Determinations of Uranium(VI) Binding Properties with some Metalloproteins (Transferrin, Albumin, Metallothionein and Ferritin) by Fluorescence Quenching

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 phy-siologically relevant conditions of pH. Results showed that fluorescence from the four metalloproteins was quenched by UO 2 2+ . Stoichiometry reactions, fluorescence quenching mechanisms and complexing properties of metalloproteins, i.e. binding constants and binding sites densities, were deter-mined 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 (L 1 and L 2). Results showed that the U(VI)–Apo-HTf complexation con-stant values (log K 1 =7.7, log K 2 =4.6) were slightly higher than those observed for U(VI)–HSA complex (log K 1 =6.1, log K 2 =4.8), U(VI)–MT complex (log K 1 =6.5, log K 2 =5.6) and U(VI)–Apo-EqsF complex (log K 1 =5.3, log K 2 =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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Jérôme Michon, Sandrine Frelon, Cédric Garnier, Frédéric Coppin. Determinations of Uranium(VI) Binding Properties with some Metalloproteins (Transferrin, Albumin, Metallothionein and Ferritin) by Fluorescence Quenching. Journal of Fluorescence, Springer Verlag, 2010, 20, pp.581-590. 〈10.1007/s10895-009-0587-3〉. 〈hal-01096837〉

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