A new peptide-based fluorescent probe selective for zinc(<scp>ii</scp>) and copper(<scp>ii</scp>)

Abstract

A novel metal ion-sensitive fluorescent peptidyl-probe has been designed based on the most common five-residue repeat in mammalian histidine rich glycoproteins (HRGs). A dansyl-amide moiety at the N-terminus and a tryptophan residue at the C-terminus of the peptide were added as they can act as a FRET (fluorescence resonance energy transfer) pair. The dansyl fluorophore was chosen also because it frequently shows strong CHEF (chelation enhanced fluorescence) and solvatochromic effects. The designed peptide, dansyl-HPHGHW-NH₂ (dH3w), showed a selective fluorescence turn-on response to Zn²⁺ in aqueous solutions at pH 7.0 when excited at both 295 nm and 340 nm, thus indicating that both FRET and CHEF or solvatochromic effects are active in the metal/peptide complex. Steady-state fluorescence and isothermal titration calorimetry (ITC) measurements demonstrated that two peptide molecules bind to one zinc ion with an association constant K_a = 5.7 × 10⁵ M^-1 at 25 °C and pH 7.0. The fluorescence response to Zn²⁺ was not influenced by Pb²⁺, Cd²⁺, Mn²⁺, Fe²⁺, Fe³⁺, Mg²⁺, Ca²⁺, K⁺ and Na⁺ ions and only slightly influenced by Co²⁺ and Ni²⁺. Copper(ii), at concentrations as low as 5 μM, caused a strong quenching of both free and Zn²⁺ complexed dH3w. The determination of the binding parameters for Cu²⁺ has shown that one copper ion binds to one dH3w molecule with an association constant of 1.2 × 10⁶ M^-1 thus confirming the higher affinity of peptide for Cu²⁺ than for Zn²⁺. Finally, we demonstrated that dH3w can penetrate into HeLa cells and could thus be used for the determination of intracellular Zn²⁺ and Cu²⁺ concentrations.