title: Fluorescence Quenching Determination of Uranium (VI) Binding Properties by Two Functional Proteins: Acetylcholinesterase (AChE) and Vitellogenin (Vtg).
abstract: The interactions between uranium and two functional proteins (AChE and Vtg) were investigated using fluorescence quenching measurements. The combined use of a microplate spectrofluorometer and logarithmic additions of uranium into protein solutions allowed us to define the fluorescence quenching over a wide range of [U]/[Pi] ratios (from 1 to 3235) at physiologically relevant conditions of pH. Results showed that fluorescence from the two functional proteins was quenched by UO2 (2+). Stoichiometry reactions, fluorescence quenching mechanisms and complexing properties of proteins, i.e. binding constants and binding sites densities, were determined using classic fluorescence quenching methods and curve-fitting software (PROSECE). It was demonstrated that in our test conditions, the protein complexation by uranium could be simulated by two specific sites (L1 and L2). The obtained complexation constant values are log K1 = 5.7 (±1.0), log K2 = 4.9 (±1.1); L1 = 83 (±2), L2 = 2220 (±150) for U(VI) - Vtg and log K1 = 8.1 (±0.9), log K2 = 6.6 (±0.5), L1 = 115 (±16), L2 = 530 (±23) for U(VI)-AChE (Li is expressed in mol/mol of protein).
authors.affiliation: Laboratoire de Biogéochimie, Biodisponibilité et Transfert des Radionucléides, Institut de Radioprotection et Sûreté Nucléaire, Cadarache, Bât 186, BP3, 13115, Saint-Paul-lez-Durance Cedex, France, frederic.coppin@irsn.fr.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Coppin
authors.forename: Jérôme
authors.initials: J
authors.lastname: Michon
authors.forename: Cédric
authors.initials: C
authors.lastname: Garnier
authors.forename: Sandrine
authors.initials: S
authors.lastname: Frelon
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D005453:Fluorescence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D012815:Signal Processing, Computer-Assisted; D012984:Software; D013050:Spectrometry, Fluorescence / Q000379:methods*; D017974:Uranium Compounds / Q000737:chemistry* / Q000378:metabolism; D014819:Vitellogenins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D017974:Uranium Compounds; D014819:Vitellogenins; D000110:Acetylcholinesterase; C012597:uranium dioxide