Comparison of the kinetics of S-S bond, secondary structure, and active site formation during refolding of reduced denatured hen egg white lysozyme

To investigate the role of some tertiary interactions, the disulfide bonds, in the early stages of refolding of hen lysozyme, we report the kinetics o f reoxidation of denatured and reduced lysozyme under the same refolding co nditions as those previously used to investigate the kinetics of regain of its circular dichroism (CD), fluorescence, and activity. At different stage s of the refolding, the oxidation of the protein was blocked by alkylation of the free cysteines with iodoacetamide and the Various oxidation states p resent in the samples were identified by electrospray-mass spectrometry. Th us, it was possible to monitor the appearance and/or disappearance of the s pecies with 0 to 4 disulfide bonds. Using a simulation program, these kinet ics were compared with those of regain of far-UV CD, fluorescence, and enzy matic activity and were discussed in terms of a refined model for the refol ding of reduced hen egg white lysozyme.