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NADase (DPNase) - Assay

CAS:
9032-65-9
EC:
3.2.2.5

NADase (DPNase) Assay

Method
Cyanide reacts with the quaternary nitrogen form of NAD to form an addition product with an absorbance maximum at 340 nm (Colowick et al. 1951). No such reaction occurs with nicotinamide or adenosine-diphosphate-ribose. Therefore, the reaction between cyanide and NAD before and after incubation with NADase will indicate the activity of the enzyme (Kaplan 1955; Nason et al. 1951). One unit will cleave one micromole of NAD per minute at 37°C and pH 7.5. Note: The original method used a unit which was equivalent to the splitting of 0.01 micromole of NAD in 0.5 ml in 7.5 minutes.
Reagents
  • 0.1 M Potassium phosphate buffer, pH 7.5
  • 1.0 M Potassium cyanide. Caution: Poison, handle with care! Read product label for proper handling instructions.
  • 5.4 mM NAD
Enzyme

Reconstitute vial contents with one milliliter reagent grade water. Immediately prior to use dilute further to a concentration of 0.10-0.50 mg/ml.

Procedure

Pipette into test tubes as follows:

  Test Blank
0.1 M Potassium phosphate
buffer pH 7.5		 0.3 ml 0.3 ml
5.4 mM NAD 0.1 ml 0.1 ml
1.0 M KCN 3.0 ml -----
Reagent grade water 0.1 ml -----

Incubate at 37°C for 3-5 minutes to achieve temperature equilibration. At zero time add 0.1 ml appropriately diluted enzyme to assay and blank tube. Incubate for exactly 7.5 minutes at 37°C and add 3.0 ml of 1.0 M KCN to assay tube. Cool to room temperature and read A340 of blank and assay tubes.

Calculation

Note: If [A340(Blank) - A340(Test)] approaches 0.7, a further dilution of the enzyme is required.