GHK-Cu

Molecular Identity and Classification

GHK-Cu is the copper(II) complex of the tripeptide glycyl-L-histidyl-L-lysine, written H-Gly-His-Lys-OH or GHK. Unlike most research peptides, it is not a single organic molecule but a metal–ligand assembly: one Cu²⁺ ion held by three nitrogen atoms of the peptide, with a fourth, labile position occupied by water or another weak donor. That makes GHK-Cu as much an object of bioinorganic chemistry as of peptide chemistry, and most of what is distinctive about it, from its colour to its behaviour on an HPLC column, follows from the copper centre.

PubChem records the complex as compound 71587328, with the formula C₁₄H₂₃CuN₆O₄, a formal charge of +1, an average molecular weight of 402.92 g/mol and a monoisotopic mass of 402.1077 Da. The CAS registry number of the copper complex is 89030-95-5. The metal-free peptide is a separate entry: PubChem compound 73587, CAS 49557-75-7, C₁₄H₂₄N₆O₄, 340.38 g/mol, IUPAC name (2S)-6-amino-2-[[(2S)-2-[(2-aminoacetyl)amino]-3-(1H-imidazol-5-yl)propanoyl]amino]hexanoic acid. Comparing the two formulas shows the chemistry of complex formation directly: the complex has one hydrogen fewer than the free peptide plus one copper atom (340.38 − 1.008 + 63.546 = 402.92 g/mol), because binding Cu²⁺ displaces the proton of the Gly–His amide nitrogen.

The +1 charge describes the species that dominates at neutral pH. Counting charges: Cu²⁺ contributes +2, the deprotonated amide −1, the C-terminal carboxylate −1 and the protonated lysine ε-amine +1, for a net +1. Any isolated solid therefore also contains a counterion (commonly acetate or chloride, or trifluoroacetate from purification), which the formula and molecular weight of the cation alone do not include. The CAS index name, [N²-(N-glycyl-L-histidyl)-L-lysinato(2−)]copper, describes the formally neutral complex in which the lysine amine is also deprotonated (C₁₄H₂₂CuN₆O₄, 401.91 g/mol), a form relevant only at high pH. This bookkeeping often explains molecular weights that differ between papers.

GHK carries the N-terminal "Xaa-His" copper-binding motif (histidine at position 2). It is distinct from the ATCUN motif Xaa-Xaa-His of the Asp-Ala-His-Lys (DAHK) N-terminus of human serum albumin; three nitrogen donors versus four explains most of the contrasts between them.

Structure and Chemical Features

Each residue of GHK plays a defined part in metal binding. Glycine, the N-terminal residue, has no side chain; its free α-amine is the first copper ligand. Histidine supplies two ligands: the nitrogen of the Gly–His peptide bond, which loses its proton when it binds the metal, and the imidazole nitrogen nearest the backbone (N^π, also written N-δ1). Lysine is not bound to copper in the principal solution species, but its positively charged ε-ammonium group and its C-terminal carboxylate shape the outer coordination sphere, the charge of the complex and its interactions with other molecules. Lau and Sarkar showed by potentiometry that the ε-amino group measurably enhances the stability constants of the copper complexes compared with Gly-His and Gly-His-Gly.

The three nitrogen donors form two fused chelate rings around the copper: a five-membered ring from the glycine amine and the deprotonated amide nitrogen, and a six-membered ring from the amide nitrogen and the imidazole N^π. Together they occupy three corners of a roughly square-planar equatorial plane. The fourth equatorial position is open to a weak oxygen donor, typically water in dilute solution, and one or two further molecules can bind more weakly along the axis perpendicular to the plane, the elongated geometry expected for a d⁹ Cu(II) ion.

