Effects of pH, salt and time on ligand binding properties of overexpressed melanocortin 4 receptor.
Dolby. Viveka V; Lundqvist. Andreas A; Fröberg. Thomas T; Lüllau. Elke E; Shaw. Judith J; Tjerneld. Folke F; Cronet. Philippe P
Key Findings
- Affinity for MTII is lost below pH 6 but recovers at physiological pH
- High NaCl (up to 1 M) does not significantly affect ligand binding
- At neutral pH, physiological salt, and 4 °C, ligand affinity remains stable for up to 6 days
Practical Outcomes
- Store melanotan‑I in a neutral‑pH, physiological‑salt buffer and keep it refrigerated (around 4 °C). Avoid acidic environments, as they quickly reduce activity. Under these conditions the peptide stays effective for several days, simplifying dosing schedules for users.
Summary
The study shows that the melanotan‑I peptide (MTII) sticks to its target receptor best when kept at normal body pH (around 7.4) and that its effectiveness drops in acidic conditions but comes back when the pH is corrected. Salt levels up to 1 M don’t hurt binding, and if you store the peptide in a neutral‑pH, normal‑salt solution at 4 °C, it stays active for about a week.
Abstract
The G-protein coupled melanocortin 4 receptor (MC4r) plays an important role in the energy metabolism. We overexpressed the MC4r in CHO cells and performed characterisation studies on the cell membranes to determine functional stability and ligand binding properties of the receptor. The affinity for the ligands [Nle4, d-Phe7]-alphaMSH and MTII was lost below pH 6 but could be restored by returning to physiological pH. Increasing NaCl concentration up to 1 M had little influence on the binding of either ligand. At neutral pH, physiological salt concentration and 4 degrees C the ligand affinity of the receptor was stable for up to 6 days. These findings will facilitate design of purification methods for the receptor.
Study Information
pubmed
2004
2004-03-31T00:00:00.000Z
10.1016/j.jbbm.2003.10.007