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Determining Metal Ion Complexation Kinetics with Fluorescent Ligands by Using Fluorescence Correlation Spectroscopy
Authors:Arjun Sharma  Aranyak Sarkar  Dr Dibakar Goswami  Dr Arunasis Bhattacharyya  Dr Jörg Enderlein  Dr Manoj Kumbhakar
Institution:1. Radiation & Photochemistry Division, Bhabha Atomic Research Centre, Mumbai, 400085 India

Chemical Sciences, Homi Bhabha National Institute, Mumbai 400094, India;2. Chemical Sciences, Homi Bhabha National Institute, Mumbai 400094, India

Bio-organic Division, Bhabha Atomic Research Centre, Mumbai, 400085 India;3. Chemical Sciences, Homi Bhabha National Institute, Mumbai 400094, India

Radiochemistry Division, Bhabha Atomic Research Centre, Mumbai, 400085 India;4. III. Institute of Physics – Biophysics, Georg August University, 37077 Göttingen, Germany;5. Radiation & Photochemistry Division, Bhabha Atomic Research Centre, Mumbai, 400085 India

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.
Keywords:actinides  fluorescence correlation spectroscopy  lanthanides  metal ion complexation kinetics  quenching
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