Solid State Versus Solution

The structure of GHK-Cu has been examined in both phases, and they differ. X-ray crystallography at 1.1 Å resolution by Perkins and colleagues (1984), following an earlier structure reported by Pickart and colleagues (1980), showed that in the crystal the open equatorial site is filled by a carboxylate oxygen from a neighbouring complex, so the solid is an extended assembly of oxygen-bridged copper units rather than isolated molecules. Freedman and colleagues (1982) used optical, electron paramagnetic resonance (EPR) and electron spin-echo envelope spectroscopy to show that this bridged structure does not persist in solution: at neutral pH GHK forms a mononuclear 1:1 Cu(II) complex with two or three equatorial nitrogen donors, one of them demonstrably the histidine imidazole. Hureau and colleagues (2011) confirmed the picture with X-ray, EPR, HYSCORE, X-ray absorption and NMR data: [Cu(GHK)] is dimeric in the crystal and monomeric in solution, with the equatorial set NH₂, amidyl N and imidazole N, and a labile fourth site.

pH-Dependent Speciation

Because copper binding is coupled to deprotonation, the species present depends on pH. In the spectrophotometric titration of Freedman and colleagues, three transitions appeared with apparent pK values of 3.6, 9.2 and 11.4, involving two, two and one protons respectively. The transition at pK 3.6 is complex formation itself: below about pH 3.5 the copper is largely released, and above it the 3N complex that exists at physiological pH forms. At higher pH the copper site rearranges while the imidazole stays bound. Lau and Sarkar, working by potentiometry and visible spectroscopy in 0.15 M NaCl at 25 °C over pH 3.5 to 10.6, likewise found multiple binary species and, in the presence of L-histidine, extensive ternary complexes in which a second ligand occupies the open coordination site. The practical consequence for laboratories is that the complex is stable in neutral buffers but dissociates in acidic solutions, including the mobile phases used for reversed-phase HPLC.

With excess peptide a bis complex, [Cu(GHK)₂], forms; Hureau and colleagues attributed GHK's fast copper exchange to it, whereas DAHK, with its fourth site filled by a second amide nitrogen, exchanges copper very slowly.

Synthesis and Manufacturing Chemistry

GHK-Cu is made in two stages: synthesis of the free tripeptide, then formation of the copper complex. The peptide is assembled by solid-phase peptide synthesis (SPPS), introduced by Merrifield in 1963, in which the chain grows one residue at a time from the C-terminus on an insoluble resin. For the C-terminal free acid, lysine is anchored first to an acid-labile resin (for example 2-chlorotrityl chloride or Wang resin), then histidine and glycine are coupled in turn.

Current practice uses Fmoc chemistry, based on the 9-fluorenylmethoxycarbonyl group described by Carpino and Han in 1972. The building blocks are typically Fmoc-Lys(Boc)-OH, Fmoc-His(Trt)-OH and Fmoc-Gly-OH (or Boc-Gly-OH for the final residue), with the Fmoc group removed by 20% piperidine in dimethylformamide between couplings. Histidine is the residue that demands care in this sequence: its imidazole can catalyse epimerization at the α-carbon during carboxyl activation, so His couplings are run with low-racemization activators such as DIC/Oxyma, short pre-activation and minimal base, and the trityl (Trt) group on the imidazole further limits side reactions. Chiral analysis of the final product checks that the D-His epimer is absent. A single cleavage with trifluoroacetic acid (TFA) and scavengers such as water and triisopropylsilane releases the peptide and removes the Boc and Trt groups; the crude product is purified by preparative reversed-phase HPLC and lyophilized.

Complexation is a simple aqueous reaction but its stoichiometry matters. The purified peptide is dissolved in water and combined with a copper(II) salt such as copper(II) chloride or copper(II) acetate at a 1:1 molar ratio, and the pH is brought to the near-neutral range in which the 3N complex forms (above the pK of 3.6 noted above). Two errors are avoided. Excess copper precipitates as copper(II) hydroxide or basic salts as the pH rises and leaves unbound Cu²⁺ in the material; excess peptide produces a mixture of the 1:1 complex, the bis complex [Cu(GHK)₂] and free GHK. The solution is then filtered and lyophilized. If the peptide was purified as its trifluoroacetate salt, the counterion carries through unless it is exchanged for acetate or chloride, which is why the counterion stated on the certificate of analysis is part of the identity of a given lot.

