Studies on the relationship of disulfide bonds to the formation and maintenance of secondary structure in chicken egg white lysozyme.
The dependence of secondary structure on SS content was examined to gain further insight into the role of SS bond formation in chain folding. Lysozyme was reduced to various levels, and the products were studied with respect to remaining native structure. Reductions were carried out in the absence of denaturant. Thus, the observed changes were not attributable to a general denaturing effect, but more specifically to cleavage of SS bonds. The products of reduction were found by gel filtration chromatography to contain both polymeric and monomeric forms. By "fingerprinting", the monomers gave no indication of having undergone SS interchange, and specific cleavage of native SS bonds was indicated by a stepwise disappearance of SS-containing peptides with increasing reduction level. Ion-exchange chromatography of the carboxymethylated monomers gave the three possible reduction intermediates, containing two, four, and six carboxymethylcysteine residues per mole, in addition to the fully reduced form. The intermediates exhibited circular dichroic (CD) characteristics that were clearly nonnative, indicating alteration of the secondary structure throughout all stages of reduction. Concomitantly, there were extensive losses of enzymatic activity at intermediate levels, while no activity was found for the fully reduced form. Partially and fully reduced lysozymes were also examined for their abilities to resume native secondary structure in the absence of SS formation, as would be evidenced by a reversion to native CD characteristics. Despite literature indications of native structure in the reduced protein as observed from examination of CD behavior, none was found in the present effort, under a variety of experimental conditions. These observations are consistent with an essential role of SS bonds in forming and maintaining a major portion of the native secondary structure in lysozyme.
D000818:Animals; D002645:Chickens; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology; D004529:Egg White; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D009113:Muramidase / Q000378:metabolism; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation / Q000187:drug effects; D013696:Temperature
D004220:Disulfides; D010446:Peptide Fragments; D008623:Mercaptoethanol; D009113:Muramidase; D004229:Dithiothreitol