Glycerol dehydrogenase is a nad dependent enzyme, which mainly catalyzes the oxidation of glycerol to dihydroxyacetone (DHA), belonging to the polyol dehydrogenase family.
Application: This enzyme is useful for enzymatic determination of glycerol and of triglyceride when coupled with lipoprotein lipase in clinical analysis.
Principle:
Reagents:
A. Carbonate-bicarbonate buffer, pH11.0: 200mM (Prepare by mixing 0.2M K₂CO₃ and 0.2M NaHCO₃ to reach pH11.0)
B. Glycerol solution: 0.3M
C. Ammonium sulfate solution: 1.0M
D. NAD⁺ solution: 10mM [Weigh 143.5mg of NAD⁺ (MW=717.45) and dissolve in 18.0ml of H₂O and, after adjusting the pH to 7.0 with 0.5N KOH fill up to 20.0ml with H₂O (Should be prepared fresh)]
E. Enzyme diluent: 20mM K-phosphate buffer,pH7.5.
Procedure:
1. Prepare the following working solution, immediately before use.
30.0ml Carbonate-bicarbonate buffer,pH11.0 (A)
22.0ml Substrate solution (B)
2.0ml Ammonium sulfate solution (C)
6.0ml NAD⁺ solution (D)
Concentration in assay mixture
| Carbonate buffer | 100mM |
|---|
| Glycerol | 100mM | | NAD⁺ | 1.0mM | | Ammonium sulfate | 33mM |
|
Be sure the pH in the range(pH10.0~10.5).If not, adjust the pH to 10.5 with 1.0N KOH or 1.0N HCl, and store on ice in a brownish bottle.
2. Pipette 2.9ml of the working solution into a cuvette (d=1 cm) and equilibrate at 25°C for about 5 min.
3. Add 0.1ml of the enzyme solution* and mix by gentle inversion.
4. Record the increase of optical density at 340nm against water for 3~4minutes in a spectrophotometer thermostated at 25°C ,and calculate the △OD per minute from the initial linear portion of the curve (△OD test).
At the same time,measure the blank rate(△OD Blank)by using the same method as the test expect that the enzyme diluent is added instead of the enzyme solution.
*:Dissolve the enzyme preparation in ice-cold enzyme diluent(E)and dilute to 0.10~0.25U/ml with the same buffer and store on ice.
Calcuation:
Activity can be calculated by using the following formula:
Volume activity (U / ml)=[(△OD / min (△OD test - △OD blank ) × Vt × df)] / (6.22 × 1.0 × Vs)=△OD / min × 4.82 × df
Weight activity (U / mg)=(U / ml) × 1 / C
Vt: Total volume (3.0ml)
Vs: Sample volume (0.1ml)
6.22: Millimolar extinction coefficient of NADH (cm²/μmol)
1.0: Light path length (cm)
df: Dilution factor
C: Enzyme concentration in dissolution (c mg/ml)
ZJ26F0435010