Glycerol dehydrogenase assay principle

Glycerol dehydrogenase (GDH) is useful for enzymatic determination of glycerol and of triglyceride when coupled with lipoprotein lipase in clinical analysis. Formation of NADH from the reaction of glycerol and NAD+ is catalyzed by the enzyme glycerol dehydrogenase.

Glycerol + NAD

The appearance of NADH is measured at 340 nm by spectrophotometry.

Unit definition

One unit causes the formation of one micromole of NADH per minute under the conditions described below.

Reagents

Procedure

  1. Prepare the following working solution, immediately before use.
    30.0 mL Carbonate-bicarbonate buffer, pH 11.0 (A)
    22.0 mL Substrate solution (B)
    2.0 mL Ammonium sulfate solution (C)
    6.0 mL NAD+ solution (D)
    Be sure the pH is 10.0-10.5. If not, adjust the pH to 10.5 with 1.0 N KOH or 1.0 N HCl, and store on ice in a brown bottle.
  1. Pipette 2.9 mL of the working solution into a cuvette (d=1.0 cm) and equilibrate at 25 ℃ for about 5 minutes.
  2. Add 0.1 mL of the enzyme solution* and mix by gentle inversion.
  3. Record the increase in optical density at 340 nm against water for 3 to 4 minutes in a spectrophotometer thermostated at 25℃ and calculate the ΔOD per minute from the initial linear portion of the curve (ΔOD test).

At the same time, measure the blank rate (ΔOD blank) by using the same method as the test except that the enzyme diluent is added instead of enzyme solution.

* Dissolve the enzyme preparation in ice-cold enzyme diluent (E), dilute to 0.10-0.25U/mL with the same buffer and store on ice.

Calculation

Activity can be calculated by using the following formula:

Volume activity

Weight activity (U/mg)=(U/ml)×1/C

Materials
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