Chemical and enzymatic treatment of endothelin

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

Research output: Contribution to journalArticle

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

Fingerprint

Endothelins
Endothelin-1
Therapeutics
Trypsin
Derivatives
Fast Atom Bombardment Mass Spectrometry
Amino Acids
Peptides
Freeze Drying
Protein Sequence Analysis
Chymotrypsin
Vasoconstrictor Agents
Lactones
Enzymes
Disulfides
Mass spectrometry
Byproducts
Molecular Weight
Molecular weight
Impurities

Keywords

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

ASJC Scopus subject areas

  • Biochemistry

Cite this

Perego, R., Gozzini, L., Arlandini, E., Bolis, G., & De Castiglione, R. (1995). Chemical and enzymatic treatment of endothelin. International Journal of Peptide and Protein Research, 46(5), 341-345.

Chemical and enzymatic treatment of endothelin. / Perego, R.; Gozzini, L.; Arlandini, E.; Bolis, G.; De Castiglione, R.

In: International Journal of Peptide and Protein Research, Vol. 46, No. 5, 1995, p. 341-345.

Research output: Contribution to journalArticle

Perego, R, Gozzini, L, Arlandini, E, Bolis, G & De Castiglione, R 1995, 'Chemical and enzymatic treatment of endothelin', International Journal of Peptide and Protein Research, vol. 46, no. 5, pp. 341-345.
Perego R, Gozzini L, Arlandini E, Bolis G, De Castiglione R. Chemical and enzymatic treatment of endothelin. International Journal of Peptide and Protein Research. 1995;46(5):341-345.
Perego, R. ; Gozzini, L. ; Arlandini, E. ; Bolis, G. ; De Castiglione, R. / Chemical and enzymatic treatment of endothelin. In: International Journal of Peptide and Protein Research. 1995 ; Vol. 46, No. 5. pp. 341-345.
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