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 r eceptor (hTfR) associates isolated from human placenta. The dissociation co nstant 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 hT fR associates. Under pseudo-first-order conditions, with respect to transfe rrin, complex formation is monophasic. From these curves, association and d issociation rate constants for a reversible bimolecular binding reaction we re 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 w ere determined. From isothermal titration calorimetry experiments, we obtai ned an enthalpy change of -44.4 kJ/mol associated with the reaction, We thu s conclude that the reaction is mainly enthalpy driven.