MALATE DEHYDROGENASE from Microorganism

MAD-211

PREPARATION and SPECIFICATION
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%

PROPERTIES
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)

APPLICATIONS

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

ASSAY

Principle:

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.

Method:

Reagents
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

Procedure

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.

Calculation

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

6.22×1.0×Vs

Weight activity (U/mg)=(U/ml)×1/C

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

REFERENCES

C.J.R.Thorne and N.O.Kaplan; J.Biol.Chem., 238, 1861 (1963).
R.G.Wolfe and J.B.Neilands; J.Biol.Chem., 221, 61 (1956).
C.J.R.Thorne; Biochim, Biophys, Acta., 59, 624 (1962).
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 activity(%)	Chemical	Concn.(mM)	Residual activity(%)
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	NaN₃	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, CH₃CO; NEM, N-Ethylmaleimide; MIA, Monoiodoacetate; PCMB, p-Chloromercuribenzoate; IAA, Iodoacetamide; EDTA, Ethylenediaminetetraacetate; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammonium chloride.

To get a quote, contact us at info@toyobousa.com, or INQUIRY.