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

  • Ana Trapaidze
  • Christelle HureauEmail author
  • Wojciech Bal
  • Mathias Winterhalter
  • Peter FallerEmail author
Original Paper


The peptides Asp-Ala-His-Lys (DAHK) and Gly-His-Lys (GHK) are naturally occurring Cu(II)-chelating motifs in human serum and cerebrospinal fluid. Here, the sensitive thermodynamic technique isothermal titration calorimetry was used to study the energetics of Cu(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 2-(4-(2-hydroxyethyl)-1-piperazinyl)ethanesulfonic acid buffer] of 2.6 ± 0.4 × 10−14 M and 7.0 ± 1.0 × 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 enthalpic contribution for GHK. The thermodynamic parameters obtained correspond well to those in the literature obtained by other techniques, suggesting that the use of the weaker ligand glycine as a competitor in isothermal titration calorimetry provides accurate data for Cu(II) binding to high-affinity peptides, which cannot be accurately determined without the use of a competitor ligand.


Calorimetry Peptide Thermodynamics Albumin Bioinorganic chemistry Copper 



We would like to thank Laurent Paquereau (IPBS, Toulouse) for access to the VP-ITC microcalorimeter.

Supplementary material

775_2011_824_MOESM1_ESM.pdf (722 kb)
Supplementary material (PDF 721 kb)


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Copyright information

© SBIC 2011

Authors and Affiliations

  1. 1.Laboratoire de Chimie de CoordinationCNRSToulouseFrance
  2. 2.UPS, INPT, Laboratoire de Chimie de CoordinationUniversité de ToulouseToulouseFrance
  3. 3.Jacobs University BremenBremenGermany
  4. 4.Institute of Biochemistry and BiophysicsPolish Academy of SciencesWarsawPoland

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