Aspartate buffer and divalent metal ions affect oxytocin in aqueous solution and protect it from degradation

Research output: Research - peer-reviewJournal article

  • Christina Avanti
    Christina AvantiUniversity of Groningen, University of Groningen, Dept. of Pharmaceutical Technology and BiopharmacyNetherlands
  • Nur Alia Oktaviani
    Nur Alia OktavianiUniversity of Groningen, Groningen Biomolecular Sciences and Biotechnology InstituteNetherlands
  • Wouther L.J. Hinrichs
    Wouther L.J. HinrichsUniversity of Groningen, University of Groningen, Dept. of Pharmaceutical Technology and BiopharmacyNetherlands
  • Henderik W. Frijlink
    Henderik W. FrijlinkUniversity of Groningen, Dept. of Pharmaceutical Technology and BiopharmacyNetherlands
  • Frans A.A. Mulder
Oxytocin is a peptide drug used to induce labor and prevent bleeding after childbirth. Due to its instability, transport and storage of oxytocin formulations under tropical conditions is problematic. In a previous study, we have found that the stability of oxytocin in aspartate buffered formulation is improved by the addition of divalent metal ions (unpublished results). The stabilizing effect of Zn2+ was by far superior compared to that of Mg2+. In addition, it was found that stabilization correlated well with the ability of the divalent metal ions to interact with oxytocin in aspartate buffer. Furthermore, LC–MS (MS) measurements indicated that the combination of aspartate buffer and Zn2+ in particular suppressed intermolecular degradation reactions near the Cys1,6 disulfide bridge. These results lead to the hypothesis that in aspartate buffer, Zn2+ changes the conformation of oxytocin in such a way that the Cys1,6 disulfide bridge is shielded from its environment thereby suppressing intermolecular reactions involving this region of the molecule. To verify this hypothesis, we investigate here the conformation of oxytocin in aspartate buffer in the presence of Mg2+ or Zn2+, using 2D NOESY, TOCSY, 1H–13C HSQC and 1H–15N HSQC NMR spectroscopy. Almost all 1H, 13C and 15N resonances of oxytocin could be assigned using HSQC spectroscopy, without the need for 13C or 15N enrichment. 1H–13C and 1H–15N HSQC spectra showed that aspartate buffer alone induces minor changes in oxytocin in D2O, with the largest chemical shift changes observed for Cys1. Zn2+ causes more extensive changes in oxytocin in aqueous solution than Mg2+. Our findings suggest that the carboxylate group of aspartate neutralizes the positive charge of the N-terminus of Cys1, allowing the interactions with Zn2+ to become more favorable. These interactions may explain the protection of the disulfide bridge against intermolecular reactions that lead to dimerization.Mg or Zn, using 2D NOESY, TOCSY, H-C HSQC and H- N HSQC NMR spectroscopy. Almost all H, C and N resonances of oxytocin could be assigned using HSQC spectroscopy, without the need for C or N enrichment. H-C and H-N HSQC spectra showed that aspartate buffer alone induces minor changes in oxytocin in D O, with the largest chemical shift changes observed for Cys . Zn causes more extensive changes in oxytocin in aqueous solution than Mg. Our findings suggest that the carboxylate group of aspartate neutralizes the positive charge of the N-terminus of Cys, allowing the interactions with Zn to become more favorable. These interactions may explain the protection of the disulfide bridge against intermolecular reactions that lead to dimerization.
Original languageEnglish
JournalInternational Journal of Pharmaceutics
Volume444
Issue number1-2
Pages (from-to)139-145
Number of pages7
ISSN0378-5173
DOIs
StatePublished - 2013

See relations at Aarhus University Citationformats

ID: 52972400