Chemical and enzymatic treatment of endothelin

R. Perego, L. Gozzini, E. Arlandini, G. Bolis, R. De Castiglione

Research output: Contribution to journalArticlepeer-review

Abstract

Endothelin-1 (ET), the most potent vasoconstrictor yet discovered, is a peptide containing 21 amino acids with two intrachain disulfide bridges. With the aim of obtaining two-chain derivatives, ET was submitted to chemical and enzymatic treatments. Reaction of ET with CNBr in 70% HCOOH gave, in addition to the expected [Hse7 lactone]-7,8-seco-ET and unreacted material, a by-product whose molecular weight was 25 m.u. greater than that of ET. When the reaction mixture, after lyophilisation, was immediately quenched with NH3-saturated dry MeOH, two products could be recovered in a 5:1 ratio, both obtained by nucleophilic attack of the homoserine lactone: the expected [Hse7-NH2]-7,8-seco-ET and [Hse7]ET, resulting from competitive intramolecular reaction of the deprotonated α-amino group of the Asp8 residue. The Lys9-Glu10 bond turned out to be very resistant to enzymatic attack both by Lys-C-endopeptidase and trypsin. The 9,10-seco-ET derivative could be obtained by treatment with Lys-C-endopeptidase only by using a high enzyme/ET ratio and after a prolonged incubation time. Cleavage of the Lys9-Glu10 bond could not be achieved by treatment with trypsin, even with a high enzyme/substrate ratio. The main product was 13,14-seco-ET, deriving from the action of chymotrypsin (present as an impurity in the trypsin preparation) on Tyr13. The structure of these peptides was confirmed by amino-acid sequence analysis and fast atom bombardment mass spectrometry (FAB-MS). Nicking of the ET structure at different positions had different impact on the biological properties of the resulting derivatives.

Original languageEnglish
Pages (from-to)341-345
Number of pages5
JournalInternational Journal of Peptide and Protein Research
Volume46
Issue number5
Publication statusPublished - 1995

Keywords

  • Chymotrypsin
  • Cyanogen bromide (CNBr)
  • Endothelin-1 (ET)
  • Fast atom bombardment mass spectrometry (FAB-MS)
  • Lys-C-endopeptidase
  • Trypsin

ASJC Scopus subject areas

  • Biochemistry

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