How the tripeptide Gly-His-Lys binds Cu(II): the coordination sphere, conditional stability constants, competing ligands and redox behaviour, and what they mean for handling and analysing GHK-Cu in the laboratory.
GHK is the tripeptide glycyl-L-histidyl-L-lysine. It binds Cu(II) tightly. Most practical questions about GHK-Cu, from buffer choice to what a chromatogram shows, come back to that coordination chemistry.
Data panel
| GHK (free peptide) | GHK-Cu (1:1 complex) | |
|---|---|---|
| Sequence | Gly-His-Lys | Gly-His-Lys with Cu(II) |
| Formula | C₁₄H₂₄N₆O₄ | C₁₄H₂₂CuN₆O₄ (neutral, two protons displaced) |
| Average MW | 340.38 g/mol | 401.91 g/mol |
| Monoisotopic mass | 340.1859 Da | 401.0999 Da (⁶³Cu) |
| CAS | 49557-75-7 | Depends on the form supplied |
Salt forms change the formula weight. As a monoacetate, for example, the complex is C₁₄H₂₂CuN₆O₄·C₂H₄O₂, 461.96 g/mol. Calculate molarity from the confirmed form. (Masses calculated from atomic masses.)
The coordination sphere
In solution at neutral pH, Cu(II) sits in a three-nitrogen chelate: the N-terminal amine of glycine, the deprotonated amide nitrogen of the Gly-His bond and a histidine imidazole nitrogen (Hureau et al., 2011; Trapaidze et al., 2012). The lysine ε-amino group is not a donor; it deprotonates only at high pH, with a pK of 10.77 (Bossak-Ahmad et al., 2020).
The fourth equatorial position is left open to an external ligand (Trapaidze et al., 2012). In the crystal it is filled by a labile carboxylate oxygen and the complex is a dimer; in solution it is a monomer (Hureau et al., 2011).
Complex formation releases protons: about 1.87 per Cu(II) at pH 7.4 (Trapaidze et al., 2012). If the complex is formed in situ from GHK and a Cu(II) salt, those protons go into the buffer, and a weak buffer will drift.
How tightly it binds
| Method | Conditions | Constant |
|---|---|---|
| Isothermal titration calorimetry, glycine as competitor | pH 7.4, 80 mM HEPES, 303 K; corrected for glycine and HEPES competition | Kd = 7.0 ± 1.0 × 10⁻¹⁴ M (Trapaidze et al., 2012) |
| Potentiometry | pH 7.4, equimolar Cu(II) and GHK at 1 µM | cK = 4.17 × 10¹² M⁻¹ (Bossak-Ahmad et al., 2020) |
The ITC value converts to an association constant of about 1.4 × 10¹³ M⁻¹, so the two methods differ by a factor of three to four. Quote the method and conditions with any constant you use.
The chemistry has been mapped for decades. Early potentiometric and visible-absorption work at 25 °C in 0.15 M NaCl found several binary species between pH 3.5 and 10.6, with higher stability constants than Gly-His or Gly-His-Gly (Lau & Sarkar, 1981).
Competing ligands
The open fourth site, and the copper itself, are available to other ligands.
- Imidazole and histidine. Imidazole forms a ternary Cu(GHK)(Im) complex, cK₇.₄ = 725 ± 22 M⁻¹; a second GHK binds only weakly, cK₇.₄ = 237 ± 5 M⁻¹ (Bossak-Ahmad et al., 2020). Free L-histidine forms substantial ternary complexes (Lau & Sarkar, 1981).
- Albumin. In equilibrium dialysis at pH 7.5 and 6 °C, with albumin and GHK equimolar, GHK held about 42% of the Cu(II) (Lau & Sarkar, 1981). Albumin competes for the copper.
- Buffers. Even the ITC constant above had to be corrected for competition from glycine and HEPES (Trapaidze et al., 2012). Ligands of comparable affinity, or weaker ones in large excess, will compete for the copper, so check buffers and media for chelators before attributing an effect to the complex.
Redox behaviour
Cyclic voltammetry and ascorbate oxidation measurements show Cu(II)GHK to be inert at moderate redox potentials. It can be reduced to Cu(I) at about −0.62 V vs Ag/AgCl, after which the copper is released (Hureau et al., 2011).
Glutathione does reduce it, through a transient GSH–Cu(II)GHK intermediate, and ternary ligands tune the rate (Ufnalska et al., 2021). A thiol in the buffer or medium is not a neutral additive.
