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, at St. Louis.
2. Scope
This procedure applies to all products that have a specification for the enzymatic assay of D Alanine Racemase.
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, 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, 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, Product Number N7004. PREPARE FRESH.
7.3.4 1L-Alanine Dehydrogenase Enzyme Solution (L-AlaDH)
Use 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, 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 50ml beaker:
Mix by stirring and adjust pH to 10.5 with 5 N NaOH at 30°C.
Mix by inversion and equilibrate to 30°C. Monitor the A340nm until constant, using a suitably thermostatted spectrophotometer. Then add:
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
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
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 |
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