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

Published online by Cambridge University Press:  01 December 1999

PASCALE ROUX ,
MARGHERITA RUOPPOLO ,
ALAIN-FRANÇOIS CHAFFOTTE  and
MICHEL E. GOLDBERG
PASCALE ROUX
Affiliation:
Unité de Biochimie Cellulaire (Centre National de la Recherche Scientifique: CNRS URA 1129), Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France
MARGHERITA RUOPPOLO
Affiliation:
Dipartimento di Chimica, Università di Salerno, via Salvatore Allende, 84081 Baronissi, Salerno, Italy Centro Internazionale di Servizi di Spettrometria di Massa, Via Pansini, 5 80131 Napoli, Italy
ALAIN-FRANÇOIS CHAFFOTTE
Affiliation:
Unité de Biochimie Cellulaire (Centre National de la Recherche Scientifique: CNRS URA 1129), Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France
MICHEL E. GOLDBERG
Affiliation:
Unité de Biochimie Cellulaire (Centre National de la Recherche Scientifique: CNRS URA 1129), Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France
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Abstract

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.

Keywords

foldingkineticslysozymemass spectroscopyoxidationsecondary structure
Type
Research Article
Information
Protein Science , Volume 8 , Issue 12 , December 1999 , pp. 2751 - 2760
Copyright
© 1999 The Protein Society

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