Malate dehydrogenase (MDH) catalyzes the mutual conversion of oxaloacetate and malate, and is associated with the oxidation/reduction of dinucleotide coenzymes.
ORIGIN: Thermus sp.
CATALYSIS: L-Malate + NAD⁺ ⇄ Oxaloacetate + NADH + H⁺
SPECIFICATIONS:
Activity (MDH-3): ≥100 U/mg enzyme powder
Appearance: White powder, lyophilized
Contaminants:
NADH oxidase: ≤4 × 10⁻⁴%
GOT: ≤3 × 10⁻³%
GPT: ≤1.5 × 10⁻³%
α-KGDH: ≤3 × 10⁻³%
Additive: Dextran
CHARACTERISTICS:
Molecular weight: 35,000 (SDS-PAGE)
Isoelectric point: 4.8
Km value:
5.6 × 10⁻⁵M (Oxaloacetate)
6.6 × 10⁻⁴M (NADH)
Opt. pH: 7.8
Opt. temperature: 70℃
Stable pH range: 8.0 – 9.0 (70℃, 30 min.)
Stable temp. range: below 80℃ (pH8.5, 30 min.)
APPLICATIONS:
This enzyme is used for the enzymatic determination of L-malate and glutamate oxaloacetate transaminase (GOT) in clinical diagnosis.
Assay Method
Principle
The consumption of NADH is measured at 340 nm by spectrophotometry.
Unit Definition
One unit is defined as the enzyme quantity which oxidizes one μ mole of NADH per minute under the conditions described below.
Reagents
A. 0.1M Tris-HCl Buffer (pH 7.8)
B. 0.01M Phosphate Buffer (KH₂PO₄-NaOH, pH 7.0)
C. Triton X-100 Solution (50 mg/ml)
Weigh 5.0 g of Triton X-100 and dissolve in 30 ml of deionized water with heating. After cooling, fill up to 100 ml with deionized water.
D. 0.01 M Phosphate Buffer, containing 0.1% Triton X-100 (KH₂PO₄-NaOH, pH 7.0)
Dilute 20 ml of Triton X-100 Solution (C) with 0.01 M Phosphate Buffer (B). Fill up to 1,000 ml with 0.01 M Phosphate Buffer (B).
E. NADH Solution
Weigh 9 mg of NADH and dissolve in 0.1M Tris-HCl Buffer (A). Fill up to 50 ml with 0.1M Tris-HCl Buffer (A). (Expires after 5 days at 2–8℃)
F. Substrate Solution
Weigh 11 mg of oxaloacetic acid and dissolve in 0.1 M Tris-HCl Buffer (A). Fill up to 50 ml with 0.1 M Tris-HCl Buffer (A). (Make a fresh solution for each use.)
G. Enzyme Solution
Weigh approx. 20 mg of Malate Dehydrogenase and dissolve in chilled 0.01M Phosphate Buffer (D).
Enzyme Solution should be prepared so that the value of ΔOD/minute becomes in the range of 0.025±0.010.
Procedure
Pipette 2.0 ml of NADH Solution (E) and 0.90 ml of Oxaloacetic Acid Solution (F) respectively into a cuvette (d=10 mm), and keep at 25 ± 0.1℃ for 5 minutes. Then, pipette 0.10 ml of Enzyme Solution (G) into the cuvette and mix well immediately. Keep the reaction mixture at 25 ± 0.1℃.Exactly at 2 minutes and 5 minutes after the addition of Enzyme Solution (G), measure the absorbances of the reaction mixture at 340 nm (A2 and A5).As a blank, pipette 0.01M Phosphate Buffer (D) into another cuvette (d=10 mm) instead of the Enzyme Solution (G) and take the same procedure described above (Ab2 and Ab5).
Calculation
3: Reaction time
6.22: Millimolar extinction coefficient of NADH at 340 nm
3.0: Final volume of the reaction mixture
0.1: Volume of Enzyme Solution
Dm: Dilution multiple of Enzyme Solution
K2519474