Department of Pharmaceutical Technology and Biopharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands.
Int J Pharm. 2013 Feb 28;444(1-2):139-45. doi: 10.1016/j.ijpharm.2013.01.051. Epub 2013 Feb 1.
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that the stability of oxytocin in aspartate buffered formulation is improved by the addition of divalent metal ions (unpublished results).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 Zn(2+) was by far superior compared to that of Mg(2+). 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 Zn(2+) in particular suppressed intermolecular degradation reactions near the Cys(1,6) disulfide bridge. These results lead to the hypothesis that in aspartate buffer, Zn(2+) changes the conformation of oxytocin in such a way that the Cys(1,6) 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 Mg(2+) or Zn(2+), using 2D NOESY, TOCSY, (1)H-(13)C HSQC and (1)H-(15)N HSQC NMR spectroscopy. Almost all (1)H, (13)C and (15)N resonances of oxytocin could be assigned using HSQC spectroscopy, without the need for (13)C or (15)N enrichment. (1)H-(13)C and (1)H-(15)N HSQC spectra showed that aspartate buffer alone induces minor changes in oxytocin in D2O, with the largest chemical shift changes observed for Cys(1). Zn(2+) causes more extensive changes in oxytocin in aqueous solution than Mg(2+). Our findings suggest that the carboxylate group of aspartate neutralizes the positive charge of the N-terminus of Cys(1), allowing the interactions with Zn(2+) to become more favorable. These interactions may explain the protection of the disulfide bridge against intermolecular reactions that lead to dimerization.
催产素是一种用于引产和预防产后出血的肽类药物。由于其不稳定性，在热带条件下运输和储存催产素制剂存在问题。在之前的研究中，我们发现添加二价金属离子可以提高天冬氨酸缓冲制剂中天冬氨酸缓冲制剂中天冬氨酸缓冲制剂中催产素的稳定性（未发表结果）。与 Mg(2+)相比，Zn(2+)的稳定效果要好得多。此外，还发现稳定作用与二价金属离子在天冬氨酸缓冲液中与催产素相互作用的能力密切相关。此外，LC-MS（MS）测量表明，特别是天冬氨酸缓冲液与 Zn(2+)的组合抑制了 Cys(1,6) 二硫键附近的分子间降解反应。这些结果导致了这样的假设，即在天冬氨酸缓冲液中，Zn(2+)改变了催产素的构象，使 Cys(1,6) 二硫键免受其环境的影响，从而抑制了涉及该分子区域的分子间反应。为了验证这一假设，我们在此使用 2D NOESY、TOCSY、(1)H-(13)C HSQC 和 (1)H-(15)N HSQC NMR 光谱研究了在存在 Mg(2+)或 Zn(2+)的情况下，天冬氨酸缓冲液中催产素的构象。使用 HSQC 光谱，无需 (13)C 或 (15)N 富集，几乎可以对催产素的所有 (1)H、(13)C 和 (15)N 共振进行分配。(1)H-(13)C 和 (1)H-(15)N HSQC 光谱表明，天冬氨酸缓冲液本身在 D2O 中使催产素发生较小的变化，最大的化学位移变化发生在 Cys(1)。Zn(2+)在水溶液中引起的催产素变化比 Mg(2+)更广泛。我们的研究结果表明，天冬氨酸的羧酸盐中和了 Cys(1)的 N-末端的正电荷，使与 Zn(2+)的相互作用变得更加有利。这些相互作用可能解释了对二硫键的保护，防止导致二聚化的分子间反应。
