The interaction between retinol-binding protein and transthyretin analyzed by fluorescence anisotropy.
The retinol carrier retinol-binding protein (RBP) forms in blood a complex with the thyroid hormone carrier transthyretin (TTR). The interactions of retinoid-RBP complexes, as well as of unliganded RBP, with TTR can be investigated by means of fluorescence anisotropy. RBP represents the prototypic lipocalin, in the internal cavity of which the retinol molecule is accommodated. Due to the tight binding of retinol within a substantially apolar binding site, an intense fluorescence emission characterizes the RBP-bound vitamin. The addition of TTR to the retinol-RBP complex (holoRBP) causes a marked increase in the fluorescence anisotropy of the RBP-bound retinol within the system, due to the formation of the holoRBP-TTR complex, which allows the interaction between the two proteins to be monitored. The fluorescence anisotropy technique is also suitable to study the interaction of TTR with apoRBP and RBP in complex with non-fluorescent retinoids. In the latter cases, the fluorescence signal is provided by a fluorescent probe covalently linked to TTR rather than by RBP-bound retinol. We report here on the preparation of recombinant human RBP and TTR, the covalent labeling of TTR with the fluorescent dansyl probe, and fluorescence anisotropy titrations for RBP and TTR.
Folli
Claudia
C
Department of Biochemistry and Molecular Biology, University of Parma, Via G.P. Usberti 23/A, Parma, Italy.
Favilla
Roberto
R
Berni
Rodolfo
R
D000042:Absorption; D001059:Apoproteins / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D003619:Dansyl Compounds / Q000737:chemistry; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D011228:Prealbumin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012177:Retinol-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014801:Vitamin A / Q000378:metabolism
D001059:Apoproteins; D018076:DNA, Complementary; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D011228:Prealbumin; D011994:Recombinant Proteins; D012177:Retinol-Binding Proteins; D014801:Vitamin A