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Hydroxysteroid Dehydrogenase - Assay

Source:
Pseudomonas testosteroni
CAS:
9028-56-2
9015-81-0
EC:
1.1.1.51
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Hydroxysteroid Dehydrogenase Assay

Method
The assay is that of Marcus and Talalay (1956) in which the reaction velocity is measured as an increase in absorbance at 340 nm resulting from the reduction of NAD. One unit reduces one micromole NAD per min at 25°C and pH 9.0, using androsterone or testosterone as a substrate under the specified conditions.
Reagents
  • 0.03 M Tris⋅HCl buffer, pH 7.2 with 0.001 M EDTA
  • 0.166 M Sodium pyrophosphate buffer, pH 9.0
  • 0.0043 M NAD in reagent grade water. Note: NAD may vary in salt form and degree of hydration. Care should be exercised to use an analytical grade and the correct molecular weight.
  • 0.015% Androsterone. Prepare by dissolving 15 mg androsterone in 100 ml absolute methanol.
  • 0.015% Testosterone. Prepare by dissolving 15 mg testosterone in 100 ml absolute methanol.
Enzyme

Dissolve the purified enzyme at a concentration of 1 mg/ml in 0.03 M Tris⋅HCl pH 7.2 buffer with 0.001 M EDTA. Further dilutions are also made with this buffer.

Crude enzyme: Extraction of the enzyme from the cells can be accomplished by sonication of a 50 mg/ml cell suspension in 0.03 M Tris⋅HCl buffer, pH 7.2 with 0.001 M EDTA. Clarify by centrifugation and assay the supernatant.

For assay, dilute the enzyme to obtain a rate of 0.02-0.04 ΔA/min.

Procedure

Adjust the spectrophotometer to 340 nm and 25°C.

Pipette into each cuvette as follows:

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 spectrophotometer for 3-4 minutes to achieve temperature equilibration and establish blank rate, if any. At zero time, add 0.01 ml testosterone solution. Record A340 for 3-4 minutes. Calculate ΔA340 per minute from the initial linear portion of the curve. Repeat, using androsterone as substrate.

Calculation

Hydroxysteroid Dehydrogenase Products

Description
Activity
Code
Cat. #
Size
Price
Description
Hydroxysteroid Dehydrogenase
Source:
Pseudomonas testosteroni
A lyophilized powder obtained from induced cells. Contains both alpha and beta activities.
Store at -20°C.
…
Ice Pack required
Activity
≥0.03 units per mg dry weight
Code
STDH
LS004915
1 gm
$87.00
LS004916
5 gm
$349.00
LS004918
Bulk
---
The minimum amount for bulk packaging/pricing for this product is 100 x 1 gm. Please contact custservice@worthington-biochem.com to request a quote for smaller amounts.
Description
Hydroxysteroid Dehydrogenase
Source:
mutant: Pseudomonas testosteroni
A purified powder obtained from adapted cells of a mutant strain. Activity on androsterone only, no activity exhibited on testosterone.
Store at -20°C.
…
Ice Pack required
Activity
≥0.5 units per mg dry weight
Code
STDHMP
LS004908
10 un
$108.00
LS004910
50 un
$385.00
LS004911
Bulk
---
The minimum amount for bulk packaging/pricing for this product is 1000 x 1 un. Please contact custservice@worthington-biochem.com to request a quote for smaller amounts.
Description
Hydroxysteroid Dehydrogenase
Source:
Pseudomonas testosteroni
A purified powder obtained from induced cells. Contains both alpha and beta activities.
Store at -20°C.
…
Ice Pack required
Activity
≥0.5 units per mg dry weight
Code
STDHP
Minimum order quantity
5000
LS004922
Bulk
---
The minimum amount for bulk packaging/pricing for this product is 5000 x 1 un. Please contact custservice@worthington-biochem.com to request a quote for smaller amounts.