⚠️ For In-Vitro Laboratory Research Use Only. This document serves as an analytical reference guide compiled for qualified biochemical researchers. It is not intended for diagnostic procedures, clinical trials, or human administration.
Chemical Profile & Coordination Chemistry
GHK-Cu (glycyl-L-histidyl-L-lysine:copper(II) complex) is a naturally occurring metallopeptide comprising the carrier tripeptide Gly-His-Lys coordinated with a divalent cupric ion (Cu²⁺) in a 1:1 molar ratio. Discovered in 1973 by Dr. Loren Pickart in human plasma fraction IV, GHK-Cu exhibits high affinity for Cu²⁺, serving as a classical metallopeptide model.
In human physiology, native plasma concentrations of GHK-Cu decline significantly with age: healthy adults (age ~20) average ~200 ng/mL (0.5 µM), declining to ~80 ng/mL (0.2 µM) by age 60. In cellular and molecular biology research, GHK-Cu is investigated for:
- Tissue Remodeling: Upregulating pro-collagen and elastin expression while modulating matrix metalloproteinases (MMPs) and TIMPs.
- Anti-Inflammatory Signaling: Downregulating TNF-α and IL-6 while suppressing NF-κB nuclear translocation.
- Antioxidant Defense Research: Inducing protective antioxidant enzymes, including copper-zinc superoxide dismutase (SOD-1) and catalase.
| Chemical Property | Parameter / Value |
|---|---|
| Systematic Name | Glycyl-L-histidyl-L-lysine:copper(II) complex |
| CAS Registry Number | 49557-75-7 |
| Molecular Weight (with Cu) | 403.93 Da |
| Molecular Weight (without Cu) | 340.38 Da |
| Sequence | Gly-His-Lys (tripeptide) |
| Category | Copper Peptides |
| Coordination Ratio | 1:1 molar complex (GHK : Cu²⁺) |
| Physical Appearance | Deep royal blue crystalline powder |
Critical Chelation Chemistry & Handling Precautions
Because GHK-Cu is an organometallic coordination complex, laboratory handling requires specific chemical safeguards:
- Avoid Competitive Chelators (EDTA, EGTA): The stability constant of GHK-Cu is log K ≈ 16.4. Strong chelators like EDTA (log K ≈ 18.8), EGTA, and citrate strip Cu²⁺ from histidine, converting GHK-Cu into unchelated GHK base and free metal chelates. Never add EDTA to cell harvesting, lysis, or assay buffers.
- pH Sensitivity: The complex is stable between pH 6.0 and 7.5. In acidic media (pH < 5.0), histidine protonation (pKa ≈ 6.0) severs coordinate bonds, releasing free Cu²⁺. In alkaline solutions (pH > 8.0), insoluble cupric hydroxide precipitates.
- Redox Sensitivity: Strong reducing agents (DTT, β-mercaptoethanol, ascorbate) reduce Cu²⁺ to Cu⁺, initiating Fenton reactions that induce oxidative peptide cleavage.
- Visual Verification: Coordinated Cu²⁺ exhibits an absorbance band at 600–650 nm, creating an intense royal blue solution. A colorless or cloudy solution indicates complex dissociation.
Solvent Compatibility & Stability
| Solvent Vehicle | Cell Culture Use | Preservation | Shelf Life (4°C) | Key Laboratory Considerations |
|---|---|---|---|---|
| Bacteriostatic Water (0.9% Benzyl Alcohol) | Unsuitable (cytotoxic) | High | 28–35 days | Recommended for analytical standards, HPLC calibration, and non-cellular assays. |
| Sterile Water (SWFI) | High (in media) | None | 7–14 days | Standard choice for in-vitro cell assays; eliminates ion interference. |
| 0.9% Normal Saline | High (isotonic) | None | 7–14 days | Maintains physiological osmolarity (308 mOsm/L) for explant assays. |
| PBS (pH 7.4, EDTA-free) | High | None | 7–10 days | Buffers against pH drift; ensure no chelating preservatives are present. |
Master Stock Reconstitution Tables
GHK-Cu dissolves readily in water (>50 mg/mL). Commercial research vials commonly contain 10 mg, 50 mg, or 100 mg lyophilized cake.
