Determining Metal Ion Complexation Kinetics with Fluorescent Ligands by Using Fluorescence Correlation Spectroscopy

Abstract

Fluorescence correlation spectroscopy (FCS) has been extensively used to measure equilibrium binding constants (K) or association and dissociation rates in many reversible chemical reactions across chemistry and biology. For the majority of investigated reactions, the binding constant was on the order of ∼100 M−1, with dissociation constants faster or equal to 103 s−1, which ensured that enough association/dissociation events occur during the typical diffusion‐determined transition time of molecules through the FCS detection volume. However, complexation reactions involving metal ions and chelating ligands exhibit equilibrium constants exceeding 104 M−1. In the present paper, we explore the applicability of FCS for measuring reaction rates of such complexation reactions, and apply it to binding of iron, europium and uranyl ions to a fluorescent chelating ligand, calcein. For this purpose, we exploit the fact that the ligand fluorescence becomes strongly quenched after binding a metal ion, which results in strong intensity fluctuations that lead to a partial correlation decay in FCS. We also present measurements for the strongly radioactive ions of 241Am3+, where the extreme sensitivity of FCS allows us to work with sample concentrations and volumes that exhibit close to negligible radioactivity levels. A general discussion of the applicability of FCS to the investigation of metal‐ligand binding reactions concludes our paper.

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

“…The key descriptor for a complex-formation reaction in biological and supramolecular system is the equilibrium constant ( K ), which is the ratio of association and dissociation rates. Fluorescence correlation spectroscopy (FCS), having a dynamic range from picoseconds to seconds, is an ideal tool to investigate such reactions with single-molecule sensitivity under equilibrium conditions and most importantly, without any external perturbation. − Modern day confocal setups allow FCS measurements with very small sample volumes of low concentrations, and this has been implicitly employed to investigate complexation reactions involving miniscule amounts of radioactive metal ions in regular laboratory scenarios . The binding of metal ions by chelating ligands generally results in highly stable complexes, as reflected by their high K values (≥10 5 M –1 ) and negligible dissociation rates (≤10 –3 s –1 ).…”

“…The binding of metal ions by chelating ligands generally results in highly stable complexes, as reflected by their high K values (≥10 5 M –1 ) and negligible dissociation rates (≤10 –3 s –1 ). Thus, the direct determination of rate constants of such interactions with FCS is rather impossible because the time scale of dissociation is by orders of magnitude longer than the typical diffusion time of a complex through the confocal detection volume (typically less than a millisecond in aqueous media). , However, the equilibrium constant for such reactions can be easily extracted by measuring concentration changes (i.e., correlation amplitude) ,, or changes of hydrodynamic radius (i.e., diffusion coefficient) − ,, of the fluorescent chelator as a function of analyte concentration. Although a change in correlation amplitude is reported for many fluorescent chelators upon binding of metal ions (as a result of a binding-induced change of fluorescence intensity), such an approach to determine K requires specific chemistry and photophysics of the chelator.…”

“…In this context, FCS can be a powerful tool for obtaining relevant data for the binding of metals and metal–Tf complexes to metal-free Tf (apoTf) and TfR in the presence of sequestrating chelators, respectively. Such data will be important for devising new and efficient strategies of handling radioactive contamination …”

“…1−10 Modern day confocal setups allow FCS measurements with very small sample volumes of low concentrations, and this has been implicitly employed to investigate complexation reactions involving miniscule amounts of radioactive metal ions in regular laboratory scenarios. 11 The binding of metal ions by chelating ligands generally results in highly stable complexes, as reflected by their high K values (≥10 5 M −1 ) and negligible dissociation rates (≤10 −3 s −1 ). Thus, the direct determination of rate constants of such interactions with FCS is rather impossible because the time scale of dissociation is by orders of magnitude longer than the typical diffusion time of a complex through the confocal detection volume (typically less than a millisecond in aqueous media).…”

“…Such data will be important for devising new and efficient strategies of handling radioactive contamination. 11 Tf is composed of two homologous lobes, the N-and the Clobe, with a well-characterized binding-cleft. Each metal ion is tightly bound in a distorted octahedral geometry through phenolate oxygen atoms from two tyrosine residues, carboxylic oxygen atom from one aspartate, imidazole nitrogen atom from one histidine, and a bidentated synergistic carbonate anion at physiological pH.…”