REFOLDING OF UREA-DENATURED OVALBUMIN THAT COMPRISES NONNATIVE DISULFIDE ISOMERS

Ovalbumin, which contains one cystine disulfide (Cys73-Cys120) and fou r cysteine sulfhydryls (Cys11, Cys30, Cys367, and Cys382) in the nativ e state, undergoes intrachain sulfhydryl-disulfide exchanges at high c oncentrations of urea, generating many non-native disulfide isomers [E . Tatsumi, N. Takahashi, and M. Hirose (1994) J. Biol. Chem. 269,; 280 62-28067]. The refolding of ovalbumin from the urea-denatured state wa s investigated. When the denatured protein was diluted 20-fold with a refolding buffer (pH 8.2), an initial burst intermediate I-N was produ ced within the 20 ms instrumental dead time; I-N showed about 60% of t he native CD ellipticity at 222 nm. The intrinsic tryptophan fluoresce nce of I-N showed the same peak (338 nm), but with decreased intensity (57%), as compared to the native protein. After the rapid formation o f I-N, most of the ovalbumin molecules correctly refolded into the nat ive state with slow biphasic kinetics, as evaluated by far-UV CD, tryp tophan fluorescence, and trypsin-resistance analyses. Furthermore, a p eptide-mapping analysis revealed that sulfhydryl/disulfide exchange re actions occurred during the refolding, thereby increasing the formatio n of the native disulfide. The integrity of overall refolding was conf irmed by a differential scanning calorimetry analysis. These data were consistent with the view that most, if not all, of the mispaired disu lfide isomers in the urea-denatured ovalbumin can correctly refold int o the native state via intrachain disulfide rearrangements.