| Vial Mass | Solvent Volume | Final Concentration (mg/mL) | Final Concentration (mcg/mL) | Molarity (mM) |
|---|---|---|---|---|
| 10 mg | 1.0 mL | 10.0 mg/mL | 10,000 mcg/mL | 24.76 mM |
| 10 mg | 2.0 mL | 5.0 mg/mL | 5,000 mcg/mL | 12.38 mM |
| 50 mg | 2.5 mL | 20.0 mg/mL | 20,000 mcg/mL | 49.51 mM |
| 50 mg | 5.0 mL | 10.0 mg/mL | 10,000 mcg/mL | 24.76 mM |
| 100 mg | 5.0 mL | 20.0 mg/mL | 20,000 mcg/mL | 49.51 mM |
| 100 mg | 10.0 mL | 10.0 mg/mL | 10,000 mcg/mL | 24.76 mM |
Serial Dilution Math & In-Vitro Working Concentrations
In cell assays, active working concentrations typically span 1.0 to 10.0 mcg/mL (approx. 2.48 to 24.76 µM). A two-tier dilution protocol ensures volumetric precision.
Dilution Equation
C₁ × V₁ = C₂ × V₂ ⟹ V₁ = (C₂ × V₂) ÷ C₁
(where C₁ is stock concentration, V₁ is stock volume to pipette, C₂ is target concentration, and V₂ is target final volume)
Intermediate Working Stock (100 mcg/mL)
To prepare 10 mL of intermediate stock (100 mcg/mL) from a 10.0 mg/mL primary stock:
V₁ = (100 mcg/mL × 10 mL) ÷ 10,000 mcg/mL = 0.10 mL = 100 µL
Add 100 µL of 10.0 mg/mL stock to 9.90 mL sterile PBS or culture medium. Invert gently 5 times.
Working Concentrations Matrix (from 100 mcg/mL Intermediate)
| Target Conc. (C₂) | Molarity | Intermediate Stock (V₁) | Diluent Volume | Final Volume (V₂) | Typical Assay Application |
|---|---|---|---|---|---|
| 1.0 mcg/mL | 2.48 µM | 100 µL | 9,900 µL | 10 mL | Keratinocyte migration assays |
| 2.5 mcg/mL | 6.19 µM | 250 µL | 9,750 µL | 10 mL | Fibroblast scratch wound models |
| 5.0 mcg/mL | 12.38 µM | 500 µL | 9,500 µL | 10 mL | Pro-collagen Type I/III induction |
| 10.0 mcg/mL | 24.76 µM | 1,000 µL | 9,000 µL | 10 mL | MMP and antioxidant enzyme assays |
Concentration Calculator Reference: Use the PepDad Reconstitution Calculator for custom vial masses and aliquot volumes.
Reconstitution Steps & Storage Guidelines
Reconstitution Protocol
- Equilibrate vial to room temperature (20–25°C) for 30 minutes before unsealing.
- Disinfect rubber septum with 70% isopropyl alcohol and let air-dry.
- Inject diluent slowly against inner vial wall.
- Gently swirl for 60 seconds until dissolved; avoid vigorous shaking.
- Verify complete clarity and deep royal blue coloration.
Storage & Stability Matrix
| Material Form | Storage Temp | Recommended Vessel | Shelf Life | Stability Notes |
|---|---|---|---|---|
| Lyophilized Cake | -20°C to -80°C | Amber borosilicate vial | 24–36 months | Desiccated; protected from light. |
| Primary Stock (Frozen) | -80°C | Polypropylene cryovials | 6–9 months | Single-use aliquots; avoid freeze-thaw. |
| Primary Stock (4°C) | 2°C to 8°C | Sealed amber vial | 21–35 days | 21 days in SWFI/saline; 35 days in BAC water. |
| Assay Media Dilutions | 2°C to 8°C | Sterile culture vessel | 24–48 hours | Highly dilute peptides adsorb to plastic; prepare fresh. |