pH and Solvent titrations of enkephalins by carbon-13 nuclear magnetic resonance spectroscopy: complete assignments of resonances

Abstract

Carbon-13 magnetic resonances of two enkephalins, [Met⁵]- and [Leu⁵]-enkephalin, have been assigned in water, dimethyl sulphoxide, and trifluoroethanol, and assignments of [D-Ala²]-enkephalin-amide have been made in water. The assignments of the Gly₂ carbon atoms have been made by comparison with [D-Ala²]-enkephalin amide. pD Titrations were followed to assign all the carbon resonances in D₂O first, and then the assignments of resonances in other solvents were obtained by solvent–solvent titrations. An unusual titration behaviour of the Tyr₁ aromatic γ-atom at a higher pH was found, and this has been suggested to be due to a conformational change in the Tyr₁ C^α–C^β bond. Conformational features of enkephalins in solution are briefly considered in relation to previous studies.