Ternary Cu(II) Complex with GHK Peptide and Cis-Urocanic Acid as a Potential Physiologically Functional Copper Chelate

Abstract

The tripeptide NH₂-Gly-His-Lys-COOH (GHK), cis-urocanic acid (cis-UCA) and Cu(II) ions are physiological constituents of the human body and they co-occur (e.g., in the skin and the plasma). While GHK is known as Cu(II)-binding molecule, we found that urocanic acid also coordinates Cu(II) ions. Furthermore, both ligands create ternary Cu(II) complex being probably physiologically functional species. Regarding the natural concentrations of the studied molecules in some human tissues, together with the affinities reported here, we conclude that the ternary complex [GHK][Cu(II)][cis-urocanic acid] may be partly responsible for biological effects of GHK and urocanic acid described in the literature.

Keywords: copper; imidazole ligands; ternary complex.

Scheme 1

UV-radiation-induced isomerisation of trans-urocanic acid (UCA) and cis-UCA [10].

Figure 1

The conditional Cu(II) binding constant ^cK_7.4 = [Cu(GHK)₂]/([Cu(GHK)][GHK]). (A) UV-vis, (B) circular dichroism (CD), and (C) electron paramagnetic resonance (EPR) spectra of Cu/GHK 0.5:n (0.63 ≤ n ≤ 30 mM) at pH 7.4, 25 °C. (D) Speciation diagram obtained by decomposition of all UV-vis (diamonds), CD (squares), and EPR (circles) spectra (Figures S4–S6). The solid lines were calculated using the binding constant derived from (E) and log ^cK₁ = [Cu(GHK)]/([GHK][Cu]) = 12.62 derived from potentiometry. (E) Determination of the binding constant ^cK_7.4 using least squares regression. The margin of error derives from the 95% confidence interval (dashed lines).

Figure 2

UV-vis spectra of 4 mM cis-UCA and 0.8 mM CuCl₂ titrated in a pH-metric manner. (A) Spectra are colour coded with reds (the lowest pH) to purples (the highest pH). (B) Species distribution of Cu(II) complexes of cis-UCA (red, Cu(cis-UCA); blue, Cu(cis-UCA)₂) at 25 °C, calculated for concentrations from UV-vis data (presented on panel A), based on protonation and stability constants shown in Table 1. Left-side axis represents molar fractions of Cu(II) complexes, right side axes stand for absorbance at 550 and 840 nm.

Figure 3

The conditional Cu(II) binding constant ^cK_7.4 = [Cu(GHK)(Im)]/([Cu(GHK)][Im]). (A) UV-vis, (B) CD, and (C) EPR spectra of Cu/GHK/Im 0.5:0.63:n (0 ≤ n ≤ 25 mM) at pH 7.4, 25 °C. (D) Speciation of GHK complexes obtained from decomposition of UV-vis, CD, and EPR spectra in dependence of Im concentration (Figures S8–S11). (E) Determination of the binding constant ^cK_7.4 using least squares regression. The margin of error derives from the 95% confidence interval (dashed lines).

Figure 4

spectra of 0.95 mM GHK, 0.8 mM CuCl₂, and 6 mM cis-UCA (A) or trans-UCA (B) in pH-metric manner. Arrows indicate the spectral changes from low to high pH. The ellipticity at 650 nm was read from panels (A) and (B) and shown for cis-UCA (C) or trans-UCA (D) (navy squares) juxtaposed to data obtained via pH-metric titration of 0.95 mM GHK with 0.8 mM CuCl₂ (magenta circles).

Figure 5

The conditional Cu(II) binding constant ^cK_7.4 = [Cu(GHK)(cis-UCA)]/([Cu(GHK)][cis-UCA]). (A) UV-vis, and (B) CD spectra of Cu/GHK/cis-UCA 0.8:0.95:n (0 ≤ n ≤ 22 mM) at pH 7.4, 25 °C. (C) Speciation of GHK complexes obtained from decomposition of UV-vis and CD spectra in dependence of cis-UCA concentration. (D) Determination of the binding constant ^cK_7.4 using least squares regression. The margin of error derives from the 95% confidence interval dashed lines).

Figure 6

Influence of variable concentrations of cis-UCA on the apparent affinity of Cu/GHK/cis-UCA system. Values of CI_7.4 and CI_6.5 in the presence of cis-UCA (blue and green solid line) are valid for [Cu] = [GHK] = 1 µM, at pH 7.4 and 6.5, respectively. The dotted lines represent the reference CI_7.4 and CI_6.5 levels calculated for solutions without cis-UCA.

Figure 7

Species distribution simulated for natural moisturizing factor (NMF), 0.9 µM Cu²⁺ ions, and different concentrations of GHK, at pH 7.4 (representing the possible conditions in wounds). The lowest concentration of GHK (0.6 µM) is equal to the concentrations found in human plasma. Higher concentrations (6 and 60 µM) of GHK may occur in wounds and/or after application of cosmetics or other products with GHK as the active substance. The protonation constants and stability constants for Cu(II) complexes were taken from the literature [47,48] and this paper. The conditional constant for the ternary complexes formation of Cu(GHK) with carboxylates (68 M⁻¹) [20] was also included in calculations. Concentrations taken for calculations are 43 mM serine, 30 mM glycine, 23 mM pyroglutamic acid, 18 mM alanine, 14 mM lactic acid, and 14 mM cis-UCA [11,12]. All ternary complexes of Cu(GHK) with aforementioned compounds with carboxylic groups were combined in the “Cu(GHK)(carboxylates)”. Only the species exceeding 1% of all Cu(II) species are shown for clarity.