Enzymatic Assay of Malic Dehydrogenase
1. OBJECTIVE
To standardize a procedure for the enzymatic assay of Malic Dehydrogenase.
2. SCOPE
This procedure applies to all products that have a specification for the enzymatic activity of Malic Dehydrogenase. M1006 has been deleted.
3.1. Purified water – water from a deionizing system, resistivity ~ 18MΩ•cm @ 25ºC
3.2. MDH – Malic Dehydrogenase
3.3. β-NAD+ - β-Nicotinamide Adenine Dinucleotide, Oxidized Form
3.4. β -NADH - β -Nicotinamide Adenine Dinucleotide, Reduced form
3.5. Unit Definition – One unit will convert 1.0 µMole of Oxalacetate and β -NADH to L-Malate and β-NAD+ per minute at pH 7.5 at 25ºC.
4. DISCUSSION
Oxalacetate + β-NADH MDH >L-Malate + β - NAD+
5. RESPONSIBILITIES
It is the responsibility of all trained Analytical Services laboratory personnel to follow this procedure as written.
6. SAFETY
Refer to the Material Safety Data Sheet (MSDS) for hazards and appropriate handling precautions.
7.1. CONDITIONS
T = 25°C, pH = 7.5, A340nm, Light path = 1cm
7.2. METHOD
Continuous Spectrophotometric Rate Determination
7.3. REAGENTS
7.3.1. 100mM Potassium Phosphate Buffer, pH 7.5 at 25ºC (Buffer)
Prepare a 13.6 mg/mL solution in purified water using Potassium Phosphate such as Sigma-Aldrich Product Number P5379. Adjust to pH 7.5 at 25°C with 1.0 N KOH.
7.3.2. 0.14mM β-Nicotinamide Adenine Dinucleotide Solution (β-NADH)
Prepare a 0.14mM solution, corrected for water content, in Reagent 7.3.1 (Buffer), using β-Nicotinamide Adenine Dinucleotide Solution (reduced form), such as Sigma-Aldrich Product Number N8129. Prepare Fresh.
7.3.3. 7.6mM Oxalacetic Acid (OAA)
Immediately prior to use, prepare a 1.0 mg/mL solution in Reagent 7.3.1 (Buffer), using Oxalacetic Acid, such as Sigma-Aldrich Product Number O4126 . Product is not stable once in solution. Prepare Fresh For Each Kinetics Run.
7.3.4. Malic Dehydrogenase (MDH)
Immediately before use, prepare a 0.2-0.5 units/mL solution in cold Reagen 7.3.1 (Buffer). Prepare Fresh.
7.4. TEST METHOD
7.4.1. Pipette (in milliliters) the following reagents into suitable cuvettes:
| Test | Blank | |
| Reagent 7.3.2 (β-NADH) | 2.80 | 2.80 |
7.4.2. Mix by inversion and equilibrate to 25°C using a suitably thermostatted Spectrophotometer.
7.4.3. Then add:
| Test | Blank | |
| Reagent 7.3.3 (OAA) | 0.10 | 0.10 |
| Reagent 7.3.1 (Buffer) | 0.10 | ---- |
| Reagent 7.3.4 (MDH) | ---- | 0.10 |
Immediately mix by inversion and record the increase in A340nm for approximately 5 minutes. Obtain the A340nm /minute using the maximum linear rate for both the Test and Blank using a minimum of 4 data points over a one minute time interval.
7.5 CALCULATIONS
| 7.5.1. | Units/ml enzyme = | (ΔA340nm/min Test - ΔA340nm/min Blank)(Vf)(df) |
| (6.22)(Ve) |
where:
VF = Total Volume (in milliters) of Assay
df = Dilution factor
6.22 = Millimolar extinction coefficient of β -NADH at 340nm
VE = Volume (in milliliters) of enzyme used
| 7.5.2. | Units/mg solid = | Units/mL enzyme |
| mg solid/mL enzyme |
| 7.5.3. | Units/mg protein = | Units/mg Solid |
| mg protein/mg Solid |
7.6 FINAL ASSAY CONTENTRATION
In a 3.00 mL Reaction Mixture, the final concentrations are 100mM Potassium Phosphate, 0.13mM β-Nicotinamide Adenine Dinucleotide Solution, 0.25mM Oxalacetic Acid, and 0.02-0.05 units Malic Dehydrogenase.
8.1. Bergmeyer, H.U. (1965) Methods of Enzymatic Analysis, Vol. 1, 2nd ed., 485-486.
8.2. Replaces SPOXAL01
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