Malate Dehydrogenase
PREPARATION and SPECIFICATION
PROPERTIES
APPLICATIONS
This enzyme is used in diagnostic tests for the determination of aspartate aminotransferase or in applications for citric and acetic acid testing
UNIT DEFINITION
One unit (U) is defined as the amount of enzyme which consumes 1 μmol of NADH per min under the conditions described below.
REAGENTS PREPARATION
Reagent I: 100 mM pH 7.5 potassium phosphate buffer.
Reagent II: 15 mM oxaloacetic acid dissolved by Reagent I.
Reagent III: 6.0 mM NADH.
Enzyme diluent: 100 mM pH 7.5 potassium phosphate buffer, contains 0.2% BSA.
Sample: The enzyme was diluted to 0.05-0.5 U/mL with enzyme diluent.
PROCEDURE
- Add 2.8 mL Reagent I, 0.1mL Reagent II and 0.1 mL Reagent III in a cuvette and preincubate the reaction mixture at 30℃ for 5 min.
- Add 50 μL the diluted enzyme solution and mix.
- Record the ΔAs at 340 nm in 1 minute in a spectrophotometer thermo-stated at 30℃
At the same time, measure the blank rate ΔAb by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution.
∆A = ∆As – ∆Ab
Vt: Total volume (3.05 mL)
Vs: Enzyme volume (0.05 mL)
1.0: Light path length (cm)
df: Dilution factor
C: Enzyme concentration (mg/mL)
6.22: Millimolar extinction coefficient of NADH under 340 nm (cm2/μmol)
REFERENCES
- C.J.R. Thorne and N.O. Kaplan; J. Biol. Chem., 238, 1861 (1963).
- R.G. Wolfe and J.B. Neilands; J. Biol.
- C.J.R. Thorne; Biochim, Biophys, Acta., 59, 624 (1962).
- D.J. Blonde et al; Can. J. Biochem., 45, 641 (1967). Chem., 221, 61 (1956)