To investigate the role of some tertiary interactions,
the disulfide bonds, in the early stages of refolding of
hen lysozyme, we report the kinetics of reoxidation of
denatured and reduced lysozyme under the same refolding
conditions as those previously used to investigate the
kinetics of regain of its circular dichroism (CD), fluorescence,
and activity. At different stages of the refolding, the
oxidation of the protein was blocked by alkylation of the
free cysteines with iodoacetamide and the various oxidation
states present in the samples were identified by electrospray-mass
spectrometry. Thus, it was possible to monitor the appearance
and/or disappearance of the species with 0 to 4 disulfide
bonds. Using a simulation program, these kinetics were
compared with those of regain of far-UV CD, fluorescence,
and enzymatic activity and were discussed in terms of a
refined model for the refolding of reduced hen egg white
lysozyme.