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Total synthesis, purification, and characterization of human [Phe(p-CH2SO 3Na)52, Nle32,53,56, Nal55]-CCK20-58, [Tyr52, Nle32,53,56, Nal55]-CCK-58, and [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK-58.

Publication ,  Journal Article
Miranda, MT; Craig, AG; Miller, C; Liddle, RA; Rivier, JE
Published in: J Protein Chem
October 1993

The synthesis of [Phe(p-CH2SO3Na)52, Nle32,53,56 Nal55]-CCK20-58, [Tyr52, Nle32,53,56, Nal55]-CCK-58 and of [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK-58 using the (9-fluorenylmethyloxy)-carbonyl (Fmoc) strategy on a 2,4-DMBHA resin is described. The crude peptide preparations were extremely complex when analyzed by RP-HPLC, capillary zone electrophoresis (CZE), and ion-exchange chromatography (IE-FPLC). We found that the most effective strategy for purification included cation-exchange chromatography followed by a RP-HPLC desalting step. The highly purified peptides (purity greater than 90%) were characterized by RP-HPLC, size exclusion HPLC (SEC), IE-FPLC, CZE, mass spectrometry, amino acid analysis, and Edman sequence analysis (for [Tyr52, Nle32,53,56, Nal55]-CCK-58). The results demonstrate the applicability of the 2,4-DMBHA resin for Fmoc solid-phase synthesis of long peptides amides (58 residues in length in this case) as well as the efficacy of an FPLC/RP-HPLC approach for the purification of very long, heterogeneous crude peptides, allowing a true assessment of the biological properties of these analogs to be carried out. [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK20-58 was less than 1% as potent as CCK-8 while [Tyr52, Nle32,53,56, Nal55]-CCK-58 and [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK-58 were inactive at the doses tested (< 0.01%).

Duke Scholars

Published In

J Protein Chem

DOI

ISSN

0277-8033

Publication Date

October 1993

Volume

12

Issue

5

Start / End Page

533 / 544

Location

United States

Related Subject Headings

  • Rats
  • Pancreas
  • Molecular Sequence Data
  • Indicators and Reagents
  • Humans
  • Chromatography, Liquid
  • Chromatography, High Pressure Liquid
  • Cholecystokinin
  • Biophysics
  • Biological Assay
 

Citation

APA
Chicago
ICMJE
MLA
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Miranda, M. T., Craig, A. G., Miller, C., Liddle, R. A., & Rivier, J. E. (1993). Total synthesis, purification, and characterization of human [Phe(p-CH2SO 3Na)52, Nle32,53,56, Nal55]-CCK20-58, [Tyr52, Nle32,53,56, Nal55]-CCK-58, and [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK-58. J Protein Chem, 12(5), 533–544. https://doi.org/10.1007/BF01025118
Miranda, M. T., A. G. Craig, C. Miller, R. A. Liddle, and J. E. Rivier. “Total synthesis, purification, and characterization of human [Phe(p-CH2SO 3Na)52, Nle32,53,56, Nal55]-CCK20-58, [Tyr52, Nle32,53,56, Nal55]-CCK-58, and [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK-58.J Protein Chem 12, no. 5 (October 1993): 533–44. https://doi.org/10.1007/BF01025118.

Published In

J Protein Chem

DOI

ISSN

0277-8033

Publication Date

October 1993

Volume

12

Issue

5

Start / End Page

533 / 544

Location

United States

Related Subject Headings

  • Rats
  • Pancreas
  • Molecular Sequence Data
  • Indicators and Reagents
  • Humans
  • Chromatography, Liquid
  • Chromatography, High Pressure Liquid
  • Cholecystokinin
  • Biophysics
  • Biological Assay