A New Strategy to Stabilize Oxytocin in Aqueous Solutions: I. The Effects of Divalent Metal Ions and Citrate Buffer

Abstract

In the current study, the effect of metal ions in combination with buffers (citrate, acetate, pH 4.5) on the stability of aqueous solutions of oxytocin was investigated. Both monovalent metal ions (Na+ and K+) and divalent metal ions (Ca2+, Mg2+, and Zn2+) were tested all as chloride salts. The effect of combinations of buffers and metal ions on the stability of aqueous oxytocin solutions was determined by RP-HPLC and HP-SEC after 4 weeks of storage at either 4°C or 55°C. Addition of sodium or potassium ions to acetate- or citrate-buffered solutions did not increase stability, nor did the addition of divalent metal ions to acetate buffer. However, the stability of aqueous oxytocin in aqueous formulations was improved in the presence of 5 and 10 mM citrate buffer in combination with at least 2 mM CaCl2, MgCl2, or ZnCl2 and depended on the divalent metal ion concentration. Isothermal titration calorimetric measurements were predictive for the stabilization effects observed during the stability study. Formulations in citrate buffer that had an improved stability displayed a strong interaction between oxytocin and Ca2+, Mg2+, or Zn2+, while formulations in acetate buffer did not. In conclusion, our study shows that divalent metal ions in combination with citrate buffer strongly improved the stability of oxytocin in aqueous solutions.

A New Strategy To Stabilize Oxytocin in Aqueous Solutions: II. Suppression of Cysteine-Mediated Intermolecular Reactions by a Combination of Divalent Metal Ions and Citrate

“…In the presence of divalent metal ions, the formation of metal−citrate adducts reduces the concentration of free citrate, which reduces the driving force for citrate adduct formation with oxytocin. 15 A second possibility is that binding of a divalent metal ion to oxytocin may change the position of the N-terminal amino group from the cysteine residue rendering it less accessible for binding with citrate.…”

Analysis of argentinated peptide complexes using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry: Peptide = oxytocin, arg8‐vasopressin, bradykinin, bombesin, somatostatin, neurotensin

“…Unfortunately, OT is unstable at temperatures ≥30°C, and metal salts along with a suitable buffer are used as stabilizing agents. Both monovalent (Na + and K + ) and divalent (Ca 2 + , Mg 2 + , Zn 2 + ) ions were used to stabilize OT; however, Zn 2 + was reported to be more effective . The structural and thermo‐chemical properties of OT and its metal ion complexes [OT+M] + were determined by the DFT method.…”

17β-estradiol upregulates oxytocin and the oxytocin receptor in C2C12 myotubes

“…1). OXT peptide was dissolved in 10 nM citrate buffer to increase its stability at high temperature (Avanti et al, 2011). Day 7 was chosen to harvest the differentiated MTs and to conduct the transcriptomic analysis because spontaneous contraction of MTs was observed, which is commonly associated with the maturation of MTs in culture.…”