Degradation of aspartic acid and asparagine residues in human growth hormone-releasing factor
Por:
BONGERS J., HEIMER E.P., LAMBROS T., Pan Y.-C.E., CAMPBELL R.M., FELIX A.M.
Publicada:
1 ene 1992
Resumen:
Products of the degradation of human growth hormone-releasing factor (GRF) in aqueous solutions (15-200 ?M) have been isolated and fully characterized. The cleavage product, GRF(4-44)-NH2, and the isomerization product, [?-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 [?-Asp3]GRF(1-29)-NH2, from the degradation of GRF(1-29)-NH2 have been isolated and characterized. The products, [?-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, [?-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 pK(a) of the Asp3 side-chain (pH < 4), cleavage at Asp3-Ala4 is the major route of degradation. For pH > 4, isomerization of Asp3 to ?-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 ?-Asp8 and Asp8 in about a 4:1 ratio, presumably via a cyclic imide intermediate, occurs at pH ? 5 and is general base-catalyzed. Evidence was also obtained for direct hydrolysis of protonated Asn8 in GRF(1-29)-NH2 at pH ? 2 to give exclusively [Asp8]GRF(1-29)-NH2. The deamidation of Asn8 in GRF(1-29)-NH2 at pH 8.0, 22-55°C, is relatively insensitive to temperature for T < 37°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.
Filiaciones:
BONGERS J.:
Departments of Peptide Research, Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey, United States
Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey, United States
HEIMER E.P.:
Departments of Peptide Research, Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey, United States
Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey, United States
LAMBROS T.:
Departments of Peptide Research, Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey, United States
Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey, United States
CAMPBELL R.M.:
Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey, United States
FELIX A.M.:
Departments of Peptide Research, Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey, United States
Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey, United States
Roche Research Center, Hoffmann-La Roche Inc, Nutley, New Jersey, United States
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