Structural and Functional Characteristics of Partially Disulfide-reduced Intermediates of Ovotransferrin N Lobe CYSTINE LOCALIZATION BY INDIRECT END-LABELING APPROACH AND IMPLICATIONS FOR THE REDUCTION PATHWAY

摘要: Ovotransferrin N lobe contains six intrachain disulfides (SS-I/Cys10-Cys45; SS-II/Cys20-Cys36; SS-III/Cys115-Cys197; SS-IV/Cys160-Cys174; SS-V/Cys171-Cys182; SS-VI/Cys228-Cys242) in a single polypeptide chain of 332 amino acid residues. Upon the protein disulfide reduction with dithiothreitol under nondenaturing conditions, intermediate species four, three, and two were generated. The partially disulfide-reduced intermediates isolated, localization intact was determined by an indirect end-labeling method. This method included S-cyanocysteine-specific fragmentation, followed gel electrophoresis immunochemical visualization C terminus-intact fragments using antiserum raised against non-cysteine C-terminal fragment (Ser280-Arg332). Results clearly showed that first SS-IV SS-V, second SS-III, then SS-VI are cleaved. No observed for SS-I SS-II employed reducing conditions. conclusion confirmed peptide mapping analyses same reverse phase high performance liquid chromatography. Transverse urea gradient visible absorption spectra revealed four-disulfide intermediate, but not three- or two-disulfide retains essentially iron-binding function as native protein. By far-UV CD analyses, residual conformation found to decrease increased number reduced disulfides. Implications disulfide-reductive unfolding pathway ovotransferrin discussed.