What Is GHK-Cu? Chemistry of the GHK Copper Peptide

GHK-Cu is the copper(II) complex of the tripeptide glycyl-L-histidyl-L-lysine (Gly-His-Lys, or GHK). The copper ion is held by three nitrogen atoms of the peptide: the N-terminal amine, the deprotonated amide nitrogen between glycine and histidine, and a nitrogen of the histidine imidazole ring. The neutral 1:1 complex has the formula C14H22CuN6O4, a molecular weight of 401.91 g/mol and the CAS number 89030-95-5, and its coordination chemistry has been characterised in detail by crystallography and spectroscopy.

This page explains what the term "copper peptide" means at the molecular level, how GHK binds copper(II), why the complex is blue, and how a GHK-Cu sample is characterised.

GHK and GHK-Cu: the peptide and the complex

GHK is a tripeptide in which glycine, histidine and lysine are joined by two peptide bonds. Its N-terminus carries a free amine and its C-terminus a free carboxyl group, and both chiral residues are L-amino acids. On its own it is a colourless, metal-free peptide. GHK-Cu forms when that peptide binds a copper(II) ion, one ion per peptide.

The complex's formula has two fewer hydrogen atoms than the peptide's. In the neutral complex the peptide carries two negative charges, one from the carboxylate and one from the amide nitrogen that loses its proton on binding copper, and these balance the Cu2+ ion to give a neutral complex.

Why the formula is written in more than one way

In PubChem the CAS number 89030-95-5 is attached to several records of the same complex that differ in charge and protonation state, and to a 2:1 bis-complex, Cu(GHK)2. At neutral pH the lysine side-chain amine is normally protonated, so a solid GHK-Cu material may be isolated as a salt with a counter-ion such as acetate or chloride. Its gross formula and weight then differ from the registry value. A certificate should say which form the batch is, because every mass-based figure, from molar quantities to copper content, depends on it. For the neutral complex, copper accounts for 15.8% of the mass and one mole of complex corresponds to one mole of GHK. A salt form carries extra counter-ion mass, so the copper fraction of the supplied solid is correspondingly lower. The same principle applies to any peptide salt, as explained in peptide purity versus net peptide content.

How GHK binds copper(II)

The coordination chemistry of GHK-Cu has been worked out by X-ray crystallography, EPR (electron paramagnetic resonance) and NMR spectroscopy, optical spectroscopy and calorimetry. The main findings are consistent across these methods.

• Stoichiometry. At neutral pH GHK forms a mononuclear 1:1 complex with copper(II) in solution (Freedman et al., 1982; Trapaidze et al., 2012).
• Solid state versus solution. In crystals the fourth equatorial site is taken by a carboxylate oxygen from a neighbouring complex, giving oxygen-bridged dimeric or polymeric arrangements. In solution the complex is monomeric, with the three nitrogen donors and a labile fourth ligand (Hureau et al., 2011).
• pH dependence. Optical measurements across the pH range show transitions with apparent pK values of 3.6, 9.2 and 11.4. The complex present at neutral pH forms at the lowest of these, with release of two protons (Freedman et al., 1982).
• Affinity. Calorimetry gives a conditional dissociation constant of 7.0 ± 1.0 × 10⁻¹⁴ M at pH 7.4, so GHK binds copper(II) very tightly (Trapaidze et al., 2012).
• Redox behaviour. The complex is inert at moderate potentials. It can be reduced to copper(I) at around −0.62 V versus Ag/AgCl, and the copper ion is then released (Hureau et al., 2011).

The open fourth position is the defining feature. It lets GHK-Cu form ternary complexes with small ligands such as glycine or histidine, and copper exchanges quickly between GHK molecules, an exchange that has been attributed to the presence of a Cu(GHK)2 complex.

The pH dependence also matters whenever GHK-Cu is analysed. Because the complex forms above about pH 3.6, the pH of the analytical medium decides whether the copper stays bound: in the acidic eluents of reversed-phase HPLC it is expected to be released, while near-neutral buffers used for spectroscopy keep it in place. Chelating components compete for the metal as well. The calorimetry study corrected its binding data for the contributions of glycine and the buffer, and the crystallography work notes that the GHK tag cannot be used with chelators such as imidazole or EDTA in the buffer.

Copper peptides: where GHK-Cu fits

"Copper peptide" is a broad label for short peptides that bind copper through backbone and side-chain donor atoms. How tightly and in what geometry they do so depends strongly on where histidine sits relative to the free N-terminus. GHK is often compared with DAHK (Asp-Ala-His-Lys), which has histidine at position 3 and belongs to the ATCUN family of amino-terminal copper- and nickel-binding motifs.

The same chemistry has found a use in structural biology. Mehr et al. (2020) attached the GHK sequence to the N-terminus of proteins as a crystallisation and phasing tag: residues from neighbouring molecules in the crystal completed the copper coordination sphere, and the anomalous scattering of the bound copper was used to solve the structures. That work gives a clear picture of the open coordination positions described above.

Why GHK-Cu is blue

Copper(II) has a d⁹ electron configuration, and its partly filled d orbitals absorb visible light through d–d transitions. For GHK-Cu the absorption band is centred in the orange part of the spectrum, so the complex appears blue, while the free GHK peptide is colourless. The colour is only a first, qualitative sign that copper(II) is present. EPR spectroscopy gives a firmer fingerprint: copper(II) is paramagnetic, and its EPR spectrum reflects the nitrogen donors in the equatorial plane (Freedman et al., 1982).

How GHK-Cu is analysed

Because GHK-Cu is a peptide and a metal complex at once, a full characterisation combines peptide methods with element-specific ones.

Copper has two stable isotopes, 63Cu and 65Cu, with natural abundances of 69.15% and 30.85%. Any copper-containing ion therefore appears as a pair of peaks 2 mass units apart, the heavier roughly 45% as intense as the lighter. That pattern distinguishes the complex from the free peptide at a glance. Two cautions apply to electrospray spectra of the complex. Copper(II) can be partly reduced to copper(I) in the ion source, which gives a companion ion, [Cu(GHK)]+, one mass unit higher (m/z 403.115 for 63Cu). And some of the complex can dissociate during ionisation, so the size of the free-peptide signal does not show how much peptide was unbound in the sample. More on reading such spectra is in mass spectrometry for peptide identity, and on chromatographic purity in HPLC peptide purity.

Certificates, specification and storage

For our GHK-Cu, the purity specification is ≥99% (HPLC, supplier specification). Certificate status is shown on every product page, and certificates of analysis are published in the COA Library where available. When reading one, check the batch number and analysis date against the vial, the identity data against the masses above, and whether the form of the material (neutral complex or a named salt) is stated.

GHK-Cu is supplied as a lyophilised solid in a sealed vial. Store the sealed vial at 2–8 °C for short-term storage, or at −20 °C and below for long-term storage, protect it from light and avoid repeated freeze-thaw cycles.

GHK-Cu sits in the Melanocortin & Copper Peptides research area, which groups two structurally distinct families of short peptides. Terms such as monoisotopic mass and chelate are defined in the glossary.