title: Reversible denaturation of disulfide-reduced ovalbumin and its reoxidation generating the native cystine cross-link.
abstract: The authors in a previous report (Klausner, R. D., Kempf, C., Weinstein, J. N., Blumenthal, R., and van Renswoude, J. (1983) Biochem. J. 212, 801-810) have argued that native folding of ovalbumin occurs during translation, but not in a renaturation system of the denatured form. To re-examine the possibility, we searched for the conditions of correct oxidative refolding of denatured disulfide-reduced ovalbumin. Data of trypsin resistance, CD-spectrum, and selective reactivity of cysteine sulfhydryls revealed that the fully denatured protein can refold into the native conformation under disulfide-reduced conditions. The interconversion between the native and denatured forms was fully reversible with a free energy change for unfolding of 6.6 kcal/mol at 25 degrees C. Subsequent reoxidation under a variety of redox conditions generated only one disulfide bond in the reduced refolded protein with six cysteine sulfhydryls. Furthermore, the regenerated disulfide was found by peptide analyses to correspond to the native disulfide pairing, Cys73-Cys120. We, therefore, concluded that co-translational folding, if any, is not requisite for the correct oxidative folding of ovalbumin.
authors.affiliation: Research Institute for Food Science, Kyoto University, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Takahashi
authors.forename: M
authors.initials: M
authors.lastname: Hirose
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000378:metabolism*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011489:Protein Denaturation; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D004220:Disulfides; D013438:Sulfhydryl Compounds; D010047:Ovalbumin; D014357:Trypsin; D003545:Cysteine