{'fraction_non_alphanumeric': 0.04579207920792079, 'fraction_numerical': 0.0297029702970297, 'mean_word_length': 5.341176470588235, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
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.