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Enzymatic Assay Of Alcohol Dehydrogenase Attached To Agarose (A2529)

1. Objective

The objective of this procedure is to standardize the enzymatic assay of Alcohol Dehydrogenase attached to Agarose, Sigma Product Number (A2529) , at Sigma-Aldrich St. Louis.

2. Scope

The scope of this procedure includes activity assays of Alcohol Dehydrogenase attached to Agarose, Sigma Product Number (A2529).

3. Definitions

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

4. Discussion

Ethanol + b-NAD   Alcohol Dehydrogenase   >Acetaldehyde + b-NADH

5. Responsibilities

It is the responsibility of all Analytical Services laboratory personnel to follow this procedure as written

6. Safety

Refer to Safety Data Sheets (SDS) for hazards and appropriate handling precautions.

7. Procedure

7.1 CONDITIONS:
T = 25°C, pH = 8.8 , A340nm, Light path = 1 cm

7.2 METHOD:
Spectrophotometric Stop Reaction

7.3 REAGENTS:

7.3.1    22mM sodium pyrophosphate, 3.2%(v/v) ethanol, 7.5 mM b-Nicotinamide Adenine Dinucleotide, 0.3mM sodium phosphate, 0.003%(w/v) bovine serum albumin, pH 8.8 @ 25°C (Cocktail) (Dissolve 0.981G of Sodium Pyrophosphate, Decahydrate,Sigma Product Number (S9515) , Dissolve 4.14 mg of Sodium Phosphate, Monobasic, Anhydrous, Sigma-Aldrich Product Number S9638 , 3.3 mg of Albumin, Bovine, Sigma Product Number A9647 , and 559 mgs of b- Nicotinamide Adenine Dinucleotide ,Sigma-Aldrich Product Number N6522 in 90ml deionized water. Immediately adjust the pH to 8.8 with 8% (v/v) Phosphoric Acid or 1 N NaOH @ 25°C. Add 3.2 mls of 200 Proof USP Ethyl Alcohol, Sigma Product Number Z5130 with stirring. Readjust the pH to 8.8 with 8% (v/v) Phosphoric Acid or 1 N NaOH @ 25°C and dilute to 100 mls with deionized water.)

7.3.2    10 mM Sodium Phosphate Buffer, pH 7.5 at 25°C (Enzyme Diluent-1) (Dissolve 710 mg of Sodium Phosphate Dibasic, Anhydrous, Sigma-Aldrich Product Number S9763 , in 500 ml of deionized water. Adjust to pH 7.5 at 25°C with Reagent 7.3.3.)

7.3.3    10 mM Sodium Phosphate Solution, (Enzyme Solution) (Dissolve 480 mg of Sodium Phosphate Monobasic, Anhydrous, Sigma-Aldrich Product Number S9638 , in 480 ml of deionized water.)

7.3.4    10M Sodium Phosphate Buffer with 0.1% (w/v) Bovine serum Albumin, pH 7.5 @ 25°C (Enzyme Diluent-2) (Dissolve 100 mg of Albumin, Bovine, Sigma Product Number A9647 , in 100 mls of Reagent 7.3.2. Adjust pH to 7.5 at 25°C with 8% Phosphoric or 1N NaOH.)

7.3.5    Alcohol Dehydrogenase Solution(Enzyme Solution) (Obtain a “Evergreen” 3mm X 1mm column with porous filter disc and flexible rubber closure. Add 0.10 ml of deionized water to each column and mark the column at this level with a “Sharpie”. Keep repeating this process until the column is graduated to 1.50 ml. Empty the column and dry with forced air. Accurately weigh 250 mgs of A2529 into column with closure on. Add 5.0 mls of cold deionized water and swirl. Allow to swell on ice for one hour. Immediately prior to pipetting into reaction cocktail, measure swelled, gel volume in milliliters. After removing bottom closure proceed to wash the sample with 50 volumes of cold deionized water (50 times packed gel volume). Record the volume of packed/washed gel and add the appropriate volume of Reagent 7.3.2 (Enzyme Diluent-1) to obtain a concentration of 10 Units of Alcohol Dehydrogenase / ml of suspension. Carefully invert the suspension until all of packed gel is resuspended and immediately remove an aliquot and dilute to 1 to 2 Units / ml with Reagent 7.3.4 (Enzyme Diluent-2).

7.4 ENZYMATIC ASSAY:

Pipette (in milliliters) the following reagents into a 100 ml beaker with a stir bar

7.4.1    Pipette (in milliliters) the following reagents into a suitable containers:

  Test Blank
Reagent 7.1.1 (Cocktail) 29.9 29.9

 

7.4.2    Mix by inversion and allow to stand at 25ºC for a minimum of five minutes and no longer than six minutes.

  Test Blank
Reagent 7.1.4 (Enzyme Diluent –2) ----- 0.10
Reagent 7.1.5 (Enzyme Solution) 0.10 -----

 

Immediately start timer and filter an aliquot from each reaction mixture using a 0.45mm filter. Record time in minutes and A340nm (AI). After exactly six minutes, remove another aliquot and filter an aliquot from each reaction mixture using a 0.45m filter. Record the time in minutes and A340nm (AF).

8. Calculations

ΔA340nm/min (Blank) = [AF (Blank) – AI (Blank)] / ( Time Final in minutes – Time Initial in minutes)
ΔA340nm/min (Test) = [AF (Test) – AI (Test)] / ( Time Final in minutes – Time Initial in minutes)

Units/ml packed gel = (ΔA340nm/min Test - ΔA340nm/min Blank) (30.0)(df-1)(df-2)
(6.22) (0.10) (mls of packed gel)

 

30.0= Total Volume (in milliliters) of enzymatic assay
6.22 = Millimolar extinction coefficient of b-NADH at 340nm
df-1 = Dilution Factor-1
df-2 = Dilution Factor-2
0.1 = Volume (in milliters) of diluted ezyme used in enzyme reaction mixture

Units/gram of solid = (ΔA340nm/min Test - ΔA340nm/min Blank) (30.1)(df-1)(df-2)
(6.22) (0.10) (grams of solid)

 

30.0= Total Volume (in milliliters) of enzymatic assay
6.22 = Millimolar extinction coefficient of b-NADH at 340nm
df-1 = Dilution Factor-1
df-2 = Dilution Factor-2
0.1 = Volume (in milliters) of diluted ezyme used in enzyme reaction mixture
Units / gram of Agarose = (Units / gram of solid ) X 9.708
9.708 = gram of solid / gram of Agarose

9. Emergency Shutdown

NA

10. References

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

11. Notes

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

2. In a 30.0ml reaction mix, the final concentrations are 22mM sodium pyrophosphate, 3.2%(v/v) ethanol, 7.5mM b-Nicotinamide Adenine Dinucleotide, 0.3mM sodium phosphate, 0.003%(w/v) bovine serum albumin, and 0.1 to 0.2 unit alcohol dehydrogenase.

12. Approval

  Print Name Sign Name Title Date
Prepared by: Marvin Rice Marvin Rice Originator 02/10/04
Approved by: David Lintz David Lintz Supervisor, Analytical Services 02/13/04
Approved by: Gene McNaughton Gene McNaughton Quality Assurance 02/17/04
Approved by: Adam Pocius Adam Pocius Production 02/13/04

Materials

     
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