Immunochemical pulsed-labeling characterization of intermediates during hen lysozyme oxidative folding.
Previous studies have shown that reduced hen egg white lysozyme refolds and oxidizes according to a linear model, in which the number of disulfide bonds increases sequentially. In this study, we describe the kinetics of native tertiary structure formation during the oxidative-renaturation of reduced hen egg white lysozyme, as monitored using an immunochemical pulsed-labeling method based on enzyme-linked immunosorbent assay (ELISA) in conjunction with two monoclonal antibodies (mAb). Each of these antibodies recognizes a separate face of the native lysozyme surface and, more importantly, each epitope is composed of discontinuous regions of the polypeptide chain. Renaturation kinetics were studied under the same refolding conditions as previous investigations of the kinetics of the regain of far-UV CD, fluorescence, enzymatic activity, and disulfide bonds. Comparison of our results with the results from those studies showed that the immunoreactivity (i.e., the native fold) of the alpha-domain appeared in intermediates containing two SS bonds only (C6-C127 and C30-C115), while the immunoreactivity of the beta-domain appeared together with the formation of the third SS bond (C64-C80). Thus, the alpha-domain folds before the beta-domain during the oxidative folding of reduced lysozyme.
Jarrett
Nicole M
NM
Unité de Repliement et Modélisation des Protéines (CNRS URA 2185), Institut Pasteur, 75254 Paris Cedex 15, France.
Djavadi-Ohaniance
Lisa
L
Willson
Richard C
RC
Tachibana
Hideki
H
Goldberg
Michel E
ME
D000818:Animals; D000911:Antibodies, Monoclonal; D002645:Chickens; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D019791:Guanidine / Q000737:chemistry; D007120:Immunochemistry / Q000379:methods; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary
D000911:Antibodies, Monoclonal; D000939:Epitopes; C421864:hen egg lysozyme; D009113:Muramidase; D019791:Guanidine