title: Alexa and Oregon Green dyes as fluorescence anisotropy probes for measuring protein-protein and protein-nucleic acid interactions.
abstract: The fluorescence properties of Alexa 488, Oregon Green 488, and Oregon Green 514 (Molecular Probes (Eugene, OR)) are compared when conjugated to biomolecules and as model compounds free in solution. We show that these relatively new, green fluorescence probes are excellent probes for investigation of the thermodynamics of protein-protein and protein-nucleic acid interactions by fluorescence anisotropy. Unlike fluorescein, the emission of these dyes has minimal pH dependence near neutrality and is significantly less susceptible to photobleaching. Steady-state and time-resolved fluorescence anisotropy data are compared for two interacting proteins of different size and for the association of a transcription factor with a DNA oligonucleotide containing a specific binding site. The temperature dependence of the fluorescence lifetimes of the probes is reported, and the effects of molecular size and probe motion on steady-state anisotropy data are discussed. The critical interplay among correlation time, fluorescence lifetime, and the observed steady-state anisotropy is evaluated.
authors.affiliation: Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA.
authors.forename: Elena
authors.initials: E
authors.lastname: Rusinova
authors.forename: Vira
authors.initials: V
authors.lastname: Tretyachenko-Ladokhina
authors.forename: Oana E
authors.initials: OE
authors.lastname: Vele
authors.forename: Donald F
authors.initials: DF
authors.lastname: Senear
authors.forename: J B
authors.initials: JB
authors.lastname: Alexander Ross
mesh_terms: D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism; D015942:Factor VIIa / Q000737:chemistry / Q000378:metabolism; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D009841:Oligonucleotides / Q000737:chemistry* / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics; D013925:Thromboplastin / Q000737:chemistry / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D009841:Oligonucleotides; D011994:Recombinant Proteins; D012097:Repressor Proteins; D013925:Thromboplastin; D015942:Factor VIIa