Analytical Characterization

A metal–peptide complex poses two analytical questions: is the peptide correct, and is the copper present in the right amount and bound the right way?

Reversed-phase HPLC sees the peptide, not the complex. This is the most important analytical point about GHK-Cu. Conventional peptide HPLC uses water–acetonitrile gradients containing 0.1% TFA or formic acid, at a pH near 2. That is well below the pK of 3.6 for copper binding, so the complex dissociates on the column: GHK elutes as the free, protonated peptide and Cu²⁺ passes through near the void volume. The HPLC purity on a certificate of analysis is therefore the purity of the GHK peptide component. Detection is at 210–220 nm, where the amide bonds and the histidine imidazole absorb; GHK contains no tryptophan, tyrosine or phenylalanine, so absorbance at 280 nm is negligible. As a short, highly polar and basic peptide (calculated XLogP −4.4 for free GHK), it is weakly retained on C18 columns and is typically analysed with a shallow gradient from very low organic content, an ion-pairing acid, or a polar-embedded or HILIC column.

Mass spectrometry. Under the acidic conditions of LC-MS the free peptide dominates, and positive electrospray gives [M+H]⁺ at m/z 341.193, [M+2H]²⁺ at m/z 171.100 and, with three basic sites (N-terminal amine, imidazole and lysine), [M+3H]³⁺ at m/z 114.403. Direct infusion at near-neutral pH, for example from ammonium acetate, can preserve the complex, which already carries a +1 charge and so appears as the intact cation at m/z 402.107 (with ⁶³Cu) rather than as a protonated adduct; additional protonation gives [M+H]²⁺ at m/z 201.557. Copper's isotopes give the complex an unmistakable signature: ⁶³Cu (69.15%) and ⁶⁵Cu (30.85%) produce a pair of peaks at m/z 402.107 and 404.105 in a ratio of about 100:45, plus a small contribution from ¹³C. Tandem MS of the free peptide yields b₂ (Gly-His) at m/z 195.088, y₁ (Lys) at 147.113, y₂ (His-Lys) at 284.172 and the histidine immonium ion at 110.071.

Copper content. Because HPLC is blind to the metal, copper is quantified independently, most often by inductively coupled plasma optical emission or mass spectrometry (ICP-OES, ICP-MS) or by atomic absorption spectroscopy after acid digestion. The expected copper mass fraction is a useful worked check. For the cation alone it is 63.546 / 402.92 = 15.8%. With one acetate counterion (C₁₄H₂₃CuN₆O₄·C₂H₃O₂, 461.97 g/mol) it falls to 13.8%; with chloride (438.37 g/mol) it is 14.5%; with trifluoroacetate (515.94 g/mol) 12.3%. Bound water lowers each of these further. A measured copper fraction well above the value expected for the stated salt indicates unbound copper; a value well below indicates free peptide or the bis complex, whose copper fraction is roughly half that of the 1:1 complex.

Spectroscopic fingerprints of the copper site. UV–visible spectroscopy shows a broad, weak d–d band in the visible region near 600 nm, characteristic of Cu(II) with three equatorial nitrogen donors and responsible for the blue colour; its position shifts with pH and with ternary ligands, which is how Lau and Sarkar and Freedman and colleagues followed speciation. X-band EPR gives an axial Cu(II) spectrum with g_∥ > g_⊥ and resolved copper hyperfine splitting, and the parameters distinguish a 3N site from the 4N site of ATCUN complexes. Paramagnetic Cu(II) broadens nearby NMR signals, so peptide NMR is run on the metal-free form.

Purity versus content. HPLC purity (the fraction of UV-absorbing material that is GHK) is not peptide content: the weighed powder also contains copper, counterion and water, so molar amounts are calculated from the salt's formula weight or a measured copper content.

