Enzymatic Assay of L-Lactic Dehydrogenase (EC 1.1.1.27)

Document History

Replaces Enzymatic Procedure SPPYRU01. See CR SOP DEK ENZ30

1. Objective

To standardize a procedure for the assay of L-Lactic Dehydrogenase for all sources from heart muscle.

2. Scope

This procedure applies to all products from heart muscle that have a specification for L-Lactic Dehydrogenase activity.

3. Definitions

3.1 Purified Water = Water from a deionizing system, resistivity > or = 18MΩ.cm @ 25°C.

3.2 β-NAD = β-Nicotinamide Adenine Dinucleotide, Oxidized Form

3.3 β-NADH = β-Nicotinamide Adenine Dinucleotide, Reduced Form

3.4 Unit Definition = One unit will reduce 1.0 μmole of pyruvate to L-lactate per minute at pH 7.5 at 37°C.

4. Discussion

4.1 Pyruvate + β-NADH     L-Lactic Dehydrogenase   > L-Lactate + β-NAD

4.2 Method = Spectrophotometric Rate Determination, Temperature (T) = 37°C, pH = 7.5, A340nm, Light Path = 1cm

5. Responsibilities

It is the responsibility of trained 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    REAGENTS

7.1.1     100 mM Sodium Phosphate Buffer, pH 7.5 at 37°C

7.1.1.1    Prepare a 12 mg/ml solution in purified water using Sodium Phosphate, Anhydrous, Monobasic, Sigma-Aldrich Product Number S0751.

7.1.1.2    Adjust to pH 7.5 at 37°C with 1 M NaOH.)

7.1.2    0.13 mM β-Nicotinamide Adenine Dinucleotide, Reduced Form Solution (β NADH)

7.1.2.1     Prepare a 0.102 mg/ml solution in cold Reagent 7.1.1 using β-Nicotinamide Adenine Dinucleotide, Reduced Form, Disodium Salt, Sigma-Aldrich Product Number N8129.

7.1.2.2    PREPARE FRESH.

7.1.2.3    Specification for this product is a white to light yellow powder; if powder appears yellow or clumpy, please do not proceed with assay until fresh N8129 is available.)

7.1.3    34 mM Sodium Pyruvate Solution (Pyruvate)

7.1.3.1    Prepare a 3.74 mg/ml solution in cold Reagent 7.1.1 using Pyruvic Acid, Sodium Salt, Sigma-Aldrich Product Number P2256.

7.1.3.2    Use freshest lot of Sodium Pyruvate available.

7.1.4    1.0% (w/v) Bovine Serum Albumin Solution (BSA) Prepare a 10 mg/ml solution in Reagent 7.1.1 using Albumin, Bovine, Sigma Prod. No. A-4503.

7.1.5    L-Lactic Dehydrogenase Enzyme Solution (Enzyme) Immediately before use, prepare a solution containing 0.15 - 0.50 unit/ml of L Lactic Dehydrogenase in cold Reagent 7.1.4.

7.2    ASSAY

7.2.1    Pipette (in milliliters) the following reagents into suitable cuvettes:

  Test Blank
Reagent 7.1.2 (β-NADH) 2.80 2.80
Reagent 7.1.3 (Pyruvate) 0.10 0.10

 

7.2.2    Mix by inversion and equilibrate to 37°C. Monitor the A340nm until constant, using a suitably thermostatted spectrophotometer. Then add:

Reagent 7.1.4 (BSA) ---- 0.10
Reagent 7.1.5 (Enzyme) 0.10 ----

 

7.2.3    Immediately mix by inversion and record the decrease in A340nm for approximately 5 minutes.

7.2.4    Obtain the ΔA340nm/minute using the maximum linear rate for both the Test and Blank.

7.3    CALCULATIONS

7.3.1 Units/ml enzyme =   (ΔA340nm/min Test - ΔA340nm/min Blank) (3) (df)
  (6.22) (0.1)

 


     3 = Total volume (in milliliters) of assay
    df = Dilution factor
    6.22 = Millimolar extinction coefficient of β-NADH at 340 nm
    0.1 = Volume (in milliliters) of enzyme used

7.3.2 Units/mg solid =   units/ml enzyme
  mg solid/ml enzyme

 

7.3.3 Units/mg protein =   units/ml enzyme
  mg protein/ml enzyme

 

7.3.4    Final Assay Concentration In a 3.00 ml reaction mix, the final concentrations are 100 mM sodium phosphate, 0.12 mM β-nicotinamide adenine dinucleotide, 1.1 mM pyruvate, 0.03% (w/v) bovine serum albumin, and 0.015 - 0.050 unit L-lactic dehydrogenase.

8. References & Attachments

Bergmeyer, H.U., and Bernt, E., (1974) In Methods of Enzymatic Analysis; Bergmeyer, H.U., 2nd ed.; Academic Press: New York, NY, Volume II, 574-579

9. Approval

Review, approvals and signatures for this document will be generated electronically using DocCompliance (QUMAS). Print a “For Use” copy if hardcopy with signature verification is required.

Materials

     
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