Appearance Slightly yellowish amorphous powder, lyophilized
Activity GradeⅡ 40U/mg-solid or more
Contaminants Glutamate oxaloacetate transaminase ≤1.0×10⁻³%
Lactate dehydrogenase ≤1.0×10⁻³%
NADH oxidase≤1.0×10⁻³%
Stability Stable at -20°C for at least One year (Fig.1)
Molecular weight ¹ ⁾ approx. 140,000
Isoelectric point ² ⁾ pH 4.8±0.1
Michaelis constants ³ ⁾ 5.4×10⁻⁵M (L-Malate), 5.0×10⁻⁶M (Oxaloacetate),
8.1×10⁻⁶M (NADH)
Structure 4 subunits per enzyme molecule
Inhibitors Hg⁺⁺
Optimum pH 8.0(Fig.2)
Optimum temperature 70°C(Fig.3)
pH Stability pH 3.0-9.0 (25°C, 20hr)(Fig.4)
Thermal stability below 70°C (pH 7.5, 15min)(Fig.5)
Effect of various chemicals (Table 1)


This enzyme is useful for enzymatic determination of L-malate and of glutamate oxaloacetate transaminase (GOT) in clinical analysis.



malate dehydrogenase

Oxaloacetate+NADH+H⁺                                                   L-Malate+NAD⁺

The disappearance of NADH is measured at 340nm by spectrophotometry.

Unit definition:

One unit causes the oxidation of one micromole of NADH per minute under the conditions described below.


A. K-phosphate buffer, pH 7.5 0.1M
B. Oxaloacetate solution 15mM[2.0mg oxaloacetic acid (MW=132.1)/ml of ice-cold K-phosphate buffer (A). This reagent is rather unstable and should be stored in an ice-bath during use](Should be prepared fresh)
C. NADH solution 6.0mM[4.25mg NADH・Na₂ (ORIENTAL YEAST, MW=709.4)/ml of H₂O] (Should be prepared fresh)
D. Enzyme diluent 0.1M K-phosphate buffer, pH 7.5 contg. 0.2% BSA


Concentration in assay mixture
K-Phosphate buffer 97 mM
Oxaloacetate 0.49mM
NADH 0.20mM

1. Prepare the following reaction mixture in a cuvette (d=1.0cm) and equilibrate at 30°C for about 5 minutes.

2.80 ml K-phosphate buffer, pH 7.5 (A)
0.10 ml Oxaloacetate solution (B)
0.10 ml NADH solution (C)

2. Add 0.05ml of the enzyme solution* and mix by gentle inversion.

3. Record the decrease in optical density at 340nm against water for 3 to 4 minutes in a spectrophotometer thermostated at 30°C, and calculate theΔOD per minute from the initial linear portion of the curve (ΔOD test).
At the same time, measure the blank rate (ΔOD blank) by using the same method as the test expect that the enzyme diluent is added instead of the enzyme solution.

* Dissolve the enzyme preparation in ice-cold enzyme diluent (D), dilute to 0.05−0.5U/ml with the same buffer and store on ice.


Activity can be calculated by using the following formula :

ΔOD/min (ΔOD test-ΔOD blank)×Vt×df

Volume activity (U/ml) =                                                               = ΔOD/min×9.807×df


Weight activity (U/mg)=(U/ml)×1/C

: Total volume (3.05ml)
: Sample volume (0.05ml)
: Millimolar extinction coefficient of NADH under the assay condition (㎠/micromole)
: Light path length (cm)
: Dilution factor
: Enzyme concentration in dissolution (c mg/ml)


  1. C.J.R.Thorne and N.O.Kaplan; J.Biol.Chem., 238, 1861 (1963).
  2. R.G.Wolfe and J.B.Neilands; J.Biol.Chem., 221, 61 (1956).
  3. C.J.R.Thorne; Biochim, Biophys, Acta., 59, 624 (1962).
  4. D.J.Blonde et al; Can.J.Biochem., 45, 641 (1967).
Table 1. Effect of Various Chemicals on Malate dehydrogenase
[The enzyme solution dissolved in 0.1M K-phosphate buffer, pH 7.5 contg. 0.2% of BSA (17U/ml) was incubated with each chemical at 25°C for 1hr.]
Chemical Concn.(mM) Residual
Chemical Concn.(mM) Residual
None 100 2-Mercaptoethanol 2.0 102
Metal salt 2.0   PCMB 0.1 100
MgCl₂   100 IAA 2.0 99
CaCl₂   100 Hydroxylamine 2.0 98
Ba(OAc)₂   101 EDTA 5.0 99
FeCl₃   102 o-Phenanthroline
2.0 99
CoCl₂   100 α,α′-Dipyridyl 2.0 100
MnCl₂   102
Borate 5.0 99
ZnSO₄   99
NaF 2.0 98
Cd(OAc)₂   94 NaN
2.0 98
NiCl₂   100 Triton X-100 0.10% 99
CuSO₄   99 Brij 35 0.10% 98
Pb(OAc)₂   99 Tween 20 0.10% 98
AgNO₃   98 Span 20 0.10% 97
HgCl₂   0 Na-cholate 0.1% 98
MEM 2.0 100 SDS
0.05% 95
NIA 2.0 99 DAC 0.05% 96

Ac, CHCO; NEM, N-Ethylmaleimide; MIA, Monoiodoacetate; PCMB, p-Chloromercuribenzoate; IAA, Iodoacetamide; EDTA, Ethylenediaminetetraacetate; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammonium chloride.

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