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Malate Dehydrogenase (MDH)

(L-Malate NAD+ oxidoreductase; EC

Malate dehydrogenase (MDH) catalyzes the following reaction using nicotinamide adenine dinucleotide (NAD+) as a coenzyme:

L-Malate + NAD+ ------> Oxaloacetate + NADH + H+
All eukaryotic cells contain two distinct isozymes of MDH, the mitochondrial form and the cytoplasmic form, while the prokaryotes contain only a single form. MDH has a molecular weight of 70,000.

MDH can be used as an indicator enzyme for the determination of the activity of glutamate oxaloacetate transaminase, ATP - citrate lyase and citrate synthase. Monitoring of MDH activity in serum and cerebrospinal fluid has been shown to be of clinical significance (Sharpe, D.M., Wilcock, A.R. and Goldberg, D.M., Clin. Chem., 19, 240, 1973). MDH is also widely used in coupled systems for determination of biological metabolites.

1.Source: Porcine Heart Mitochondria
   Form: 70% Ammonium sulfate suspension in phosphate buffer, pH 7 
   Solubility: Soluble in distilled water or dilute buffer 
   Stability: Stable when stored at 4C. Do not freeze 
   Activity (approx.): 1000-1800 U/mg protein 
   Protein (approx.): 10 mg/ml (biuret) 
   Contaminants (approx.): Fumarase 0.001%, Glutamate pyruvate transaminase 0.01%, Glutamate oxaloacetate transaminase 0.01%, Lactate dehydrogenase 0.01% 
   Catalog No.: 098B1800 
2.Source: Porcine Heart Mitochondria
   Form: Freeze-dried 
   Solubility: Soluble in distilled water or dilute buffer 
   Stability: -20 C; -4 F 
   Activity (approx.): 50-100 U/mg solid 
   Protein (approx.): 10% Protein/mg solid (biuret) 
   Contaminants (approx.): Fumarase <0.01%, Glutamate pyruvate transaminase <0.01%, Glutamate oxaloacetate transaminase <0.01%, Lactate dehydrogenase <0.01% 
   Catalog No.: 098A0050 
3.Source: Porcine Heart Mitochondria
   Form: Glycerol solution 
   Solubility: Soluble in distilled water or dilute buffer 
   Stability: Store at 4 C (39 F); Do not freeze 
   Activity (approx.): 1000-1800 U/mg protein 
   Protein (approx.): 10 mg/ml (biuret) 
   Contaminants (approx.): Fumarase <0.001%, Glutamate pyruvate transaminase <0.001%, Glutamate oxaloacetate transaminase <0.001%, Lactate dehydrogenase <0.001% 
   Catalog No.: 098C1800 

The amount of enzyme which will convert one micromole of oxaloacetate and NADH per minute at 25°C, pH 7.5 in 0.1 M sodium phosphate buffer.

The rate of decrease in the absorbancy at 340 nm, resulting from the oxidation of NADH, is a measure of MDH activity.

  1. 0.1 M Sodium phosphate buffer, pH 7.5.
  2. NADH solution (5 mg/ml). Dissolve 5 mg NADH sodium salt in 1.0 ml of 0.1 M phosphate buffer, pH 7.5. Always prepare fresh.
  3. Cis-Oxaloacetic acid (1 mg/ml) in 0.1 M phosphate buffer, pH 7.5. Prepare fresh for each run.
  4. 1% Bovine serum albumin (BSA) solution. Dissolve 1.0 g BSA in 100 ml distilled water. Albumin should be of highest purity, otherwise it could inhibit the activity of MDH.
  5. MDH Solution (0.1-0.2 U/ml). Dilute 0.1 ml enzyme suspension to 5 ml with cold 1% BSA solution. Use an aliquot of this solution and dilute to a final concentration of 0.1-0.2 U/ml with cold 1% BSA. This solution must be used as soon as it is prepared and must be made fresh for each run.

  1. Set spectrophotometer (equipped with strip chart recorder and temperature control) at 340 nm and 25°C.
  2. Into the cuvette, pipette the following:
    0.1 M Phosphate buffer, pH 7.5 2.9 ml
    Cis-oxaloacetic acid 0.1 ml
    NADH 0.05 ml
  3. Mix and incubate at 25°C for five minutes.
  4. Transfer the cuvette to the spectrophotometer and record the blank rate at 340 nm for 2-3 minutes.
  5. Pipette 0.02 ml fresh MDH (0.1-0.2 U/ml) solution. Mix and monitor the reaction for 5-10 minutes at 340 nm.
  6. Calculate µE340nm/min

Activity (U/mg) = (ΔE340nm/min)(Total Vol.)(Enz. Diln.)
(6.22)(Enz. Vol.)(mg Enz./ml)

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