Alcohol Dehydrogenase Assay

Method: The reaction velocity is determined by the method of A Vallee and Hoch (1955) in which the rate of absorbance at 340 nm resulting from reduction of NAD is measured. One unit reduces one micromole of NAD per minute at 25°C under the specified conditions.

Reagents

• 0.1 M Sodium pyrophosphate buffer, pH 9.2
• 2 M Ethanol. Dilute 12.12 ml of 95% ethanol to 100 ml with reagent grade water.
• 0.025 M NAD. 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.
• 0.1 M Phosphate buffer, pH 7.5
• 0.1% Albumin (BSA)

Enzyme

Dissolve lyophilized enzyme at one mg/ml in 0.1 M phosphate buffer, pH 7.5.

Immediately prior to use, dilute all enzymes to a concentration of 0.05-0.25 units/ml in 0.1% albumin.

For protein determinations, dissolve enzyme in 0.1 M phosphate buffer, pH 7.5.

Procedure

Set spectrophotometer to 340 nm and 25°C.

Pipette into each cuvette:

0.1 M Pyrophosphate buffer	1.5 ml
2.0 M Ethanol	0.5 ml
0.025 M NAD	1.0 ml

Incubate in spectrophotometer for 3-4 minutes at 25°C to achieve temperature equilibrium and establish blank rate, if any. At zero time, add 0.1 ml of appropriately diluted enzyme to the cuvette and record the A₃₄₀ for 3-4 minutes. Calculate the ΔA₃₄₀/minute from the initial linear portion of the curve.

Calculation