Phosphodiesterase II Assay

Method: A modification of the assay as described by Hilmoe (1961). One unit is defined as a change in absorbance of 0.2 at 37°C and pH 6.5 under the specified conditions.


  • 5.9 mM Uranyl acetate in 2.5% perchloric acid. Prepare by dissolving 250 mg uranyl acetate and 3.55 ml 70% perchloric acid in 80 ml reagent grade water. Dilute to a final volume of 100 ml with reagent grade water.
  • 0.25 M Sodium succinate⋅HCl, pH 6.5
  • Worthington RNA Core (Code:RNAC). Dissolve at a concentration of 17 mg/ml in reagent grade water. Adjust pH to 7.0.


Dissolve contents of vial with one ml reagent grade water. Prior to use, dilute to 2-8 units/ml with reagent grade water.


Prepare a 37°C water bath.

Pipette into tubes as follows:

  Test Blank
Sodium succinate buffer 0.4 ml 0.4 ml
RNA core 0.5 ml 0.5 ml
Reagent grade water 1.0 ml 1.1 ml
Incubate tubes in 37°C water bath for 5 minutes to achieve temperature equilibration. At timed intervals add:
Enzyme dilution 0.1 ml ------

Incubate at 37°C for 30 minutes. Stop the reaction at timed intervals by adding 2.0 ml uranyl acetate/perchloric acid solution. Remove to an ice bath and chill for 10 minutes. Centrifuge at high speed in a clinical centrifuge for 15 minutes. Transfer 0.2 ml aliquot of each supernatant to a clean dry test tube and add 7.8 ml reagent grade water. Mix well and read A260 vs water.



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