Degradation of aspartic acid and asparagine residues in human growth hormone-releasing factor.
Bongers. J J; Heimer. E P EP; Lambros. T T; Pan. Y C YC; Campbell. R M RM; Felix. A M AM
Key Findings
- GRF‑1‑29 is most stable at pH 4‑5; below pH 4 it cleaves at Asp3‑Ala4, above pH 5 it undergoes isomerization and deamidation at Asp3 and Asn8.
- Degradation follows simple first‑order kinetics and is faster at higher temperatures, but the peptide is relatively temperature‑stable up to body temperature when kept at the right pH.
- All identified degradation products have much lower biological activity than the original peptide, meaning loss of effectiveness if storage conditions are poor.
Practical Outcomes
- Store GRF‑1‑29 lyophilized and reconstitute it in a mildly acidic solution (around pH 4‑5) just before use. Keep reconstituted peptide refrigerated and avoid exposing it to high pH buffers or prolonged heat. Use acid‑adjusted saline or proprietary acidic carrier solutions to maintain stability, and discard any solution that has been left at room temperature for extended periods.
Summary
The study shows that the short growth‑hormone‑releasing peptide GRF‑1‑29 (also called sermorelin) breaks down quickly when the solution is too acidic or too basic, especially at the Asp3 and Asn8 spots, and that this breakdown makes the peptide far less active. Keeping the peptide in a mildly acidic environment (pH 4‑5) and cool temperatures preserves its potency, while higher pH or heat speeds up harmful changes.
Abstract
Products of the degradation of human growth hormone-releasing factor (GRF) in aqueous solutions (15-200 microM) have been isolated and fully characterized. The cleavage product, GRF(4-44)-NH2, and the isomerization product, [beta-Asp3]GRF(1-44)-NH2, from the degradation of GRF(1-44)-NH2 in acidic solution and the corresponding products, GRF(4-29)-NH2 and [beta-Asp3]GRF(1-29)-NH2, from the degradation of GRF(1-29)-NH2 have been isolated and characterized. The products, [beta-Asp8]GRF(1-44)-NH2 and [Asp8]GRF(1-44)-NH2, from the deamidation of GRF(1-44)-NH2 at pH 8.0 and the corresponding products, [beta-Asp8]GRF(1-29)-NH2 and [Asp8]GRF(1-29)-NH2, from the deamidation of GRF(1-29)-NH2 have been isolated and characterized. All the degradation products of GRF(1-44)-NH2 and GRF(1-29)-NH2 were evaluated for biological activity and found to have much lower in vitro potencies than the parent peptides. Degradation occurs at Asp3 and Asn8 and the kinetics of these various transformations versus pH and temperature have been studied. GRF is most stable at pH 4-5. At pH below the pKa of the Asp3 side-chain (pH less than 4), cleavage at Asp3-Ala4 is the major route of degradation. For pH greater than 4, isomerization of Asp3 to beta-Asp3 (iso-Asp3) predominates. The rates of cleavage and isomerization are simple first order and vary with pH, independent of buffer concentration, such that the protonated (COOH) form of Asp3 undergoes cleavage while the ionized (COO-) form isomerizes. The more rapid deamidation of Asn8 to generate beta-Asp8 and Asp8 in about a 4:1 ratio, presumably via a cyclic imide intermediate, occurs at pH greater than or equal to 5 and is general base-catalyzed. Evidence was also obtained for direct hydrolysis of protonated Asn8 in GRF(1-29)-NH2 at pH less than or equal to 2 to give exclusively [Asp8]GRF(1-29)-NH2. The deamidation of Asn8 in GRF(1-29)-NH2 at pH 8.0, 22-55 degrees C, is relatively insensitive to temperature for T less than 37 degrees C, possibly due to conformational constraints. Asp25 and Asn35 are sterically, conformationally, or otherwise hindered with respect to these changes as no degradation at these sites was observed under the conditions employed.
Study Information
pubmed
1992
10.1111/j.1399-3011.1992.tb01596.x