Thermodynamic study of Cu2+ binding to the DAHK and GHK peptides by isothermal titration calorimetry (ITC) with the weaker competitor glycine

Abstract

The peptides Asp-Ala-His-Lys (DAHK) and Gly-His-Lys (GHK) are naturally occurring copper(II)-chelating motives in human serum and cerebrospinal fluid. Here the sensitive thermodynamic technique isothermal titration calorimetry (ITC) has been used to study the energetics of copper(II) binding to DAHK and GHK peptides in the presence of the weaker ligand glycine as a competitor. DAHK and GHK bind Cu(II) predominantly in a 1:1 stoichiometry with conditional dissociation constants (i.e. at pH 7.4, in the absence of the competing chelators glycine and HEPES buffer) of 2.6 +/-0.4 x 10 -14 M and of 7.0 +/-1.0 x 10 -14 M, respectively. Furthermore, the apparent ΔH values were measured and the number of protons released upon Cu(II) binding was determined by performing experiments in different buffers. This allowed us to determine the conditional ΔG, ΔH, and ΔS, i.e. corrected for the contributions of the weaker ligand glycine and the buffer at pH 7.4. We found that the entropic and enthalpic contributions to the Cu(II)-binding to GHK and DAHK are distinct, with a higher enthalpic contribution for GHK. The obtained thermodynamic parameters correspond well to those in the literature obtained by other techniques, suggesting that the use of the weaker ligand glycine as a competitor in ITC provides accurate data for Cu(II)-binding to high affinity peptides, which cannot be accurately determined without the use of a competitor ligand.3

Dissecting ITC data of metal ions binding to ligands and proteins

“…An alternative method to maintain the metal ions solubility is to include a small chelating ligand (e.g. glycine) with known metal binding thermodynamic parameters [19,42,43]. However, caution is urged to select a chelator and buffer pair that will not compete with each other for the metal ion; this may result in undesirable mixed ligand species that are difficult to account for.…”

X‐ray and Solution Structures of CuIIGHK and CuIIDAHK Complexes: Influence on Their Redox Properties

“…Thus differences in the coordination of the apical position might explain differences seen between the reactivity of [Cu II -A C H T U N G T R E N N U N G (DAHK)] and Cu-albumin. [8,28] Although the affinity of GHK for Cu II is on the same order of magnitude as that of DAHK, [21] it exchanges much faster and hence could serve as a fast Cu II supplier. The structural reason for that is that the fourth equatorial binding site on Cu II is available to bind a transfer partner.…”

Calorimetric investigation of copper(II) binding to Aβ peptides: thermodynamics of coordination plasticity

“…A major concern with these values is the potential formation of ternary complexes with the buffer or competing ligand which, if not accounted for, can alter the buffer-independent association constant considerably [10,34,35]. A recent study by Trapaidze et al [46] provided thermodynamic parameters for two well-defined peptides using glycine as a competing ligand. Their values were in agreement with literature values, and the use of different concentrations of glycine allowed the determination of a ternary complex in one peptide but not the other.…”