Amide hydrogen exchange in a highly denatured state. Hen egg-white lysozyme in urea.

The amide hydrogen exchange behaviour of hen egg-white lysozyme denatured in 8 M urea has been studied at pH 2.0, 20 degrees C. The observed exchange rates have been compared with those predicted for the same residues in a random coil conformation using recently published parameters for side-chain inductive and temperature effects on exchange catalysis. The protection factors for exchange obtained in this way were found to be close to unity, with 41 of the 61 residues that could be followed having protection factors less than 2. No protection factor was greater than 5. In addition, previous data for hen lysozyme denatured thermally and for a three-disulphide derivative, CM6-127 lysozyme, denatured at pH 2.0 have been reanalysed using the new reference parameters, and the protection factors were found to be similar to those of hen lysozyme denatured in 8 M urea. Thus, although 1H NMR and far UV CD spectroscopy suggest that considerable deviations from random coil behaviour occur in these denatured states, such residual structure is insufficient to protect amide hydrogens significantly against exchange. This behaviour contrasts with that of a partly folded state of hen lysozyme denatured in trifluoroethanol and with that of the molten globule state of a homologous protein, guinea pig alpha-lactalbumin. Here protection factors for many amide hydrogens exceed 30 and belong to residues located in continuous regions of the amino acid sequence, indicating the presence of persistent structure. The study of hydrogen exchange in substantially denatured states of a protein, therefore, provides a basis for the interpretation of protection factors in partially folded states.