title: Detection of phosphopeptides by fluorescence polarization in the presence of cationic polyamino acids: application to kinase assays.
abstract: We have studied the interaction of several phosphopeptides with cationic polyamino acids such as polyarginine and polylysine by fluorescence polarization. The phosphopeptides used were labeled with fluorescein, and their net charges at the experimental pH of 7. 5 were 0, -1, -2, and -3. These phosphopeptides represent the products of enzymatic phosphorylation reactions of the corresponding nonphosphorylated precursors by the protein kinase A, Akt1 (protein kinase Balpha), and protein kinase C. We found that these phosphopeptides bind more strongly to the cationic polyamino acids studied than their nonphosphorylated analogs. This preferential binding of the phosphorylated peptides could be conveniently detected by an increase in the fluorescence polarization signal of the attached fluorescein residue. We have exploited this observation to develop a new approach for the detection of kinase activity that does not require radioactivity or separation of substrate from product. We have successfully used this method to perform K(m) determinations of the kinase enzymes for their substrates and K(i) determinations of one of their inhibitors. This method for measuring kinase activity might be particularly useful for high-throughput screening applications.
authors.affiliation: Caliper Technologies Corp., 605 Fairchild Drive, Mountain View, California 94043, USA.
authors.forename: J
authors.initials: J
authors.lastname: Coffin
authors.forename: M
authors.initials: M
authors.lastname: Latev
authors.forename: X
authors.initials: X
authors.lastname: Bi
authors.forename: T T
authors.initials: TT
authors.lastname: Nikiforov
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D019793:Fluorescein; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D010748:Phosphopeptides / Q000032:analysis* / Q000737:chemistry
chemical_list: D000596:Amino Acids; D005456:Fluorescent Dyes; D010748:Phosphopeptides; D019793:Fluorescein