LDH (D-Lactate dehydrogenase)
Lactate dehydrogenase (LDH) is an important enzyme of the anaerobic metabolic pathway. It belongs to the class of oxidoreductases, with an enzyme commission number EC 1.1. 1.27. The function of the enzyme is to catalyze the reversible conversion of lactate to pyruvate with the reduction of NAD+ to NADH and vice versa. This enzyme is used for the removal of pyruvate in determinations working with NADH (triglycerides, lipase, aldolase, aspartate aminotransferases, glutamate dehydrogenase).
Preparation and specification
|
Appearance |
White amorphous powder,lyophilized |
|
Purity ( SDS-PAGE) |
≥90.0% |
|
Enzyme powder specific activity |
≥420.0 U/mg |
|
NADH/NADPH oxidase |
≤0.01% |
|
Malate dehydrogenase |
≤0.005% |
|
Glutamate dehydrogenase |
≤0.003% |
|
Pyruvate kinase |
≤0.03% |
Properties
|
EC number |
1.1.1.28(Recombinant from microorganism) |
|
|
Molecular weight |
45 kDa (SDS-PAGE) |
|
|
Isoelectric point |
4.5 |
|
|
Michaelis constants |
2.2× 10-6 M(Pyruvate) |
|
|
Inhibitors |
Ag+, Hg2+ |
|
|
Optimum pH |
6.5-7.0 |
Fig. 1 |
|
Optimum temperature |
45 ℃ |
Fig. 2 |
|
pH stability |
pH 5.5- 10.0 (25℃, 16 h) |
Fig. 3 |
|
Thermal stability |
Below 50℃ (pH 7.0, 30 min) |
Fig. 4 |
|
Storage stability |
At least one year at -25 ~- 15 ℃ |
Fig. 5 |
|
Stabilizers |
BSA |
|
Storage conditions
Store at -20 ℃, valid for 1 year.
Figures
Assay principle
Reagents preparation
Reagent I: Add 0.04 g sodium pyruvate in 0. 1 M, pH 7.7 Tris-HCl buffer and set to 100 mL.
Reagent II: Add 2. 11 g CAPS in 80 mL pure water and adjust to pH 9.7, then add 0. 12g NADH and set to 100 mL.
Enzyme diluent: 10mM, pH 7.0 potassium phosphate, contains 0. 1% BSA.
Sample: The enzyme was diluted to 1-2 U/mL with Enzyme diluent.
Procedure
1. Add 1mL Reagent I and 0.02mL enzyme solution to the cuvette.
2. Preincubate the reaction mixture at 37 ℃ for5 min.
3. Add 0.2 mL Reagent II and mix, record the ΔAs at 340nm in 1 minute in a spectrophotometer thermostated at 37 ℃ .
At the same time, measure the blank rate ΔAb by using the same method as the test except that the enzyme diluent is added in stead of the enzyme solution.
∆A = ∆As – ∆Ab
Calculation
1.22: Total volume (mL)
0.02: Enzyme volume (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)
Notes
The material is only used in invitro diagnostic products, not in human or in vivo experiments.
