Enzymatic Assay of L-Lactic Dehydrogenase (EC 1.1.1.27)
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 μmol 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
Analytical services laboratory personnel should 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 (Product No. S0751). 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 (Product No. 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 (Product No. 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 (Product 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:
7.2.2 Mix by inversion and equilibrate to 37°C. Monitor the A340nm until constant, using a suitably thermostatted spectrophotometer. Then add:
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
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
7.3.4 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.1
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