Determinations of Uranium(VI) Binding Properties with some Metalloproteins (Transferrin,Albumin, Metallothionein and Ferritin) by Fluorescence Quenching |
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Authors: | Jér?me?Michon Sandrine?Frelon Cédric?Garnier Frédéric?Coppin |
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Institution: | 1.Laboratoire de Radioécologie et Ecotoxicologie,Institut de Radioprotection et S?reté Nucléaire,Saint-Paul-lez-Durance Cedex,France;2.Groupe de Physico Toxico Chimie des Systèmes Naturels, Institut des Sciences Moléculaires, (ISM–UMR CNRS 5255),Université Bordeaux I,Talence Cedex,France;3.Laboratoire PROTEE,Université du Sud Toulon-Var,La Garde,France |
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Abstract: | The interactions between uranium and four metalloproteins (Apo-HTf, HSA, MT and Apo-EqSF) 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 0.05 to 1150) at physiologically
relevant conditions of pH. Results showed that fluorescence from the four metalloproteins was quenched by UO22+. Stoichiometry reactions, fluorescence quenching mechanisms and complexing properties of metalloproteins, 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 metalloprotein complexation by uranium could be simulated
by two specific sites (L1 and L2). Results showed that the U(VI)–Apo-HTf complexation constant values (log K1 = 7.7, log K2 = 4.6) were slightly higher than those observed for U(VI)–HSA complex (log K1 = 6.1, log K2 = 4.8), U(VI)–MT complex (log K1 = 6.5, log K2 = 5.6) and U(VI)–Apo-EqsF complex (log K1 = 5.3, log K2 = 3.9). PROSECE fitting studies also showed that the complexing capacities of each protein were different: 550 moles of U(VI)
are complexed by Apo-EqSF while only 28, 10 and 5 moles of U(VI) are complexed by Apo-HTf, HSA and MT, respectively. |
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