NMR studies of neurohypophyseal peptide hormones
Abstract (summary)
The chemistry and conformational behavior of the neurohypophyseal peptide hormones oxytocin and arginine vasopressin (AVP) have been studied by one and two-dimensional $\sp1$H NMR methods. The standard COSY, ROESY and TOCSY pulse sequences have been modified to eliminate the water resonance using the Water Attenuation by Transverse Relaxation (WATR) method and applied to the first assignment of the resonances of the $\sp1$H NMR spectrum of reduced AVP.
Using these WATR-modified $\sp1$H NMR experiments, minor resonances in the spectra of oxytocin, AVP and lysine vasopressin (LVP) have been assigned to peptides having the cis conformation about the cysteine$\sp6$-proline peptide bond. Relative populations of the cis and trans isomers, resonance assignments and amide proton resonance temperature coefficients for both isomers of the native and dithiol forms of these hormones are reported. Trans/cis isomerization rate constants were measured and activation parameters obtained from Arrhenius plots. The rate of interconversion of the trans isomer to the cis isomer in oxytocin and AVP is much faster in methanol than in aqueous solution.
The aqueous solution conformations of the oxidized and reduced oxytocin, AVP, tocinoic and pressinoic acids are compared. Data obtained from $\sp1$H NMR experiments, including $\sp1$H and $\sp{15}$N chemical shifts, coupling constants, amide proton temperature coefficients and NOE's obtained from ROESY experiments are presented and interpreted in terms of the conformational differences of these molecules. Comparison of the conformations of the disulfide and dithiol forms of the molecules indicates the structural constraints imposed by the disulfide bond on the conformations available to the hexapeptide ring.
The interaction of oxytocin and AVP with macromolecular components of human plasma and with plasma albumin in D$\sb2$O solution has been studied using the CPMG pulse sequence to eliminate the broad envelope of protein resonances. $\sp1$H NMR results are presented which show that oxytocin and AVP bind to proteins in human plasma, and to human and bovine serum albumin in solution at neutral pH, with the interaction reduced as the pH is lowered. From the relative broadening of the various peptide resonances, the binding appears to involve the hexapeptide ring while the tripeptide tail remains free and more mobile.