Development of a scintillation proximity assay for human cytomegalovirus protease using 33phosphorous

A scintillation proximity assay (SPA) using 33phosphorous is described for human cytomegalovirus (HCMV) UL80 protease. This is the first demonstration that 33phosphorous is compatible with the SPA system. The peptide substrate used in the assay contains an HCMV protease cleavage site and is biotinylated at its amino terminus. The peptide also contains a site for protein kinase A, enabling radiolabeling at its carboxy terminus with [gamma-33P]ATP. Peptide is incubated with protease, followed by binding to streptavidin-coated SPA beads via biotin. Cleavage of the peptide by the protease results in a decrease in the radioactive signal, which is prevented by the presence of a protease inhibitor. This methodology is applicable to other proteases whose cleavage site is known.