Enzymatic Assay of Alcohol Dehydrogenase (EC 1.1.1.1)

1. OBJECTIVE
The objective of this procedure is to standardize the enzymatic rate assay of Alcohol dehydrogenase.

2. SCOPE
The scope of this procedure includes main activity assays of Alcohol dehydrogenase, but is not to be used to assay Alcohol dehydrogenase, Insoluble Enzyme attached to beaded agarose, Sigma Prod. No. A2529.

3. DEFINITIONS

3.1 One unit will convert 1.0 mmole of ethanol to acetaldehyde per minute at pH 8.8 @ 25° C.

4. DISCUSSION
Ethanol + b-NAD    ^{Alcohol Dehydrogenase}   > Acetaldehyde + b-NADH

5. RESPONSIBILITIES
N/A.

6. SAFETY
Refer to Material Safety Data Sheets (MSDS) for hazards and appropriate handling precautions.

7. PROCEDURE

7.1 Reagents:

7.1.1     50 mM Sodium Pyrophosphate Buffer, pH 8.8 @ 25°C (Buffer)
(Dissolve 2.23 g of Sodium Pyrophosphate, Decahydrate, Sigma Product Number S9515, in 100 mL deionized water. Adjust the pH to 8.8 with 8% (v/v) Phosphoric Acid).

7.1.2    7.1.2 95% (V/V) Ethanol (ETOH)
(Dilute 9.5 ml of 200 Proof USP Ethyl Alcohol, Sigma Product Number Z5130, to a final volume of 10 mL with deionized water).

7.1.3    15 mM b-Nicotinamide Adenine Dinucleotide Solution (b-NAD)
(Prepare 50 mL in deionized water using the corrected Formula Molecular Weight of b-NAD, Sigma Product Number N6522. Prepare Fresh).

7.1.4    10 mM Sodium Phosphate Solution (NaPM)
(Dissolve 480 mg of Sodium Phosphate, Monobasic, Anhydrous, Sigma Product Number S9638, in 400 mL of deionized water).

7.1.5    10 mM Sodium Phosphate Buffer, pH 7.5 @ 25°C (NaPD)
(Dissolve 710 mg of Sodium Phosphate, Dibasic, Anhydrous, Sigma Product Number S9763, in 500 mL of deionized water. Adjust to pH 7.5 @ 25° C with NaPM.)

7.1.6    10 mM Sodium Phosphate Buffer with 0.1% (w/v) Bovine Serum Albumin(Enzyme Diluent)
(Dissolve 500 mg of Albumin, Bovine, Sigma Product Number A9647, in 500 mL of NaPD. Adjust to pH 7.5 @ 25° C with 1 N HCl).

7.1.7    Alcohol Dehydrogenase Enzyme Stock Solution1 (Enzyme Stock Solution)
(Immediately before use, carefully weigh, on an appropriate micro balance, 15-16 mg solid and dilute to 15-16 mL (1mg/mL) with cold (NaPD).

7.1.8    Alcohol Dehydrogenase Enzyme Working Solution1 (Enzyme Working Solution)
(Immediately before use, dilute 0.05 mL of Alcohol Dehydrogenase Enzyme Stock Solution) to 25.0 mL in cold Enzyme Diluent. If this enzyme concentration yields a DA/min greater than 0.15, dilute 0.05 mL of (Enzyme Stock Solution) to 50.0 mL in cold Enzyme Diluent. **Do not deviate from this dilution scheme**)2

7.2 Rate Assay:
Into appropriate cuvettes, accurately pipette the following (in milliliters)

Mix by inversion and equilibrate to 25° C. Monitor the A_340nm until constant, using a suitably thermostatted spectrophotometer. Then add:

Immediately mix by inversion and record the increase in A_340nm for approximately 6 minutes. Obtain the A_340nm/minute using the one to six minute range for both the Test and Blank.

7.3 Calculations:

3.0 = Total Volume (in milliliters) of assay
df = Dilution Factor
6.22 = Millimolar extinction coefficient of b-NADH at 340nm
0.1 = Volume (in milliliters) of enzyme used

8. EMERGENCY SHUTDOWN
N/A

NOTES

1. Alcohol Dehydrogenase is unstable in solution and should be assayed immediately following preparation of solutions.

2. Sigma Product Number A9589 is a crude product and should be regarded as an exception to this dilution scheme rule. This product must be run at 2-10 mg solid/mL as dictated by the expected result.

3. This assay calls for 100uL of Enzyme working solution to be pipetted into every test reaction mix. This is in contrast to standard Sigma procedure that typically calls for triple-tiered enzyme solution volumes in replicate test reaction mixes.

4. In a 3.00 mL reaction mix, the final concentrations are 22 mM sodium pyrophosphate, 3.2% (v/v) ethanol, 7.5 mM b-nicotinamide adenine dinucleotide, 0.3 mM sodium phosphate, 0.003% (w/v) bovine serum albumin, and 0.05 to 0.10 unit alcohol dehydrogenase.

9. REFERENCES

Kagi, J.H.R. and Vallee, B.L. (1960) Journal of Biological Chemistry 235, 3188-3192.

10. APPROVAL

Materials