CHEMICAL AND ENZYMATIC TREATMENT OF ENDOTHELIN

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 [Hse(7) 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 [Hse(7)-NH2]-7,8-seco-ET and [Hse(7)]ET, resulting from competitive intramolecular reaction of the deprotonated alpha-amino group of the Asp(8) residue. The Lys(9)-Glu(10) 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 Lys(9)-Glu(10) 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 chymotripsin (present as an impurity in the trypsin preparation) on Tyr(13). The structure of these peptides was confirmed by amino-acid sequence analysis and fast atom bombardment mass spectrometry (FAR-MS). Nicking of the ET structure at different positions had different impact on the biological properties of the resulting derivatives. (C) Munksgaard 1995.