Conformational Studies of Oxytocin, Lysine Vasopressin, Arginine Vasopressin, and Arginine Vasotocin by Carbon-13 Nuclear Magnetic Resonance Spectroscopy

Abstract

Oxytocin, arginine vasopressin, lysine vasopressin, arginine vasotocin, as well as their cyclic and acyclic analogs, were studied by carbon-13 nuclear magnetic resonance spectroscopy in deuterium oxide and deuterated dimethylsulfoxide. Fourier-transformed spectra were obtained at 25. 16 MHz. The resonances of all carbon atoms have been assigned in both solvent systems; this includes tentative assignments of the carbonyl carbons. The spectra of arginine vasopressin and lysine vasopressin are essentially identical when compared in D20 or dimethylsulfoxide, but they differ from those of oxytocin. The spectrum of arginine vasotocin in D20 is intermediate between those of oxytocin and the vasopressins. These spectral differences are not only due to variations in constituent amino acids but are also a reflection of conformational differences of oxytocin, arginine vasotocin, and the vasopressins. All hormones are sensitive to changes in hydrogen ion concentration in both solvents; this was not observed with deamino analogs, which lack the terminal amino group.Conformational analysis of data from proton nuclear magnetic resonance ('H NMR) spectroscopy has contributed significantly to elucidation of the preferred solution conformations of oxytocin (1, 2) and lysine vasopressin ([Lys8]VP) (3-7). Nevertheless, the conformational analysis involves several approximations: (1) the relationship between the dihedral angle 0 (NH-CaH) and the measured three-bond coupling constant 3JHNCH (8, 9)-a spectral parameter most useful when considered in combination with potential energy maps (10, 11); (2)

Relative conformational rigidity in oxytocin and (1-penicillamine)-oxytocin: a proposal for the relationship of conformational flexibility to peptide hormone agonism and antagonism.

“…2); the aromatic carbons(not shown) were also assigned. The assignments were made by comparison of the spectrum of [Pen'loxytocin with oxytocin (15), [Penl, Leu2joxytocin, and several deuterated derivatives of oxytocin (V. J. Hruby, J.-P. Meraldi (12). This indicates that these two protons are shielded from solvent over a wide temperature range and could be involved in intramolecular hydrogen bonding (26).…”

13C nuclear magnetic resonance studies of the interactions of bovine neurophysins with (1‐hemi‐[1‐13C]cystine)oxytocin and (1‐hemi‐[1‐13C]cystine, 8‐arginine)vasopressin

“…The chemical shift of this carbonyl carbon in the hormones is very sensitive to the ionization state of the adjacent amino group [8]. We have found the 13C resonance of the enriched carbon to be easily observed in the 1: 1 (molar ratio) hormone-protein complex and this has enabled us to monitor the ionization state and pK, value of the hormone's o-amino group in the hormone-protein complex.…”

Conformational Studies on Arginine Vasopressin and Arginine Vasotocin by Proton Magnetic Resonance Spectroscopy

“…AVP, AVT, and [Ala']AVP were from the same batch used in a previous study (18) Cc and CO protons on the same residue. After the COH resonance assignments had been obtained, the NH resonances for all residues were uniquely assigned by spin decoupling of vicinal amide and C' protons on the same residue.…”

“…In the initial report on some assignments of proton resonances in LVP it was speculated "that the peptide NH resonance furthest downfield in the spectrum may be that of Phe3 and not that of Cys6, as in oxytocin" (6), inasmuch as the observed stacking (5) Turning to AVT, it appears that its ring conformation is very similar to that of oxytocin. The evidence for this contention-while not yet complete, but nevertheless convincingcan be summarized as follows: (a) the values of 6, AWNH/AT, and kHDX for corresponding protons of residues located in the 20-membered cyclic moiety of AVT and oxytocin are quite similar; and (b) the '3C NMR spectra of atoms comprising the tocin ring of AVT and oxytocin are almost identical (18).…”

“…Since the initial conformational analysis by 'H nuclear magnetic resonance (NMR) spectroscopy of oxytocin dissolved in dimethylsulfoxide Me2SO (1-3), neurohypophyseal hormones have been extensively investigated by this technique (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15), by "IC NMR spectroscopy (16)(17)(18)(19)(20), by conformational energy calculations (14,21,22), and by model building (22). The ultimate goal of these types of studies is to relate the biological activity of peptide hormones to their solution conformation.…”