Assay Procedure for Glycerol Dehydrogenase

Principle

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

Unit definition

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

Method

Reagents

 

 

A. Carbonate-bicarbonate buffer, pH 11.0
:0.2M (Prepare by mixing 0.2M K2CO3 and 0.2M NaHCO3 to reach pH 11.0).
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 0.5 N KOH, fill up to 20.0ml with H2O](Should be prepared fresh)
E. Enzyme diluent :20mM K-phosphate buffer pH 7.5.

Procedure

  1. Prepare the following working solution, immediately before use.
    30.0ml Carbonate-bicarbonate buffer, pH 11.0 (A)
    22.0ml Substrate solution                                     (B)
    2.0ml   Ammonium sulfate solution                    (C)
    6.0ml   NAD+ solution                                          (D)
    Be sure the pH in the range (pH 10.0-10.5). If not, adjust the pH to 10.5 with 1.0 N KOH or 1.0N HCl, and store on ice in a brownish bottle.

 

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

 

  1. Pipette 2.9ml of the working solution into a cuvette (d=1.0cm) and equilibrate at 25℃ for about 5 minutes.
  2. Add 0.1ml of the enzyme solution* and mix by gentle inversion.
  3. Record the increase in optical density at 340nm against water for 3 to 4 minutes in a spectrophotometer thermostated at 25℃ and calculate theΔOD 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 (3.0ml)
Vs :Sample volume (0.1ml)
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)

 

This procedure is for informational purposes. For a current copy of Sigma’s quality control procedure contact our Technical Service Department.

Materials

     
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