Pharmaceutical Research

, Volume 11, Issue 7, pp 936–944 | Cite as

Major Degradation Products of Basic Fibroblast Growth Factor: Detection of Succinimide and Iso-aspartate in Place of Aspartate15

  • Zahra Shahrokh
  • Gert Eberlein
  • Doug Buckley
  • Mallik V. Paranandi
  • Dana W. Aswad
  • Pamela Stratton
  • Ron Mischak
  • Y. J. Wang
Article

Abstract

The degradation products of basic fibroblast growth factor (bFGF) were isolated by ion exchange HPLC (HP-IEC) and characterized. The predominant product at pH 5 was a succinimide in place of aspartate15 as determined by LC/MS, N-terminal sequencing, and susceptibility to degradation at pH >6.5. The rate of appearance of the succinimidyl-bFGF at 22 °C was comparable to that reported for small peptides, consistent with a high flexibility predicted for asp15-gly. Tryptic mapping together with [3H]-methylation indicated that iso-aspartate was formed at the position of asp15. Size exclusion HPLC indicated the presence of intact and truncated dimers and trimers which associated through disulfide linkages. Two truncated monomer forms were found that co-eluted by HP-IEC; the cleavages were determined to be at asp28-pro and asp15-gly using LC/MS and N-terminal sequencing. These degradation products which occurred at sites that are away from receptor or heparin binding domains of bFGF remained bioactive in a cell proliferation assay.

protein degradation succinimide iso-asp ion exchange HPLC LC/MS 

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Copyright information

© Plenum Publishing Corporation 1994

Authors and Affiliations

  • Zahra Shahrokh
    • 1
  • Gert Eberlein
    • 1
  • Doug Buckley
    • 1
  • Mallik V. Paranandi
    • 2
  • Dana W. Aswad
    • 2
  • Pamela Stratton
    • 1
  • Ron Mischak
    • 1
  • Y. J. Wang
    • 1
  1. 1.Scios Nova Inc.
  2. 2.Department of Molecular Biology and BiochemistryUC IrvineIrvine

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