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.
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)
Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.
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.
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:
2. Mix by inversion. Place cuvettes in a suitably thermostatted spectrophotometer and equilibrate to 25 °C.
3. Then add:
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.
|Units/mL enzyme =||(ΔA340/min Test – ΔA340/min Blank) (3.0) (df)
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
|Units/mg solid =||units/mL enzyme|
|mg solid/mL enzyme|