Enzymatic Assay of Alcohol Dehydrogenase (EC 1.1.1.1)

Description

This procedure may be used for alcohol dehydrogenase products, except for insoluble forms of alcohol dehydrogenase (Product No. A2529).

The continuous spectrophotometric rate determination (A₃₄₀, light path = 1 cm) is based on the following reaction:

β-NAD = β-nicotinamide adenine dinucleotide phosphate, oxidized
β-NADH = β-nicotinamide adenine dinucleotide phosphate, reduced

Unit Definition: One unit of alcohol dehydrogenase will convert 1.0 µmol of ethanol to acetaldehyde per minute at pH 8.8 at 25 °C.

Reagents and Equipment Required

Phosphoric acid (Product No. 438081)
Sodium pyrophosphate, tetrabasic, decahydrate (Product No. 221368)
Ethanol 95% (v/v) (Product  No. 493538)
β-NAD hydrate (Product No. N6522)
Sodium phosphate, monobasic, monohydrate (Product No. S9638)
5 M Sodium phosphate, monobasic solution (Product No. 74092)
Sodium phosphate, dibasic (Product No. S9763)
0.5 M Sodium phosphate, dibasic solution (Product No. 94046)
Bovine serum albumin (Product No. A9647)

Precautions

Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Preparation Instructions

Use ultrapure water (≥18 MΩ×cm resistivity at 25 °C) for the preparation of reagents.

9.5% (v/v) Phosphoric acid – Prepare a 0.095 mL/mL solution of phosphoric acid (Product No. 438081) in purified water.

50 mM Sodium-phosphate buffer, pH 8.8 (25 °C) – Prepare a 22.3 mg/mL solution of sodium pyrophosphate, tetrabasic, decahydrate (Product No. 221368) in ultrapure water. Adjust the pH to 8.8 at 25 °C with 9.5% (v/v) phosphoric acid.

Ethanol 95% (v/v) ethanol (Product No. 493538)

15 mM ß-NAD (ß-nicotinamide adenine dinucleotide) solution – Prepare a 11.6 mg/mL solution of ß-nicotinamide adenine dinucleotide hydrate (Product No. N6522) in ultrapure water.

10 mM Sodium-phosphate buffer, pH 7.5 (25 °C) – To prepare 500 mL of buffer, add 21.0 mL of stock 0.2 M sodium phosphate dibasic solution, prepared from 0.5 M Sodium phosphate, dibasic solution (Product No. 94046). Then add 4.0 mL of stock 0.2 M sodium phosphate monobasic solution, prepared from 5 M sodium phosphate, monobasic solution (Product  No. 74092). Adjust to a final volume of 500 mL with ultrapure water. Adjust to pH 7.5 at 25 °C using 1 M NaOH or 1 M HCl.

Enzyme Diluent (10 mM Sodium Phosphate Buffer, pH 7.5 with 0.1% (w/v) Bovine Serum Albumin) – Prepare a 1 mg/mL solution of bovine serum albumin (Product No. A9647) in 10 mM Sodium Phosphate Buffer, pH 7.5. Adjust pH to 7.5 (25 °C) with 1 M NaOH or 1 M HCl.

ADH Stock Solution – Prepare a 1 mg/mL solution of alcohol dehydrogenase in cold (2–8 °C) 10 mM sodium-phosphate buffer, pH 7.5.
Note: Prepare Fresh

ADH Working Solution – Immediately before use, dilute 0.050 mL of the ADH Stock Solution to 25.0 mL with cold Enzyme Diluent.
Note: Prepare Fresh

Note: If this enzyme concentration yields a ΔA₃₄₀/minute >0.15, dilute 0.050 mL of the ADH Stock Solution to 50.0 mL with cold Enzyme Diluent. Only deviate from the enzyme dilution scheme if assaying crude products.

Procedure

In a 3.00 mL reaction mix, the final concentrations are 22 mM sodium pyrophosphate, 3.2% (v/v) ethanol, 7.5 mM β-nicotinamide adenine dinucleotide, 0.3 mM sodium phosphate, 0.003% (w/v) bovine serum albumin, and 0.001–0.002 mg/mL of alcohol dehydrogenase.

1. Into suitable cuvettes, accurately pipette the following:

Reagent	Test 1 (mL)	Test 2 (mL)	Test 3 (mL)	Blank (mL)
50 mM Sodium Phosphate Buffer, pH 8.8	1.30	1.30	1.30	1.30
95% (v/v) Ethanol	0.10	0.10	0.10	0.10
15 mM ß-NAD Solution	1.50	1.50	1.50	1.50

2. Mix by inversion. Place cuvettes in a suitably thermostatted spectrophotometer and equilibrate to 25 °C.

3. Then add:

Reagent	Test 1 (mL)	Test 2 (mL)	Test 3 (mL)	Blank (mL)
ADH Working Solution	0.10	0.10	0.10	–
Enzyme Diluent	–			0.10

4. Immediately mix by inversion and record the increase in A₃₄₀ for ~6 minutes.

5. Obtain the A₃₄₀/minute using the one to six minute range for both the Tests and Blank.

Results

Calculations

1.

Units/mL enzyme =	(ΔA340/min Test – ΔA340/min Blank) (3.0) (df)
	(6.22) (0.1)

3.0 = Total Volume (mL) of assay
     df = Dilution Factor
     6.22 = Millimolar extinction coefficient of β-NADH at 340 nm
     0.1 = Volume (mL) of Enzyme Solution used

2.

Units/mg solid =	units/mL enzyme
	mg solid/mL enzyme

Reference

1. Kagi J, Vallee B. 1960. The Role of Zinc in Alcohol Dehydrogenase. J Biol Chem. 235(11):3188-3192.