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.
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
Analytical Services laboratory personnel should follow this protocol as written.
6. Safety
Refer to the Safety Data Sheet (SDS) for hazards and appropriate handling precautions.
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 mmol 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 millimole 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 to achieve the fastest rate/minute of the product.
12. References
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).