Oxidative regeneration and selective reduction of native disulfide bonds in the N-terminal half-molecule of ovotransferrin.
The denatured, disulfide-reduced form of the N-terminal half-molecule of ovotransferrin was reoxidized with either oxidized dithiothreitol or GSSG and analyzed for the localization of disulfide bonds. Chemical analyses of the reoxidized proteins revealed that the disulfide peptides corresponding to the six native protein disulfides (SS-I, SS-II, SS-III, SS-IV, SS-V, and SS-VI) are all regained in the reoxidized protein. The peptide recoveries from the reoxidized proteins were, however, about half of those from the native protein with respect to the two inner disulfides (SS-IV and SS-V) in the kringle bridges, but all the disulfide peptides corresponding to the remaining disulfides (SS-I, SS-II, SS-III, and SS-VI) were recovered at almost equivalent yields in the native and reoxidized proteins. In addition, on searching for a nonnative disulfide peptide, the two disulfides, Cys171-Cys174 and Cys174-Cys182, which can be accounted for by mispaired bridges of sulfhydryls in SS-IV and SS-V, were detected in the protein reoxidized with oxidized dithiothreitol. Upon disulfide reduction of the native protein with reduced dithiothreitol, both SS-IV and SS-V were selectively cleaved under the same buffer and temperature conditions as in the oxidative refolding. The lower stabilities of the two inner disulfide bonds in the kringle may be related to the lower recoveries of the disulfide peptides from SS-IV and SS-V and the generation of the nonnative disulfide bonds.
Yamashita
H
H
Research Institute for Food Science, Kyoto University, Japan.
Hirose
M
M
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003207:Conalbumin / Q000737:chemistry / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D004229:Dithiothreitol / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D011489:Protein Denaturation; D014357:Trypsin / Q000378:metabolism
D004220:Disulfides; D010446:Peptide Fragments; D003207:Conalbumin; D014357:Trypsin; D005978:Glutathione; D004229:Dithiothreitol