Interaction kinetics of tetramethylrhodamine transferrin with human transferrin receptor studied by fluorescence correlation spectroscopy.
We applied fluorescence correlation spectroscopy (FCS) to characterize the interaction dynamics of fluorescence-labeled transferrin with transferrin receptor (hTfR) associates isolated from human placenta. The dissociation constant for the equilibrium binding of TMR-labeled ferri-transferrin to hTfR in detergent free solution was determined to be 7 +/- 3 nM. Binding curves were compatible with equal and independent binding sites present on the hTfR associates. Under pseudo-first-order conditions, with respect to transferrin, complex formation is monophasic. From these curves, association and dissociation rate constants for a reversible bimolecular binding reaction were determined, with (1.1 +/- 0.1) x 10(4) M-1 s-1 for the former and (6 +/- 4) x 10(-)4 s-1 for the latter. In dissociation exchange experiments, biphasic curves and concentration-independent reciprocal relaxation times were determined. From isothermal titration calorimetry experiments, we obtained an enthalpy change of -44.4 kJ/mol associated with the reaction. We thus conclude that the reaction is mainly enthalpy driven.
Schüler
J
J
Institut für Kristallographie, Freie Universität Berlin, Germany.
Frank
J
J
Trier
U
U
Schäfer-Korting
M
M
Saenger
W
W
D002151:Calorimetry / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D017830:Octoxynol; D010920:Placenta; D011485:Protein Binding; D011990:Receptors, Transferrin / Q000737:chemistry* / Q000378:metabolism; D012235:Rhodamines / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013816:Thermodynamics
D005456:Fluorescent Dyes; D011990:Receptors, Transferrin; D012235:Rhodamines; C005358:tetramethylrhodamine; D017830:Octoxynol