PDE5A (human), (recombinant)

Assay for Measuring PDE5 Activity

Assay Overview:
cGMP/[³H]-cGMP mixture is incubated with the PDE5 preparation and the reaction is stopped. Produced GMP/[³H]-GMP is converted into guanosine/[³H]-guanosine after the phosphate group is removed by nucleotide phosphatase (from snake venom). Guanosine/[³H]-guanosine mixture does not bind to DEAE Sephadex 25, while the charged cGMP and GMP bind to the column.

Equipment Required:
Water bath at 30°C.
Heat block at 100°C.
Columns with 1.5ml bed of DEAE Sephadex 25 media: One per reaction sample. Columns should be pre-washed with 2ml of 500mM NaCl solution, 10ml of column wash buffer and 4ml of water. Drain the liquid before use of the column.
Scintillation vials and counter.

Additional Reagents Required:
Enzyme Dilution Buffer: 25mM TEA, pH 7.5, 0.2mg/ml BSA, 15mM MgCl₂ and 1mM EGTA
Assay Buffer: 25mM TEA, pH 7.5, containing 0.2mg/ml BSA, 15mM MgCl₂, 1mM EGTA, 60µM cGMP and [³H]-cGMP. Add [³H]-cGMP to 400'000-600'000 cpm/ml.
Column Wash Buffer: 25mM TRIS, pH 7.5.
Nucleotidase Reagent: Weight out several mg of snake venom from Crotalus atrox. Mark the weigh on the tube. Keep on ice.
Scintillation Fluid.

cGMP Degradation Reaction:
a) Prepare a 1:10-1:20 dilution of PDE5A (human) (recombinant) in enzyme dilution buffer.
b) Preincubate 250µl of assay buffer in a 1.5ml eppendorf tube at 30°C for 2-3 min.
c) Start the reaction by adding 1µl of diluted PDE5 to 250µl of assay buffer. Incubate the reaction for 2-5 min. at 30°C. Do not add PDE5 into one or two tubes. They will be used to determine the background (cpm_bgrnd).
d) To stop the reaction, transfer the assay tube to 100°C heating block. Incubate for 5 min. and transfer on ice. (Optional: centrifuge the sample for 5 min. at 14'000 g to precipitate the denatured protein). The sample can be stored at this time before further processing.
e) Dilute the snake venom to 10mg/ml in 25mM TEA, pH 7.5. Add 10ml of venom solution to the chilled reaction sample and transfer to 30°C. Incubate for 10-15 min. and then transfer on ice.

Guanosine Quantification:
f) Add 7ml of scintillation fluid to a scintillation vial and place under the column prepared as described above.
g) Transfer the reaction sample (260µl) onto the column. Wait 1-2 min. for the sample to completely penetrate into the column and wash the column by loading 4ml of distilled water. Collect the eluted [³H]-guanosine in the scintillation vial.
h) Shake the vial to mix the liquids and proceed to measure the amount of eluted radioactivity.
i) Determine the amount of degraded cGMP by using the following formula:

cGMP= (cpm_sample - cpm_bgrnd)/cpm_total x 15nmoles

j) The column can be regenerated by the wash cycles described above (see equipment needed).