Physicochemical Properties

The dissociation constant deserves a word of interpretation. A conditional K_d of 7.0 × 10⁻¹⁴ M at pH 7.4, measured by Trapaidze and colleagues with glycine as a weaker competing ligand in isothermal titration calorimetry, places GHK among the strongest small-peptide Cu(II) chelators, close to the albumin-derived DAHK motif (2.6 × 10⁻¹⁴ M under the same conditions). "Conditional" means the value applies to the stated pH and medium, corrected for competitor and buffer; it falls steeply in acidic solution, as the pK of 3.6 for complex formation implies. The same study found distinct enthalpic and entropic contributions for the two peptides, with an enthalpic contribution for GHK.

Thermodynamic strength is not kinetic inertness: GHK-Cu binds copper about as tightly as DAHK yet exchanges it quickly through its open fourth site. Redox behaviour follows the same structure: Hureau and colleagues found both complexes inert at moderate potentials and in ascorbate-oxidation assays, but only [Cu(GHK)] could be reduced to Cu(I), around −0.62 V versus Ag/AgCl, after which the copper is released, since the 3N peptide site does not accommodate the preferred geometry of Cu(I).

Research Context

Research on GHK-Cu joins bioinorganic chemistry (how a short peptide binds, buffers and hands over copper) with cell biology (how cultured cells respond to the complex, the free peptide or related sequences), around one recurring question: is an effect seen in culture caused by the peptide, by its copper, or by the complex as a unit?

Copper Exchange and Cellular Uptake

Pickart and colleagues (1980) noted that GHK co-isolated with roughly equimolar copper during its purification from plasma, and that its histidyl-lysyl arrangement resembles the copper-transport sites of albumin and α-fetoprotein, where Cu(II) binds a histidine next to a basic residue. They showed that the tripeptide readily forms Cu(II) complexes and increases copper uptake into cultured hepatoma (HTC4) cells, and that several tripeptides with a His-Lys linkage were nearly as active as GHK in their assays. Lau and Sarkar quantified the competition with albumin by equilibrium dialysis at pH 7.5 and 6 °C: at equimolar albumin and peptide, about 42% of the Cu(II) was bound to the peptide, and with Cu(II), albumin, L-histidine and peptide at concentrations chosen to model those of plasma, about 6% of the copper was associated with the low-molecular-weight ligands, as binary or ternary complexes.

Extracellular Matrix Synthesis in Fibroblast Cultures

The best-known cell-culture finding came from Maquart and colleagues (1988), who reported that GHK-Cu increased collagen synthesis by fibroblasts in culture. The effect began between 10⁻¹² and 10⁻¹¹ M, was maximal at 10⁻⁹ M and did not depend on a change in cell number, indicating a change in synthetic activity per cell rather than proliferation. They also pointed out that the Gly-His-Lys triplet occurs in the α2(I) chain of type I collagen, so proteolysis of matrix proteins is a possible source of the free tripeptide. In a follow-up, Wegrowski, Maquart and Borel (1992) labelled cultures of human fibroblasts with [³H]glucosamine and [³⁵S]sulfate and found that GHK-Cu increased the synthesis of glycosaminoglycans both secreted into the medium and associated with the cell layer. The response was biphasic, maximal at 10⁻⁹ to 10⁻⁸ M and returning towards control values at higher concentrations; dermatan sulfate in the medium and heparan sulfate in the cell layer were preferentially increased, while hyaluronic acid synthesis was unchanged.

In human keratinocyte cultures, Kang and colleagues (2009) reported that copper–GHK increased proliferation of monolayer cells and, in three-dimensional organotypic epidermal equivalents, increased staining for proliferating cell nuclear antigen (PCNA) and the transcription factor p63 and raised the expression of integrins α6 and β1, which they confirmed by Western blotting. Lane and colleagues (1994) approached GHK from another direction: proteolysis of the matrix protein SPARC releases peptides containing the sequence (K)GHK, from a cationic region spanning residues 113–130. SPARC and peptides from that region stimulated the formation of endothelial cords in vitro, and both SPARC 113–130 and KGHK bound Cu²⁺ with high affinity; a peptide from a second cationic region (SPARC 54–73) also bound Cu²⁺. Copper affinity and activity in a given assay need not be the same thing.

