Assignment of the complete disulphide bridge pattern in the human recombinant follitropin β-chain

A. Amoresano, S. Orrù, R. A. Siciliano, E. De Luca, R. Napoleoni, A. Sirna, P. Pucci

Research output: Contribution to journalArticle

Abstract

The chemical assessment of the complete disulphide bridge pattern in the β-chain of human recombinant follicotropin (βFSH) was accomplished by integrating classical biochemical methodologies with mass spectrometric procedures. A proteolytic strategy consisting of a double digestion of native βFSH using the broad-specificity protease subtilisin first, followed by trypsin, was employed. The resulting peptide mixture was directly analysed by FAB-MS, leading to the assignment of the first three disulphide bridges. The remaining S-S bridges were determined by HPLC fractionation of the proteolytic digest followed by ESMS analysis of the individual fractions. The pattern of cysteine couplings in βFSH was determined as: Cys3-Cys51, Cys17-Cys66, Cys20-Cys104, Cys28-Cys82, Cys32-Cys84 and Cys87-cys94, confirming the arrangement inferred from the crystal structure of the homologous βCG. A subset of the S-S bridge pattern comprising Cys3-Cys51, Cys28-Cys82 and Cys32-Cys84 constitutes a cysteine knot motif similar to that found in the growth factor superfamily.

Original languageEnglish
Pages (from-to)961-968
Number of pages8
JournalBiological Chemistry
Volume382
Issue number6
DOIs
Publication statusPublished - 2001

Keywords

  • Cysteine knot topology
  • Disulphide bridges
  • FSH
  • Gonadotropins
  • Mass spectrometry

ASJC Scopus subject areas

  • Biochemistry

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    Amoresano, A., Orrù, S., Siciliano, R. A., De Luca, E., Napoleoni, R., Sirna, A., & Pucci, P. (2001). Assignment of the complete disulphide bridge pattern in the human recombinant follitropin β-chain. Biological Chemistry, 382(6), 961-968. https://doi.org/10.1515/BC.2001.120