Phosphodiesterase [³H] cGMP SPA Enzyme Assay

Scintillation proximity assay (SPA) to measure cGMP-dependent PDE isoenzymes:

• Homogeneous assay, performed in microtitre plates or tubes
• Suitable for high throughput screening
• Ready to use buffer
• [³H] tracer included

  The assay concept from is based on the observation that linear nucleotides bind preferentially to SPA yttrium silicate beads compared to cyclic nucleotides in the presence of zinc sulphate. Therefore, under optimized conditions, the product of the enzyme reaction binds directly to the SPA beads, and the enzyme substrate will not. The binding of the radiolabelled product to the bead brings the isotope into close enough proximity to allow radiation from the tritium to excite the scintillant within the bead. Any unbound radiolabel is not close enough to the scintillant to allow this energy transfer, so no signal is generated. In addition, as the cyclic substrate does not bind effectively to the bead, the background signal generated is very low. A complex ion chelation mechanism enables the linear nucleotide to bind to the bead. Optimal concentrations of zinc sulphate enhance the binding and ensure robust and efficient capture. The addition of zinc sulphate in SPA bead suspension also has the inherent ability to terminate PDE reactions.