Two features run through this literature and shape experimental design. First, the active concentrations are low, typically picomolar to nanomolar, and responses are often biphasic, so a wide concentration series (for example 10⁻¹² to 10⁻⁶ M in half-log or log steps) is more informative than a single concentration. Second, culture media are not neutral towards copper: they contain amino acids such as histidine and often serum albumin, both of which form ternary complexes or compete for Cu(II). Well-controlled studies therefore include the metal-free peptide, a copper salt at the same copper concentration, and the complex side by side, and they report the medium composition and serum content, because these determine what species the cells actually encounter.

Discovery History

GHK entered the literature as an unidentified activity. In 1973 Pickart, Thayer and Thaler described a synthetic tripeptide, modelled on a growth-modulating factor from plasma, that increased the survival of normal liver cells and stimulated growth of hepatoma cells in culture. In 1977 Schlesinger, Pickart and Thaler reported that the growth-modulating serum tripeptide is glycyl-histidyl-lysine, settling its sequence, and in 1979 Pickart and Thaler related its molecular structure to DNA synthesis in hepatoma cells. The 1980 Nature paper connected the activity to copper, and the solution (1981, 1982) and crystal (1984) studies above followed. The modern picture of a labile 3N complex comes from Hureau (2011) and Trapaidze (2012).

GHK-Cu Compared With the Albumin DAHK Site

The albumin N-terminal DAHK sequence is the natural benchmark for GHK; the comparison shows what one extra amide nitrogen does to a copper site.

Related Compounds

The free peptide GHK, the SPARC-derived KGHK, and Gly-His and Gly-His-Gly (Lau and Sarkar's comparison ligands) are the reference compounds for separating the contributions of the peptide, the copper site and the lysine side chain. GHK-Cu is also one of the components of the multi-peptide research blend GLOW (BPC-157, TB-500 and GHK-Cu); in such a mixture, the copper isotope pattern and early elution of free GHK help locate its component. Among other short peptides with well-defined chemistry, the tripeptide KPV offers an instructive contrast: similar size and polarity, but no metal-binding motif.

Laboratory Handling and Storage

Storage of the solid. Lyophilized GHK-Cu is best kept tightly closed at −20 °C, protected from light and moisture. The powder is hygroscopic, so a vial taken from cold storage should reach room temperature in a desiccator before it is opened, to stop moisture condensing on it and adding to the weighed mass.

Preparing solutions. GHK-Cu dissolves readily in high-purity water to a clear blue solution. For molar concentrations, use the formula weight of the salt form stated on the certificate of analysis (for example 461.97 g/mol for the acetate) rather than 402.92 g/mol, or calculate from a measured copper content. As a worked example, 4.62 mg of the acetate salt dissolved to 1.00 mL gives a 10.0 mM stock, before any correction for water content. Buffers near neutral pH preserve the complex; acidic solutions below about pH 4 release copper. Stocks are usually divided into single-use aliquots and frozen at −20 °C or −80 °C, avoiding repeated freeze–thaw cycles, and filtered through a 0.22 µm membrane when destined for cell culture.

Chemical compatibility. Strong chelators such as EDTA or phenanthroline derivatives strip copper from GHK, thiol reductants (dithiothreitol, glutathione) can reduce Cu(II) and release it, and high concentrations of histidine, glycine or albumin compete for the metal. Keep these out of buffers unless they are part of the experiment.

Working with small amounts. At picomolar to nanomolar concentrations, peptide adsorption to plastic and trace copper in water and media become significant relative to the amount added; low-binding polypropylene and metal-free water help. For a trifluoroacetate salt, a counterion-matched vehicle control is worthwhile, since Cornish and colleagues showed that trifluoroacetate alone can influence cell proliferation in culture.

Verification. A shift of a solution from blue towards green, or loss of colour, suggests a change in coordination or loss of complex. LC-MS of a stock should show free GHK at m/z 341.19 under acidic conditions; near-neutral infusion shows the intact complex at m/z 402.11 with its 404.10 copper isotope partner. Each lot's certificate of analysis, linked on this page, records the analytical results for that lot.

Frequently Asked Research Questions

What is GHK-Cu?

