title: Binding stoichiometry and affinity of fluorescent dyes to proteins in different structural states.
abstract: Protocol of determination of binding stoichiometry and affinity of fluorescent dyes with proteins in different structural states is proposed. The proposed approach is based on the spectrophotometric determination of concentrations of dye bound to protein and free dye in solutions prepared by equilibrium microdialysis. This technique allows also determining spectral properties of the bound dyes. The restrictions of the use of dye fluorescence intensity for characterization of its interaction with the target protein are discussed. It is shown that the dependence of the dye fluorescence intensity on its optical density together with the data on its binding parameter can give information about the dye fluorescence quantum yield. All procedures are illustrated by interaction of 8-anilino-1-naphthalenesulfonate (ANS) with bovine serum albumin.
authors: Laboratory of Structural Dynamics, Stability and Folding of Proteins of the Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia.
authors: Anna I
authors: AI
authors: Sulatskaya
authors: Olga I
authors: OI
authors: Povarova
authors: Irina M
authors: IM
authors: Kuznetsova
authors: Vladimir N
authors: VN
authors: Uversky
authors: Konstantin K
authors: KK
authors: Turoverov
mesh_terms: D000465:Algorithms; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
chemical_list: C515594:8-anilino-1-naphthalenesulfonic acid; D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D012710:Serum Albumin, Bovine