Assay Procedure for Glycerol Dehydrogenase

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.

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

A. Carbonate-bicarbonate buffer, pH 11.0	0.2 M (Prepare by mixing 0.2 M K2CO3 and 0.2 M NaHCO3 to obtain pH 11.0).
B. Glycerol solution	0.3 M
C.Ammonium sulfate solution	1.0 M
D. NAD＋ solution	10 mM［Weigh 143.5 mg of NAD＋(MW＝717.45) and dissolve in 18.0 mL of H2O and, after adjusting the pH to 7.0 with 0.5 N KOH, fill to 20.0 mL with H2O］(Prepared fresh)
E. Enzyme diluent	20 mM K-phosphate buffer pH 7.5.

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.

Concentration in assay mixture
Carbonate buffer	0.10 M
Glycerol	0.10 M
NAD＋	1.0 mM
Ammonium sulfate	33 mM

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：

Weight activity (U/mg)＝(U/ml)×1/C

Vt	Total volume (3.0 mL)
Vs	Sample volume (0.1 mL)
6.22	Millimolar extinction coefficient of NADH (F/micromole)
1.0	Light path length (cm)
df	Dilution factor
C	Enzyme concentration in dissolution (c mg/mL)