Hg2+ and Cd2+ binding of a bioinspired hexapeptide with two cysteine units constructed as a minimalistic metal ion sensing fluorescent probe

Abstract

Hg²⁺ and Cd²⁺ complexation of a short hexapeptide, Ac-DCSSCY-NH₂ (DY), was studied by pH-potentiometry, UV and NMR spectroscopy and fluorimetry in aqueous solutions and the Hg²⁺-binding ability of the ligand was also described in an immobilized form, where the peptides were anchored to a hydrophilic resin. Hg²⁺ was demonstrated to form a 1 : 1 complex with the ligand even at pH = 2.0 while Cd²⁺ coordination by the peptide takes place only above pH ∼ 3.5. Both metal ions form bis-ligand complexes by the coordination of four Cys-thiolates at ligand excess above pH ∼ 5.5 (Cd²⁺) and 7.0 (Hg²⁺). Fluorescence studies demonstrated a Hg²⁺ induced concentration-dependent quenching of the Tyr fluorescence until a 1 : 1 Hg²⁺ : DY ratio. The fluorescence emission intensity decreases linearly with the increasing Hg²⁺ concentration in a range of over two orders of magnitude. The fact that this occurs even in the presence of 1.0 eq. of Cd²⁺ per ligand reflects a complete displacement of the latter metal ion by Hg²⁺ from its peptide-bound form. The immobilized peptide was also shown to bind Hg²⁺ very efficiently even from samples at pH = 2.0. However, the existence of lower affinity binding sites was also demonstrated by binding of more than 1.0 eq. of Hg²⁺ per immobilized DY molecule under Hg²⁺-excess conditions. Experiments performed with a mixture of four metal ions, Hg²⁺, Cd²⁺, Zn²⁺ and Ni²⁺, indicate that this molecular probe may potentially be used in Hg²⁺-sensing systems under acidic conditions for the measurement of μM range concentrations.