Proton nuclear magnetic resonance double resonance study of oxytocin in aqueous solution

Abstract

Peptide NH resonances in the 250 MHz 1H-NMR spectrum of oxytocin in H2O were assigned to specific amino acid residues by the underwater decoupling technique (i.e., decoupling from corresponding C.alpha.H resonances, which are buried beneath the intense water peak). These experiments confirm previous assignments of A. I. Brewster and V. J. Hruby and A. F. Bradbury et al. Three methods of assigning NH resonances of peptides, solvent titration, underwater decoupling and isotopic labeling, are compared. As the solvent composition is gradually changed from dimethyl sulfoxide to H2O, oxytocin undergoes a conformational change at 70-90 mol % of H2O. Exposure to solvent of specific hydrogens of oxytocin in H2O was studied by monitoring intensity changes of solute resonances when the solvent peak was saturated. Positive nuclear Overhauser effects (NOE''s) of 14 .+-. 5 and 9 .+-. 5 were observed for the Tyr o-CH and m-CH resonances, respectively. Comparative studies with deamino-oxytocin indicate that these effects result predominantly from intermolecular dipole-dipole interaction between aromatic side chain CH protons and protons of the solvent. The NOE''s therefore indicate intimate contact between H2O and the aromatic CH hydrogens of the Tyr side chain. The extent of saturation transferred by proton exchange between H2O and NH groups varies with pH in a manner which appears to reflect the acid-base catalysis of the protolysis reaction. There is no indication that any NH protons are substantially shielded from the solvent.