Interactions of Oxytocin with Bovine Neurophysins I and II. Use of ¹³C Nuclear Magnetic Resonance and Hormones Specifically Enriched with ¹³C in the Glycinamide-9 and Half-Cystine-1 Positions

The specifically ¹³C-labeled (90% ¹³C-enriched) neurohypophyseal hormone derivatives [9-[2-¹³C]glycinamidejoxytocin (1), [1-hemi-L-[2-¹³C]cystine]oxytocin (2), and [1-hemi-D-[2-¹³C]cystine]oxytocin (3) have been synthesized by solid phase methods, and the interaction of these hormones with bovine neurophysin I and bovine neurophysin II studied using ¹³C nuclear magnetic resonance at 67.9 MHz and 22.6 MHz. Studies were made at a variety of hormone and protein concentrations and temperatures, but at constant pH (6.6). Under all the conditions derivatives 1 and 2 interact strongly with both neurophysins but the diastereomer 3 apparently does not. The line widths (1/πT₂) of the labeled carbons of the hormones in a 0.9 to 1.0 hormone to protein mole ratio suggest considerable differences in the nature of the interaction(s) at the glycinamide-9 and half-cystine-1 residues of the hormones with the neurophysins, with the half-cystine-1 residue tightly bound with motional characteristics similar to those of the protein, while the glycinamide-9 residue of the hormones possesses additional motion. This extra motionof the glycinamide residue is equivalent to free rotation around one bond in the tripeptide tail of oxytocin. The half-cystine-1 α-carbon atom of oxytocin in the free state and when bound to neurophysin had a chemical-shift difference ofabout 2.7 ppm under all conditions studied, while the chemical shift for the glycinamide-9 α carbon in 1 was the same or only slightly shifted in the free hormone as compared with the complex. Of the chemical-shift difference present with 2, at most one-third can be due to a raising of the pK_a of the N-terminal half-cystine residue upon binding to neurophysin. Possible reasons for the additional shift include a conformation change of the oxytocin backbone upon binding to neurophysin, or a change in the dielectric constant of the surrounding medium. Most interestingly, the glycinamide-9 α carbon of 1 is in fast exchange (>1000 s^-1 at 37 °C) in the hormone-neurophysin I (or neurophysin II) complex, while the half-cystine-1 a carbon in 2 is in slow exchange (<15 s^-1 at 37 °C). A dynamic model of peptide hormone-neurophysin interactions is presented in which slow macroscopic exchange of the bound hormone with the bulk solution obtains, but with one or more much faster microscopic exchange rates contributing to the hormoneprotein interaction.