Binding Constant Determined from the Angstrom-Scale Change in Hydrodynamic Radius of Transferrin upon Binding with Europium Using Dual-Focus Fluorescence Correlation Spectroscopy

Monitoring the binding of a large fluorescently tagged molecule to a small solute by fluorescence correlation spectroscopy (FCS) is rather uncommon because the binding-related change in diffusion coefficient is very small. Here, we use a high-precision variant of FCS, namely, dual-focus FCS (2fFCS), for measuring the angstrom-scale change of the hydrodynamic radius of the bilobal metal transport protein transferrin (Tf) upon binding europium ions. Applying a sequential 1:2 complexation model, we use these measurements for determining the binding constants (K). Our results show a 0.7 angstrom change of the protein's hydrodynamic radius upon 1:1 Tf-Eu complex formation and a second change of 1.8 angstrom upon subsequent binding of a second europium ion. More than one unit variation in logK indicates an intrinsic dissimilarity in metal affinity of the C- and N-lobes of Tf, which agrees well with earlier reported ensemble spectroscopy results.