The Formation of Oxytocin Dimers is Suppressed by the Zinc-Aspartate-Oxytocin Complex

Abstract

ABSTRACT:The aim of this study was to investigate the effect of divalent metal ions (Ca, Mg 2+ , and Zn 2+ ) on the stability of oxytocin in aspartate buffer (pH 4.5) and to determine their interaction with the peptide in aqueous solution. Reversed-phase high-performance liquid chromatography and high-performance size-exclusion chromatography measurements indicated that after 4 weeks of storage at 55 • C, all tested divalent metal ions improved the stability of oxytocin in aspartate-buffered solutions (pH 4.5). However, the stabilizing effects of Zn 2+ were by far superior compared with Ca 2+ and Mg 2+ . Liquid chromatography-tandem mass spectrometry showed that the combination of aspartate and Zn 2+ in particular suppressed the formation of peptide dimers. As shown by isothermal titration calorimetry, Zn 2+ interacted with oxytocin in the presence of aspartate buffer, whereas Ca 2+ or Mg 2+ did not. In conclusion, the stability of oxytocin in the aspartate-buffered solution is strongly improved in the presence of Zn 2+ , and the stabilization effect is correlated with the ability of the divalent metal ions in aspartate buffer to interact with oxytocin. The reported results are discussed in relation to the possible mode of interactions among the peptide, Zn 2+ , and buffer components leading to the observed stabilization effects.

Preprint: A robust peptidomics mass spectrometry platform for measuring oxytocin in plasma and serum

“…Initially, samples contained 50 mM ZnCl 2 (10 mM aspartate buffer, pH 4.5) to stabilize OT via chelation 16 prior to subsequent sample preparation (e.g. removing proteins via protein precipitation (PPT)).…”

“…We hypothesized that strong protein binding was preventing detection of endogenous OT ((<pg/mL levels, Figure SM 4 ) due to co-precipitation during PPT. The disulfide bridge (DSB) of OT ( Figure 2a ) can engage in complexes 16 , and likely with serum albumin (buminlbumi, which contains multiple DSBs. To obstruct plasma protein binding, a reduction/alkylation (R/A) 14 step was performed which irreversibly breaks DSBs ( Figure 2b ).…”

Metal complexes of amino acids and peptides

“…292 The extent of the formation of reactive oxygen species in the presence of human and rat amylin and Cu(II) was investigated in another study. 296,297 The effect of Ca(II), Mg(II) and Zn(II) was investigated and the latter metal ion was found to be the most efficient. 293 The metal complexes of human IAPP and its fragments have been investigated by ESI-MS technique and the residues between position 22-26 were identified as the primary Cu(II) binding sites.…”

Heat-Stable Dry Powder Oxytocin Formulations for Delivery by Oral Inhalation

“…Multiple degradation pathways lead to OT instability (6,7,16). Degradation is mainly the result of chemical instability at the disulfide bond leading to dimerization and formation of sulfur-related impurities such as trisulfide, tetrasulfide, and trimers (16)(17)(18). Instability at the glutamine and asparagine groups leads to deamidation products.…”

“…Instability at the glutamine and asparagine groups leads to deamidation products. OT stability in solution can be improved by using stabilizing agents, in particular buffers and divalent cations (7,17,18). Recently, Avanti et al demonstrated the beneficial effect of combining divalent zinc and a citrate buffer that dramatically reduced the formation of dimers and other sulfur-related impurities in solution (17)(18)(19).…”