title: A fluorescence lifetime-based assay for abelson kinase.
abstract: We present a novel homogeneous in vitro assay format and apply it to the quantitative determination of the enzymatic activity of a tyrosine kinase. The assay employs a short peptidic substrate containing a single tyrosine and a single probe attached via a cysteine side chain. The structural flexibility of the peptide allows for the dynamic quenching of the probe by the nonphosphorylated tyrosine side chain. The probe responds with changes in its fluorescence lifetime depending on the phosphorylation state of the tyrosine. We use this effect to directly follow the enzymatic phosphorylation of the substrate, without having to resort to additional assay components such as an antibody against the phosphotyrosine. As an example for the application of this assay principle, we present results from the development of an assay for Abelson kinase (c-Abl) used for compound profiling. Adjustments in the peptide sequence would make this assay format suitable to a wide variety of other tyrosine kinases.
authors.affiliation: Biosyntan GmbH, Berlin, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Pritz
authors.forename: Gabriele
authors.initials: G
authors.lastname: Meder
authors.forename: Klaus
authors.initials: K
authors.lastname: Doering
authors.forename: Peter
authors.initials: P
authors.lastname: Drueckes
authors.forename: Julian
authors.initials: J
authors.lastname: Woelcke
authors.forename: Lorenz M
authors.initials: LM
authors.lastname: Mayr
authors.forename: Ulrich
authors.initials: U
authors.lastname: Hassiepen
mesh_terms: D000595:Amino Acid Sequence; D001681:Biological Assay; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D020128:Inhibitory Concentration 50; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D016315:Proto-Oncogene Proteins c-abl / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D010455:Peptides; D019000:Phosphotyrosine; D014443:Tyrosine; D016315:Proto-Oncogene Proteins c-abl