Refolding of urea-denatured ovalbumin that comprises non-native disulfide isomers.
Ovalbumin, which contains one cystine disulfide (Cys73-Cys120) and four cysteine sulfhydryls (Cys11, Cys30, Cys367, and Cys382) in the native state, undergoes intrachain sulfhydryl-disulfide exchanges at high concentrations of urea, generating many non-native disulfide isomers [E. Tatsumi, N. Takahashi, and M. Hirose (1994) J. Biol. Chem. 269, 28062-28067]. The refolding of ovalbumin from the urea-denatured state was investigated. When the denatured protein was diluted 20-fold with a refolding buffer (pH 8.2), an initial burst intermediate I(N) was produced within the 20 ms instrumental dead time; I(N) showed about 60% of the native CD ellipticity at 222 nm. The intrinsic tryptophan fluorescence of I(N) showed the same peak (338 nm), but with decreased intensity (57%), as compared to the native protein. After the rapid formation of I(N), most of the ovalbumin molecules correctly refolded into the native state with slow biphasic kinetics, as evaluated by far-UV CD, tryptophan fluorescence, and trypsin-resistance analyses. Furthermore, a peptide-mapping analysis revealed that sulfhydryl/disulfide exchange reactions occurred during the refolding, thereby increasing the formation of the native disulfide. The integrity of overall refolding was confirmed by a differential scanning calorimetry analysis. These data were consistent with the view that most, if not all, of the mispaired disulfide isomers in the urea-denatured ovalbumin can correctly refold into the native state via intrachain disulfide rearrangements.
Onda
M
M
The Research Institute for Food Science, Kyoto University.
Tatsumi
E
E
Takahashi
N
N
Hirose
M
M
D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D007536:Isomerism; D008958:Models, Molecular; D010047:Ovalbumin / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D014508:Urea / Q000737:chemistry*
D004220:Disulfides; D014364:Tryptophan; D014508:Urea; D010047:Ovalbumin