Evidence from Hydrogen-1 and Carbon-13 Nuclear Magnetic Resonance Studies That the Dissociation Rate of Oxytocin from Bovine Neurophysin at Neutral pH Is Slow

The interaction of the peptide hormone oxytocin with bovine neurophysins I and II (NPI and NPII) was studied using 270-MHz ¹H nuclear magnetic resonance (NMR) spectroscopy and observing the tyrosine-2 aromatic protons of the hormone. Using oxytocin and its diastereoisomer [l-hemi-d-cystine] oxytocin (which does not bind to the neurophysins), we investigated the Ti (line width) values of these protons as a function of temperature, protein concentration at constant hormone concentration, and protein concentration at a constant hormone protein ratio and the T₁ values of these hormones in the presence and absence of the neurophysins. The results of these studies indicate that at neutral pH most of the effects observed can be accounted for by viscosity changes and suggest that the bound hormone is in slow exchange. This was corroborated by l3C NMR studies. A total synthesis of specifically ₁₃C labeled (90% ₁₃C enrichment) [2-₁₃C]tyrosine (l and dl derivatives) was accomplished, and the enriched amino acid was incorporated into oxytocin and arginine vasopressin by total synthesis. The diastereoisomers of [2-dl-[2-₁₃C]tyrosine]oxytocin were separated and purified by partition chromatography. The interactions of these hormone derivatives with NPI and NPII were investigated at a variety of temperatures and hormone and protein concentrations but at constant pH (6.6). Under all conditions, [2-l-[2-₁₃C]tyrosine]oxytocin (1) and [2-L-[2-₁₃C]tyrosine, 8-arginine]vasopressin (3) interact strongly with NPI and NPII, and on binding, a 2.2-ppm downfield chemical shift of the labeled carbon atom is observed for both hormones at a 1:1 hormone to protein stoichiometry. These results indicate an equivalence of binding sites for both hormones to both NPI and NPII in the 1:1 complex. On the other hand, [2-d-[2-₁₃C]tyrosine]-oxytocin (2) does not significantly interact with the neurophysins. When an excess of labeled hormone 1 is added to NPI (or NPII) (e.g., a 1.4:1 hormone to protein mole ratio), free and bound peaks are observed. The spectra indicate that the hormone is in slow exchange under all conditions studied and that a 1:1 stoichiometry obtains for the oxytocin-neurophysin complex. An upper limit for the overall dissociation rate of about 2 s-l can be estimated from NMR data leading to an upper limit of about 2 × 10⁵ M^-1 s^-1 for the association rate. This small rate constant and the substantial chemical shifts associated with the binding process suggest that a substantial conformational change occurs at the tyrosine-2 position when the hormones bind to neurophysins.