Analysis
- The peptide. GHK gives [M+H]⁺ at m/z 341.19 (monoisotopic).
- The copper signature. Natural copper is 0.6915 ⁶³Cu and 0.3085 ⁶⁵Cu (CIAAW). Any copper-containing ion shows as a pair of peaks 2 Da apart, the heavier at about 45% of the height of the lighter. For the neutral 1:1 complex, [M+H]⁺ falls at m/z 402.11 (⁶³Cu) and 404.11 (⁶⁵Cu).
- Speciation follows conditions. Cu(II)–GHK speciation changes across the pH range (Lau & Sarkar, 1981), and reversed-phase separations usually run below pH 3 with TFA (Mant et al., 2007). Read an acidic chromatogram as a measure of the peptide, not proof that the complex is intact.
- Retention. Reversed-phase HPLC elutes peptides in order of increasing hydrophobicity (Mant et al., 2007). GHK has no strongly hydrophobic side chain, so expect it early in the gradient.
- Visible absorption. The complex absorbs in the visible region, so its formation can be followed spectrophotometrically, as in the original speciation work (Lau & Sarkar, 1981).
(m/z values calculated from atomic masses.)
Controls that follow from the chemistry
- Run GHK alone at the matched peptide concentration, and a Cu(II) salt alone at the matched copper concentration. Without both, an effect cannot be attributed to the complex rather than to free peptide or free copper.
- Record buffer, pH, and any chelator, thiol or albumin present. Each changes what the copper is bound to.
- Confirm the supplied form before calculating molarity: free complex or salt.
Further reading from our research series
- HPLC Purity vs Net Peptide Content Purity, peptide content and identity: three measurements, three questions.
- Peptide COA Data: Methods and Their Limits A line-by-line read of a peptide certificate, and where each result stops.
- Peptides in Cell-Based Assays: Solubility, Stability and Controls From vial to well: losses, artefacts and the controls that catch them.
References
- Bossak-Ahmad, K., Wiśniewska, M. D., Bal, W., Drew, S. C. & Frączyk, T. (2020). Ternary Cu(II) complex with GHK peptide and cis-urocanic acid as a potential physiologically functional copper chelate. International Journal of Molecular Sciences, 21(17), 6190. https://doi.org/10.3390/ijms21176190
- CIAAW, IUPAC Commission on Isotopic Abundances and Atomic Weights. (n.d.). Copper: standard atomic weight and isotopic composition. https://ciaaw.org/copper.htm
- Hureau, C., Eury, H., Guillot, R., Bijani, C., Sayen, S., Solari, P.-L., Guillon, E., Faller, P. & Dorlet, P. (2011). X-ray and solution structures of CuIIGHK and CuIIDAHK complexes: influence on their redox properties. Chemistry – A European Journal, 17(36), 10151–10160. https://doi.org/10.1002/chem.201100751
- Lau, S. J. & Sarkar, B. (1981). The interaction of copper(II) and glycyl-L-histidyl-L-lysine, a growth-modulating tripeptide from plasma. Biochemical Journal, 199(3), 649–656. https://doi.org/10.1042/bj1990649
- Mant, C. T., Chen, Y., Yan, Z., Popa, T. V., Kovacs, J. M., Mills, J. B., Tripet, B. P. & Hodges, R. S. (2007). HPLC analysis and purification of peptides. In G. B. Fields (Ed.), Peptide Characterization and Application Protocols (Methods in Molecular Biology, vol. 386, pp. 3–55). https://doi.org/10.1007/978-1-59745-430-8_1
- Trapaidze, A., Hureau, C., Bal, W., Winterhalter, M. & Faller, P. (2012). Thermodynamic study of Cu2+ binding to the DAHK and GHK peptides by isothermal titration calorimetry (ITC) with the weaker competitor glycine. Journal of Biological Inorganic Chemistry, 17(1), 37–47. https://doi.org/10.1007/s00775-011-0824-5
- Ufnalska, I., Drew, S. C., Zhukov, I., Szutkowski, K., Wawrzyniak, U. E., Wróblewski, W., Frączyk, T. & Bal, W. (2021). Intermediate Cu(II)-thiolate species in the reduction of Cu(II)GHK by glutathione: a handy chelate for biological Cu(II) reduction. Inorganic Chemistry, 60(23), 18048–18057. https://doi.org/10.1021/acs.inorgchem.1c02669