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.

Reagents

• 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.

Enzyme

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

Procedure

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.

Calculation