GHK-Cu is the copper(II) complex of the tripeptide glycyl-L-histidyl-L-lysine (H-Gly-His-Lys-OH). In solution at neutral pH it is a 1:1 complex, [Cu(GHK−H)]⁺, with the formula C₁₄H₂₃CuN₆O₄ and an average molecular weight of 402.92 g/mol.

What is the CAS number of GHK-Cu?

The copper complex is registered under CAS 89030-95-5 (PubChem CID 71587328). The metal-free peptide GHK has CAS 49557-75-7 (PubChem CID 73587).

How is copper bound in GHK-Cu?

Cu(II) is held in a square-planar arrangement by three nitrogen atoms: the N-terminal glycine amine, the deprotonated nitrogen of the Gly–His amide bond and the histidine imidazole nitrogen. The fourth equatorial position is labile and is occupied by water or another weak ligand in solution, and by a carboxylate oxygen of a neighbouring complex in the crystal.

Why is the molecular weight of GHK-Cu given as 402.92 when the free peptide is 340.38?

Complex formation adds one copper atom (63.546) and removes one proton from the amide nitrogen (1.008): 340.38 + 63.546 − 1.008 = 402.92 g/mol. This is the cation; a solid sample also contains a counterion such as acetate (giving 461.97 g/mol) or chloride (438.37 g/mol).

How tightly does GHK bind copper?

Isothermal titration calorimetry with glycine as a competitor gave a conditional dissociation constant of 7.0 ± 1.0 × 10⁻¹⁴ M at pH 7.4 (Trapaidze and colleagues, 2012), close to the 2.6 × 10⁻¹⁴ M measured for the albumin DAHK motif under the same conditions.

Why does HPLC of GHK-Cu show only the free peptide?

Reversed-phase HPLC mobile phases containing 0.1% TFA or formic acid have a pH near 2, below the pK of 3.6 for copper binding, so the complex dissociates on the column and GHK elutes as the free peptide. HPLC purity therefore describes the peptide; copper content is measured separately, for example by ICP-OES, ICP-MS or atomic absorption.

What mass should I see for GHK-Cu by mass spectrometry?

Under acidic LC-MS conditions free GHK gives [M+H]⁺ at m/z 341.193 and [M+2H]²⁺ at 171.100. When the complex is preserved, the intact cation appears at m/z 402.107 (⁶³Cu) with a ⁶⁵Cu partner at 404.105 at about 45% relative intensity.

Why is GHK-Cu blue?

The colour comes from a weak d–d electronic transition of the Cu(II) ion in its nitrogen-rich coordination environment, absorbing broadly near 600 nm in the orange-red region, so the transmitted light appears blue.

Is the copper in GHK-Cu redox active?

Hureau and colleagues (2011) found the complex inert under moderate redox conditions, including ascorbate oxidation assays. It can be reduced electrochemically to Cu(I) near −0.62 V versus Ag/AgCl, after which the copper is released from the peptide.

Which in vitro read-outs have been reported for GHK-Cu?

Reported read-outs include copper uptake into cultured hepatoma cells, collagen synthesis by fibroblasts (maximal near 10⁻⁹ M), radiolabelled glycosaminoglycan synthesis by human fibroblasts (biphasic, maximal at 10⁻⁹ to 10⁻⁸ M), keratinocyte proliferation with PCNA, p63 and integrin α6/β1 markers, and endothelial cord formation for SPARC-derived peptides (residues 113–130) containing the (K)GHK sequence.

How should GHK-Cu be stored?

As a lyophilized solid at −20 °C, tightly closed, protected from light and moisture, and warmed to room temperature in a desiccator before opening. Solutions are kept near neutral pH, free of chelators and thiol reductants, in single-use frozen aliquots.

