title: An experimental method for the determination of enzyme-competitive inhibitor dissociation constants from displacement curves: application to human renin using fluorescence energy transfer to a synthetic dansylated inhibitor peptide.
abstract: We developed a facile procedure for the determination of enzyme-competitive inhibitor dissociation constants over a wide range of potencies at any ratios of enzyme, labeled ligand, and inhibitor. The assay uses displacement curves and a fluorescent-labeled ligand to allow experimental determination of dissociation constants (Kd's) of inhibitors of human renin, a highly specific enzyme, for which numerous high affinity (up to 100 pM) inhibitors have been synthesized. The procedure involves binding a dansylated competitive inhibitor, U80215, followed by its displacement by an unlabeled inhibitor of renin. Binding of U80215 is monitored by fluorescent energy transfer from the renin tryptophans to the dansyl moiety; displacement of U80215 by an unlabeled inhibitor is monitored by a reversal of this process. The procedures may be used to determine the potencies of unlabeled inhibitors up to 100 pM affinities and to determine kinetic binding constants. The concepts described should also be useful in other protein/ligand systems.
authors.affiliation: Physical and Analytical Chemistry Research, Upjohn Company, Kalamazoo, Michigan 49001.
authors.forename: D E
authors.initials: DE
authors.lastname: Epps
authors.forename: H
authors.initials: H
authors.lastname: Schostarez
authors.forename: C V
authors.initials: CV
authors.lastname: Argoudelis
authors.forename: R
authors.initials: R
authors.lastname: Poorman
authors.forename: J
authors.initials: J
authors.lastname: Hinzmann
authors.forename: T K
authors.initials: TK
authors.lastname: Sawyer
authors.forename: F
authors.initials: F
authors.lastname: Mandel
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D004735:Energy Transfer; D004791:Enzyme Inhibitors / Q000378:metabolism*; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides; D012083:Renin / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D004791:Enzyme Inhibitors; D009842:Oligopeptides; C061582:U 80215; D012083:Renin