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Assay Procedure for L-Glutamic Dehydrogenase (NADP)

Principle

 

The disappearance of NADPH is measured at 340nm by spectrophotometry.

Unit definition

One unit causes the oxidation of one micromole of NADPH per minute under the conditions described below.

Method

Reagents

 

A. Buffer solution
:0.1M Tris-HCl buffer, pH 8.3
B. NH4Cl solution :3.3M
C. α-Ketoglutarate solution 0.225M (adjust the pH to 7.0-9.0 with NaOH)(Should be prepared fresh)
D. NADPH solution :7.5mM (Should be prepared fresh)
E. Enzyme diluent :50mM K-Phosphate buffer, pH 6.6 containing 0.2% BSA and 50mM EDTA

Procedure

  1. Prepare the following reaction mixture in a cuvette (d=1.0cm) and equilibrate at 30℃ for about 5 minutes.

    2.5ml Buffer solution                  (A)
    0.2ml NH4Cl solution                 (B)
    0.1ml α-Ketoglutarate solution (C)
    0.1ml NADPH solution               (D)
Concentration in assay mixture
Tris-HCl buffer 85 mM
α-Ketoglutarate 7.6 mM
NH4Cl .22 M
NADPH .25 mM
EDTA 0.85 mM
  1. Add 0.05ml of the enzyme solution* and mix by gentle inversion.
  2. Record the decrease in optical density at 340nm against water for 2 to 3 minutes in a spectro-photometer thermostated at 30℃ and calculate the ΔOD per minute from the 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 (E) is added instead of the enzyme solution.

* Dilute the enzyme preparation to 0.4-0.9U/ml with ice-cold enzyme diluent (E), immediately before the assay.

Calculation

Activity can be calculated by using the following formula:

 

Weight activity (U/mg)=(U/ml)×1/C

 

Vt :Total volume (2.95ml)
Vs :Sample volume (0.05ml)
6.22
:Millimolar extinction coefficient of NADPH (F/micromole)
1.0 :Light path length (cm)
df :Dilution factor

 

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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