Enzymatic Assay of D-Alanine Racemase (EC 5.1.1.1)

1. Objective

To standardize a procedure for the enzymatic assay of D-Alanine Racemase, Sigma Product Number A8936, at Sigma-Aldrich St. Louis.

2. Scope

This procedure applies to all products that have a specification for the enzymatic assay of D Alanine Racemase, Sigma Product Number A8936.

3. Definitions

3.1.    b-NAD = Beta-Nicotinamide Adenine Dinucleotide, Oxidized Form

3.2.    b-NADH = Beta-Nicotinamide Adenine Dinucleotide, Reduced Form

3.3.    Purified Water - water from a deionizing system, resistivity > or = 18 MΩ•cm @ 25°C

4. Discussion

D-Alanine   Alanine Racemase   >L-Alanine
L-Alanine + b-NAD + H2O   L-Alanine Dehydrogenase   >Pyruvate + b-NADH + NH3

5. Responsibilities

It is the responsibility of all trained Analytical Services laboratory personnel to follow this protocol as written.

6. Safety

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

7. Procedure

7.1 CONDITIONS:
T = 30°C, pH = 10.5, A340nm, Light path = 1 cm

7.2 METHOD:
Spectrophotometric Rate Determination

7.3 REAGENTS:

7.3.1    110 mM Sodium Bicarbonate Solution, pH 10.5 at 30°C(Buffer)
Prepare 200 ml in purified water using Sodium Bicarbonate, Sigma-Aldrich Product Number S6014. Adjust to pH 10.5 at 30°C with 5 N NaOH.

7.3.2    1500 mM D-Alanine Solution (D-ALA
Prepare 10.0 ml in purified water using D-Alanine, Sigma Product Number A7377.

7.3.3    75 mM b-Nicotinamide Adenine Dinucleotide, Oxidized Form Solution (b-NAD)
Prepare 5.0 ml in Reagent 7.3.1 using b-Nicotinamide Adenine Dinucleotide, Sigma-Aldrich Product Number N7004. PREPARE FRESH.

7.3.4    1L-Alanine Dehydrogenase Enzyme Solution (L-AlaDH)
Use Sigma Product Number A7189 NEAT.

7.3.5    50 mM Potassium Phosphate, pH 7.5 at 30°C (Enzyme Diluent)
Prepare 200 ml in purified water using Potassium Phosphate, Monobasic, Sigma-Aldrich Product Number P5379. Adjust to pH 7.5 at 30°C with 5 N KOH.

7.3.6    Alanine Racemase Enzyme Solution (AlaR)
Immediately before use, prepare a solution containing 0.10 to 0.20 unit/ml of L Alanine Racemase in cold Reagent 7.3.5.

7.4 REAGENT COCKTAIL

Pipette (in milliliters) the following reagents into 50 ml beaker:

 

  2Cocktail
Reagent 7.3.1 (Buffer) 13.5
Reagent 7.3.2 (D-ALA) 1.00
Reagent 7.3.3 (b-NAD) 0.5

 

Mix by stirring and adjust pH to 10.5 with 5 N NaOH at 30°C.

 

  Test Blank
Reagent Cocktail 7.4 2.80 2.80
Reagent 7.3.4 (L-AlaDH) 0.20 0.20
Reagent 7.3.5 (Enzyme Diluent) ----- 0.10

 

Mix by inversion and equilibrate to 30°C. Monitor the A340nm until constant, using a suitably thermostatted spectrophotometer. Then add:

 

Reagent 7.3.6 (AlaR) 0.10 -----

 

Immediately mix by inversion and record the increase in A340nm for approximately 5 minutes. Obtain the ΔA340nm/minute using the maximum linear rate for both the Test and Blank.

8. Calculations

 

Units/ml enzyme = (ΔA340nm/min Test - ΔA340nm/min Blank)(3.1)(df)
(6.22)(0.1)

 

3.1 = Volume (in milliliters) of enzymatic assay
df = Dilution factor
6.22 = Millimolar extinction coefficient of b-NADH at 340 nm
0.1 = Volume (in milliliter) of enzyme used

 

Units/mg solid = Units/mL enzyme

 

Units/mg protein = Units/mL enzyme
mg protein/mL enzyme

9. Unit Definition

One unit will convert 1.0 mmole of D-Alanine to L-Alanine at pH 10.5 at 30°C with a coupled assay system with L-Alanine Dehydrogenase.

10. Final Assay Contentration

In a 3.10 ml reaction mix, the final concentrations are 100 mM Sodium Bicarbonate, 90.3 mM D Alanine, 2.2 mM b-Nicotinamide Adenine Dinucleotide, ~50 units of L-Alanine Dehydrogenase, and 0.01 - 0.02 units of D-Alanine Racemase.

11. Notes

11.1    Unit definition of L-Alanine Dehydrogenase is one unit will convert one mmole of L Alanine to Pyruvate and NH3 at pH 10.0 at 25°C.

11.2    Each weigh-up of test should be run with a freshly prepared cocktail reagent.

11.3    This assay is based on the cited reference.

11.4    Reaction needs to be run one reaction level at a time in order to achieve the fastest rate/minute of the product.

12. References & Attachments

12.1    Bergmeyer, H.U. (1983) Methods of Enzymatic Analysis, 2nd edition, Volume I, 427

12.2    Unitika Ltd. New Business Division: Medical Department (2000).

13. Approval

  Print Name Sign Name Title Date
Prepared by: Carrie Cupp Carrie Cupp Originator 11/9/04
Approved by: Rodney Burbach Rodney Burbach Supervisor, Analytical Services 11/10/04
Approved by: Gene McNaughton Gene McNaughton Quality Assurance 11/10/04

Materials

     
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