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Refolding of Urea-Denatured Ovalbumin That Comprises Non-Native Disulfide Isomers

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Refolding of Urea-Denatured Ovalbumin That Comprises Non-Native Disulfide Isomers J. Biochem. 122, 83-89 (1997) Refolding of Urea-Denatured Ovalbumin That Comprises Non-Native Disulfide Isomers Maki Onda, Eizo Tatsumi,' Nobuyuki Takahashi. and Masaaki Hirocez The Research Institute for Food Science, Kyoto University, Uji, Kyoto 611 Received for publication, January 24, 1997 Ovalbumin, which contains one cystine disulfide (Cys73-Cysl20) and four cysteine sulfhy- dryls (Cysll, 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 was produced within the 20 ms instrumental dead time; I„ showed about 60 of the native CD ellipticity at 222 nm. The intrinsic tryptophan fluorescence of I,, showed the same peak (338 nm), but with decreased intensity (57%), as compared to the native protein. After the rapid formation of I„ 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-map- ping 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. Key words: disulfide rearrangement, folding intermediate, ovalbumin, protein folding, refolding. In vitro, small single-domain proteins (1-7) a well as tive species for the acquisition of the third native disulfide larger multi-domain proteins (8-11) can oxidatively refold bond (1-5). This is closely related to the inaccessible from their disulfide-reduced, denatured state into the nature of cysteine sulfhydryls in the non-productive disul- native conformation with the aid of an oxidizing reagent. fide intermediates (5). Oxidative refolding systems of disulfide proteins have been As an alternative model disulfide protein, ovalbumin has extensively utilized for the investigation of protein folding unique structural characteristics. The egg-white protein mechanisms, since major disulfide intermediates involved contain six cysteine residues (Cysll, Cys30, Cys73, in the folding pathway can be trapped in stable forms (1- 7). Cysl20, Cys367, and Cys382) in a single polypeptide chain Protein disulfide regeneration, however, includes multiple of 385 amino acid residues (13, 14). As shown in Fig. 1 chemical steps: the first step is the intermolecular attack of (15), Cys73 and Cys120 form an intrachain disulfide in the a protein sulfhydryl on an oxidizing disulfide agent, gener- native state. Our previous studies have shown that the ating a protein mixed-disulfide, and the second step is the conformational state of the disulfide-reduced ovalbumin is intramolecular attack of a second protein sulfhydryl on the almost indistinguishable from that of the disulfide-bonded mixed disulfide (12). Intrachain sulfhydryl/disulfide ex- form (16). Furthermore, the egg-white protein can correct- changes are also involved during the refolding of most of the ly refold from the urea-denatured state under disulfide- small single-domain proteins (1-7). A major problem reduced conditions, indicating the occurrence of spontane- encountered in the kinetic analysis of an oxidative refolding ous protein folding without the native disulfide bond (17). pathway is related to protein sulfhydryl accessibility to an When the disulfide-bonded ovalbumin that is denatured in oxidizing agent in the first step. In bovine pancreatic 9 M urea at pH 2.2 is refolded at near neutral pH, the trypsin inhibitor, the most extensively characterized ex- native disulfide in the acid-denatured protein undergoes ample, only the native two-disulfide intermediate [5-55; nonspecific disulfide rearrangements in an initial burst 30-51 that is produced from other native two-disulfide intermediate and then is recovered during the subsequent intermediates by disulfide rearrangements is the produc- slow refolding (18). Essentially the same intermediate is formed during the refolding of the disulfide-reduced pro- Supported by JSPS Fellowships for Japanese Junior Scientists. tein (18). These data along with other evidence are consis- z To whom correspondence should be addressed . Phone: + 81-774-38- tent with the following scheme for the refolding process of 3734, Fax: +81-774-38-3735 Abbreviation: IAEDANS, N-iodoacetyl-N'-(5-sulfo-l-naphthyl)eth- urea-denatured ovalbumin: ylenediamine. Vol. 122, No. 1, 1997 83 84 M . Onda et al. EXPERIMENTAL PROCEDURES Materials-A,-ovalbumin (diphosphorylated form) was purified by crystallization in an ammonium sulfate solution (20) and by subsequent ion-exchange chromatography as described (21). Diphenylcarbamylchloride-treated trypsin (Type XI) and chymotrypsin (Type II) were purchased from Sigma. Achromobacter protease I [EC 3.4.21.50] was obtained from Wako Pure Chemical Industries. Denaturation and Refolding of Ovalbumin-Denatured ovalbumin was prepared by incubating the native, disul fide-bonded protein at 1.0 mg/ml, 37•Žfor 30 min in Buffer A (50 mM Tris-HCl buffer, pH 8.2, 1.0 mM Na-EDTA) containing 9 M urea. Refolding was initiated at 25•Ž by 20-fold dilution of the denatured protein with Buffer A , giving a final urea concentration as low as 0.45 M. The proteins were allowed to refold at the same temperature, and then analyzed for trypsin resistance, intrinsic trypto phan fluorescence and CD spectrum. An acid-quenched equilibrium intermediate was produced by 20-fold dilution of the urea-denatured protein with 50 mM K-phosphate buffer, pH 2.2, containing 1.0 mM Na-EDTA. The buffers were degassed at reduced pressure and equilibrated under an N, atmosphere prior to the refoldinu e, nepiments CD Spectrum Measurement-The far-UV CD spectrum Fig. 1. Schematic view of ovalbumin. The figure is based on the of ovalbumin was recorded with a spectropolarimeter X-ray crystallographic data of ovalbumin (15) and was drawn using (JASCO, J-720). The temperature of the cuvette was the MolScript program (29). The numbered shaded spheres represent maintained at 25•Ž with a circulating water bath . The CD the sulfur atoms: 1, Cysll; 2, Cys30; 5, Cys367; 6, Cys382 . The sulfur atoms 3 (Cys73) and 4 (Cys120) form the native disulfide bond. data were expressed as mean residue ellipticity (degree- M2 /decimol) by using a value of 111 as the mean residue weight of ovalbumin . CD spectra at a short refolding time were determined by measuring the time-dependent increase in absolute CD ellipticity at various wavelengths and the values at a where D is the urea-denatured ovalbumin with the native refolding time of 10 s were plotted as a function of disulfide Cys73-Cys120. I, is the initial burst intermediate wavelength. with the native disulfide or without any intrachain disulfide For rapid mixing experiments, a stopped-flow rapid ki and is the competent intermediate for subsequent folding netics accessory (Applied Photophysics , RX.1000) attached into the native form N with or without the native disulfide. to the same spectropolarimeter was employed and changes I, *is the mispaired-disulfide intermediate that is formed in the CD ellipticity of the urea-denatured ovalbumin at during the refolding in the disulfide-bonded ovalbumin; 230 nm were recorded at 25•Ž after 10-fold dilution with through intrachain disulfide rearrangement reactions , 1,* Buffer A. The dead time for mixing, determined by using undergoes reversible interconversion with L. the reaction between 2,6-dichlorophenolindophenol and In the present-study,-we investigated whether or not the urea-denatured ovalbumin comprising non-native disulfide Measurement of Intrinsic Tryptophan Fluorescence- isomers, D*, can refold into N with the correct disulfide The fluorescence spectrum of ovalbumin was measured pairing. As demonstrated in a previous report (19), many with a Hitachi fluorescence spectrophotometer (Model non-native disulfide isomers that have one disulfide and F-3000). The intrinsic tryptophan residues in ovalbumin four sulfhydryls in a molecule are produced, when disul were excited at 295 run, and the emission spectrum was fide-bonded ovalbumin is denatured at high concentrations recorded at a wavelength range from 300 to 420 nm . All of urea at near neutral pH. According to the X-ray crystal- measurements were carried out at a constant temperature lographic data (15), however, only the native disulfide of 25`C. The time course of fluorescence intensity change Cys73-Cys120 can adopt the native conformation (Fig. 1). was monitored at 338 nm emission . For the spectral The refolding process of ovalbumin denatured in 9 M urea measurements at an early refolding time , the time course at pH 8.2 was investigated by using several different of fluorescence intensity changes was monitored at various structural approaches. We report here that most, if not all, emission wavelengths with excitation at 295 nm , and data of the D* species can correctly refold into the native form at a refolding time of 10 s were plotted. via disulfide
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