A new peptide-based fluorescent probe selective for zinc(ii) and copper(ii)

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 Ka = 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

Multifunctional peptide‐based fluorescent chemosensor for detection of Hg2+, Cu2+ and S2− ions

“…Fluorescence emission at 360 nm is a characteristic of Trp residues, whereas fluorescence emission at 500 nm is attributed to dansyl chromophores . When Hg 2+ was added to the D‐P5 solution, amino acid residues in D‐P5 (including imidazole group of histidine and indole group of tryptophan) can interact with Hg 2+ , therefore the peptide may fold and the distance between the dansyl fluorophore (acceptor) and Trp residue (donor) would be shortened, resulting in a decrease in emission intensity for Trp (360 nm) and an increase in emission intensity for the dansyl group (500 nm) due to the fluorescence resonance energy transfer (FRET) effect . This change suggests that DP‐5 has a selective and sensitive response to Hg 2+ and Cu 2+ using different types of response; this may be due to the formation of 2:1 coordination compounds of this sensor with Hg 2+ and Cu 2+ , respectively (Figure c,d).…”

“…Environmental pollution from mercury has received considerable attention because the accumulation of low concentrations of mercury in organisms through the food chain has caused various potential effects including motion disorders, and serious cognitive prenatal brain damage . As one of the most abundant trace metal elements in the human body, Cu 2+ plays a crucial role in metabolic homeostasis, protein regulation, and the development of living organisms . Therefore, selective and sensitive detection of Hg 2+ and Cu 2+ is needed due to fundamental application in environmental and biomedical sciences …”

Molecular Dissection of dH3w, A Fluorescent Peptidyl Sensor for Zinc and Mercury

“…All the experiments were performed in 20 mM MOPS buffer, pH 7.0. The estimation of the binding constant (where possible) was performed by plotting the fraction of bound peptide (α) as function of total metal concentration, and fitting the experimental data according to an equivalent and independent binding site model, as described in detail in [7].…”

“…Previously, we described a dansylated peptidyl sensor, dansyl-HPHGHW-NH 2 (dH3w), designed on the basis of the internal repeats of human histidine-rich glycoprotein (HPHGH) [7,8]. dH3w shows a turn-on response to Zn(II), a turn-off response to Cu(II), and a complex response to Hg(II), characterized by a turn-off phase at low Hg(II) concentrations and a turn-on phase at high concentrations.…”

“…Interestingly, dH3w shows very different binding modes for Zn(II) and Hg(II). In the presence of Zn(II), dH3w forms a dimeric complex with a stoichiometry 2:1, peptide/zinc [7]. Likely, the dimerization of the peptide causes the turn-on response by changing the exposure to the solvent of the dansyl group, a well-known solvatochromic fluorophore [9].…”