperature equilibrium and establish blank rate （ If there is ）. At zero o'clock , Join in  0.01 ml  Testosterone solution . Record  A 340 3-4  minute . Calculate every minute from the initial linear part of the curve ΔA 340 . repeat , Use androgen as substrate .