Sources & Further Reading

1. National Center for Biotechnology Information. PubChem Compound Summary for CID 71587328, glycyl-L-histidyl-L-lysine copper complex (GHK-Cu). PubChem CID 71587328.
2. National Center for Biotechnology Information. PubChem Compound Summary for CID 73587, Glycyl-L-histidyl-L-lysine. PubChem CID 73587.
3. Pickart L, Thayer L, Thaler MM. A synthetic tripeptide which increases survival of normal liver cells, and stimulates growth in hepatoma cells. Biochemical and Biophysical Research Communications 1973;54(2):562–566. doi:10.1016/0006-291X(73)91459-9
4. Schlesinger DH, Pickart L, Thaler MM. Growth-modulating serum tripeptide is glycyl-histidyl-lysine. Experientia 1977;33(3):324–325. doi:10.1007/BF02002806
5. Pickart L, Thaler MM. Growth-modulating human plasma tripeptide: relationship between molecular structure and DNA synthesis in hepatoma cells. FEBS Letters 1979;104(1):119–122. doi:10.1016/0014-5793(79)81096-0
6. Pickart L, Freedman JH, Loker WJ, Peisach J, Perkins CM, Stenkamp RE, Weinstein B. Growth-modulating plasma tripeptide may function by facilitating copper uptake into cells. Nature 1980;288(5792):715–717. doi:10.1038/288715a0
7. Lau SJ, Sarkar B. The interaction of copper(II) and glycyl-L-histidyl-L-lysine, a growth-modulating tripeptide from plasma. Biochemical Journal 1981;199(3):649–656. doi:10.1042/bj1990649
8. Freedman JH, Pickart L, Weinstein B, Mims WB, Peisach J. Structure of the glycyl-L-histidyl-L-lysine–copper(II) complex in solution. Biochemistry 1982;21(19):4540–4544. doi:10.1021/bi00262a004
9. Perkins CM, Rose NJ, Weinstein B, Stenkamp RE, Jensen LH, Pickart L. The structure of a copper complex of the growth factor glycyl-L-histidyl-L-lysine at 1.1 Å resolution. Inorganica Chimica Acta 1984;82(1):93–99. doi:10.1016/S0020-1693(00)82544-X
10. Maquart FX, Pickart L, Laurent M, Gillery P, Monboisse JC, Borel JP. Stimulation of collagen synthesis in fibroblast cultures by the tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu²⁺. FEBS Letters 1988;238(2):343–346. doi:10.1016/0014-5793(88)80509-X
11. Wegrowski Y, Maquart FX, Borel JP. Stimulation of sulfated glycosaminoglycan synthesis by the tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu²⁺. Life Sciences 1992;51(13):1049–1056. doi:10.1016/0024-3205(92)90504-I
12. Lane TF, Iruela-Arispe ML, Johnson RS, Sage EH. SPARC is a source of copper-binding peptides that stimulate angiogenesis. The Journal of Cell Biology 1994;125(4):929–943. doi:10.1083/jcb.125.4.929
13. Kang YA, Choi HR, Na JI, et al. Copper–GHK increases integrin expression and p63 positivity by keratinocytes. Archives of Dermatological Research 2009;301(4):301–306. doi:10.1007/s00403-009-0942-x
14. Hureau C, Eury H, Guillot R, et al. X-ray and solution structures of Cu^IIGHK and Cu^IIDAHK complexes: influence on their redox properties. Chemistry – A European Journal 2011;17(36):10151–10160. doi:10.1002/chem.201100751
15. Trapaidze A, Hureau C, Bal W, Winterhalter M, Faller P. 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 2012;17(1):37–47. doi:10.1007/s00775-011-0824-5
16. Merrifield RB. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. Journal of the American Chemical Society 1963;85(14):2149–2154. doi:10.1021/ja00897a025
17. Carpino LA, Han GY. 9-Fluorenylmethoxycarbonyl amino-protecting group. The Journal of Organic Chemistry 1972;37(22):3404–3409. doi:10.1021/jo00795a005
18. Cornish J, Callon KE, Lin CQ, et al. Trifluoroacetate, a contaminant in purified proteins, inhibits proliferation of osteoblasts and chondrocytes. American Journal of Physiology-Endocrinology and Metabolism 1999;277(5):E779–E783. doi:10.1152/ajpendo.1999.277.5.E779

Last reviewed: October 22, 2026