CODE No.:M1M111

MALATE DEHYDROGENASE (MDH)

[EC 1.1.1.37]
from Microorganism

FOR OXALATE → MALATE REACTION

SPECIFICATION

State	:	Lyophilized
Specific activity	:	more than 1,200 U/mg protein
Contaminants	:	(as MDH activity = 100%)
		GOT	<0.01%
		GPT	<0.01%
		NADHoxidase	<0.01%
		Glutamate dehydrogenase	<0.01%
		Fumarase	<0.01%

PROPERTIES

Molecular weight	:	ca. 72,000
Subunit molecular weight	:	ca. 36,000
Optimum pH	:	9.0	(Fig.1)
pH stability	:	5.5 - 11.0	(Fig.2)
Thermal stability	:	No detectable decrease in activity up to 50°C.	(Fig.3, 4)
Michaelis constants	:	(90mM Tris-HCl buffer, pH 9.0, at 30°C)
		Oxaloacetate	0.027mM
		NADH	0.014mM

STORAGE

stable at -20°C for at least six months

APPLICATION

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

ASSAY

Principle

The change in absorbance is measured at 340 nm according to the following reaction.

Unit Definition

One unit of activity is defined as the amount of MDH that forms 1µmol of NAD⁺ per minute at 30°C.

Solutions

1. Buffer solution ; 200mM Tris-HCl, pH 9.0
2. Oxaloacetate solution ; 15 mM (0.020 g oxaloacetate/10mL distilled water)
3. NADH solution ; 13.1 mM (0.100 g NADH disodium salt·3H₂O/10mL distilled water)

Preparation of Enzyme Solution

Dissolve the lyophilized enzyme with distilled water and dilute to 3 to 5 U/mL with 100mM Tris-HCl buffer, pH 9.0.

Procedure

1. Prepare the following reaction mixture and pipette 3.00mL of reaction mixture into a cuvette.

   Solution I	13.50mL	Solution III	0.57mL
   Solution II	1.00mL	H2O	14.93mL

2. Incubate at 30°C for about 3 minutes.
3. Add 0.01 mL of enzyme solution into the cuvette and mix.
4. Read absorbance change at 340 nm per minute (ΔAbs₃₄₀) in the linear portion of curve.

Calculation

• d.f. ; dilution factor
• 6.22 ; millimolar extinction coefficient of NADH(cm²/μmol)
• *Protein concentration ; determined by Bradford's method

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