Two-dimensional double quantum 1H NMR spectroscopy of proteins

Two-dimensional double quantum 1H NMR spectra are recorded for a protein. The application of this technique to macromolecules is shown not to be impeded by the long preparation pulse sequence essential for this experiment. The identification of spin systems by analysis of the double quantum spectrum is illustrated. Since double quantum spectra do not contain diagonal peaks, connectivities between almost degenerate signals can be detected. By analysis of remote connectivities (i) it can be established whether amide and beta-protons belong to the same spin system or not, (ii) degeneracy of beta-proton chemical shifts can be demonstrated, and (iii) glycine amide protons can be distinguished from all others.