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Proton-decoupled Fourier transform nuclear magnetic resonance of natural abundance 13C was used to obtain spectra of ribonuclease A and its oxidized derivative at a set of pH values ranging from pH 1.40 to 6.72 at 45°. Observations on the native conformation were made at pH 4.14 and 6.55, and on the denatured conformation at pH 2.12 and below. In addition, the disrupted structure obtained by performic acid oxidation of the disulfide bonds was observed at several pH values. The loss of the native structure was associated with sharpening of the resonance bands. A tentative assignment was made for the unmodified ribonuclease A at pH 1.46 based on resonance positions of free amino acids and peptides. Although a good fit to the data was obtained, further development is required for unequivocal assignment. Partially relaxed spectra were used to obtain spin-lattice relaxation times (T1) for certain carbon types. These values are traced through several pH values for both ribonuclease A and its oxidized derivatives. Pairs of effective rotational correlation times (τeff) were derived from the T1 values of certain aliphatic resonances. By considering data from fluorescence depolarization experiments and observed line widths, it was possible to exclude one of the τeff values in the majority of cases. The results were interpreted in terms of increased segmental mobility in the denatured states, especially for α carbon nuclei that appear relatively restrained in the native state.

The natural abundance carbon 13 nuclear magnetic resonance spectra of the peptides l-valyl-l-leucyl-l-seryl-l-glutamylglycine, comprising the first 5 residues of the amino terminus of sperm whale myoglobin, l-leucyl-l-seryl-l-glutamic acid, and l-seryl-l-glutamylglycine have been recorded by continuous wave and pulsed Fourier transform techniques. Signal assignments were made by comparison of the peptides with each other and with the free amino acids, by the pH dependence of the chemical shifts, and by modification of the degree of proton decoupling in certain cases. The chemical shift dependence of specific carbon centers on their position in the oligopeptide sequences and the influence of the state of ionization of various groups on neighboring carbon sites were explored.

Proton-decoupled Fourier transform nuclear magnetic resonance of natural abundance 13C was used to obtain spectra of cyanoferrimyoglobins of harbor seal (Phoca vitulina) and sperm whale (Physeter catodon). These spectra were compared with those of hen egg white lysozyme and of bovine pancreatic ribonuclease A to show differences reflecting gross composition as well as differences tentatively attributable to more subtle conformational constraints. The cyanoferrimyoglobin spectra were much alike but not identical. Carboxymethylation of the native seal protein produced barely detectable changes at the chemical shift positions of the major adducts but no systematic alterations elsewhere in the spectrum. However, extensive modification of histidine and methionine residues by carboxymethylation of seal apomyoglobin in the presence of 8 m urea yielded a denatured product, judged by circular dichroism measurements, for which the 13C spectrum was sharp and characteristic of a denatured protein. This spectrum was used to tabulate tentative assignments. Partially relaxed spectra were used to obtain spin-lattice relaxation times (T1) for certain carbon types. These values are compared for the various myoglobin preparations mentioned above. The similarities between the native forms are striking. The denatured preparation gives many evidences of increased segmental mobility, especially for those nuclei that appear relatively restrained in the native state.