Determinations of uranium(VI) binding properties with some metalloproteins (transferrin, albumin, metallothionein and ferritin) by fluorescence quenching.
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 UO(2)(2+). 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 (L(1) and L(2)). Results showed that the U(VI)-Apo-HTf complexation constant 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.
Michon
Jérôme
J
Laboratoire de Radioécologie et Ecotoxicologie, Institut de Radioprotection et Sûreté Nucléaire, Cadarache, Bât 186, BP3, 13115 Saint-Paul-lez-Durance Cedex, France.
Frelon
Sandrine
S
Garnier
Cédric
C
Coppin
Frédéric
F
D000418:Albumins / Q000737:chemistry*; D005293:Ferritins / Q000737:chemistry*; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D008668:Metallothionein / Q000737:chemistry*; D012815:Signal Processing, Computer-Assisted; D012984:Software; D013050:Spectrometry, Fluorescence / Q000379:methods; D014168:Transferrin / Q000737:chemistry*; D014501:Uranium / Q000737:chemistry*; D017974:Uranium Compounds / Q000737:chemistry
D000418:Albumins; D014168:Transferrin; D017974:Uranium Compounds; D014501:Uranium; D005293:Ferritins; D008668:Metallothionein; C012597:uranium dioxide