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