D-Lactate Dehydrogenase (LDHD) is an enzyme that catalyzes the oxidation of D-lactate to pyruvate, and it belongs to the lactate dehydrogenase family.
Application: This enzyme can be used for the enzymatic determination of various metabolites, such as ATP, ADP, glucose, creatinine, pyruvate, lactate and glycerol, as well as the enzyme activity when coupled with related enzymes including GPT, PK and CPK.
Procedure
Principle
Unit Definition
One unit causes the oxidation of one micromole of NADH per minute at pH 7.0 at 25℃.
Reagents
A. Sample diluent: Add 1 g BSA and 1.36 g potassium dihydrogen phosphate to 800 mL deionized water, adjust pH to 7.0 with sodium hydroxide (25℃), and adjust to 1 L. (Stable at 2-8℃ for 2 months)
B. Reagent 1: Add 12.1 g Tris to 800 mL deionized water, adjust pH to 7.70 with hydrochloric acid (25℃), then add 1 g sodium azide and stir until dissolved. Finally, make up to 1 L with water. To prepare, add an appropriate volume of sodium pyruvate to 0.4g/L of the solution. (Store at 2-8℃. Keep for one week.)
C. Reagent 2: Dissolve 21.1 grams of CAPS (3-cyclohexylamino propionium sulfate) in 800 mL deionized water, adjust pH to 9.70 with sodium hydroxide, then add 1.20 grams of NADH and 1 gram of sodium azide. Stir until fully dissolved and adjust to 1 liter. (Store at 2-8℃; shelf life: 1 week)
D. Sample to be tested: Before testing, weigh the required enzyme in a Weighing tube. When weighing the enzyme, at least 10mg of enzyme should be weighed to ensure the accuracy of weighing. Dissolve the enzyme in the dilution solution and adjust the volume to the preparation volume.
Procedure
The UV2450 method is as follows: Add reagent 1 (1 mL) and 0.02ml enzyme solution sequentially, preheat at 37℃ for 60 seconds, then add reagent 2 (0.2 mL). After 1 minute of reaction, measure the absorbance change rate at 340nm within 1 minute. The enzyme's linear range is (100-1000) U/L. For optimal results, use specimens with concentrations between (500-1000) U/L.
Calculation
The enzyme activity is calculated using the following formula:
Vt: Total volume of the reaction mixture (ml)
ε: Molar extinction coefficient, with a value of 6.22 (cm²/μmol)
Vs: Volume of the enzyme solution to be tested (ml)
d: Light path length of the cuvette (cm)
D: Dilution factor of the enzyme solution
△A/min: △A/min(test sample)-△A/min(blank)(ABS/min)
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