3.4. Potentiometry

Potentiometric measurements were carried out using a 907 Titrando automatic titrator (Metrohm, Herisau, Switzerland), with a Biotrode combined glass electrode (Metrohm, Herisau, Switzerland). The electrode was calibrated daily via titration of 4 mM HNO₃/96 mM KNO₃ solution. A 100 mM NaOH solution (carbon dioxide free) was used as a titrant. The 1.5 mL samples were prepared in 4 mM HNO₃/96 mM KNO₃ solution. All titrations were performed under argon atmosphere at 25 °C, in the pH range from ca. 2.7 to 11.5. Three to five titrations were used for calculations of protonation constants and Cu(II) formation constants. The obtained data were processed using SUPERQUAD and HYPERQUAD 2008 programs [35,52].

Acetate counter-ions present in the commercial GHK preparations can hinder the analysis of potentiometric data. Prior to experiments, we therefore exchanged the acetate counter-ions with TFA via two cycles of peptide dilution in ca. 0.1% TFA, followed by freeze-drying.

To calculate protonation constants and the concentration of the stock solution, we prepared samples with ca. (i) 1 mM GHK, (ii) 1–4 mM Im, and (iii) 1–4 mM cis-UCA. For each ligand, 3–4 samples were prepared in HNO₃/KNO₃.

Binary copper complex formation was studied for GHK and cis-UCA, respectively, using different molar ratios of CuCl₂ and ligands. For Cu/GHK, six samples were run in molar ratios of peptide to copper of 1:0.9 (n = 2), 1:0.5, 1:0.3 (n = 2) and 1:0.2, whereas for Cu/cis-UCA four molar ratios were studied: 3:0.8, 4:0.8, 5:0.8, and 8:0.8.

The studied molar ratios of GHK/Cu/Im were: 1:0.9:4, 1:0.9:6 and 1:0.9:8. For each molar ratio two samples were prepared. For GHK/Cu/cis-UCA five samples were prepared in ratios of 1:0.9:4; 1:0.9:5; 1:0.9:6; 1:0.9:7; and 1:0.9:8.