DEVELOPMENT OF A SCINTILLATION PROXIMITY ASSAY FOR HUMAN CYTOMEGALOVIRUS PROTEASE USING (33)PHOSPHOROUS

A scintillation proximity assay (SPA) using (33)phosphorous is describ ed for human cytomegalovirus (HCMV) UL80 protease. This is the first d emonstration that (33)phosphorous is compatible with the SPA system. T he peptide substrate used in the assay contains an HCMV protease cleav age site and is biotinylated at its amino terminus. The peptide also c ontains a site for protein kinase A, enabling radiolabeling at its car boxy terminus with [gamma-P-33]ATP. Peptide is incubated with protease , followed by binding to streptavidin-coated SPA beads via biotin. Cle avage of the peptide by the protease results in a decrease in the radi oactive signal, which is prevented by the presence of a protease inhib itor. This methodology is applicable to other proteases whose cleavage site is known. (C) 1996 Academic Press, Inc.