The Folding Pathway of Reduced Lysozyme”

on the mechanism of the glutathione regeneration of hen egg lysozyme been out. The first two stoichiometric disulfides in lysozyme are formed about 8 than the second two. Almost no enzymic activity is regained until the first two disulfides are formed, thus ruling out an all-or-none mechanism. The disulfide peptides formed early in the regeneration have been isolated and identified. The results show a limited search of folding intermediates, and outline a folding pathway. The early disulfides involve cysteinyl residues III, IV, V, and VI. At the same time cysteinyl residues I’, II, VII, and VIII are still reduced, as demonstrated by their isolation as S-alkylated derivatives. At slightly later times a peptide is found which contains the (native) disulfide between cysteinyl residues II and VII. It is likely, but as yet unproven, that formation of disulfide I-VIII completes the cross-linking of lysozyme. of acquisition of three-dimensional structure by proteins


Methods
for the purification, reduction, and assay of lysozyme have been described previously (10).

Regeneration
Reduced lysozyme at 10m6 M was regenerated using the non-enzymic glutathione buffer system of Saxena and Wetlaufer (9). This regeneration system is composed of 1 x IO-' M GSH and 1 x lo-" M GSSG in a 0.10 N Tris acetate buffer, pH 8.0, at 37". In order to inhibit the copper-catalyzed air oxidation of thiols during the regeneration, all glassware was rinsed in 6.0 M HCl to remove copper ion, all solutions were deaerated with nitrogen, and an atmosphere of nitrogen was maintained over the regeneration solution. For regenerations longer than 3.0 min, 1 x 10m3 M EDTA was included in the regeneration mixture.

Stopping
Regeneration and Blocking Thiols The regeneration was stopped and the thiols were blocked by a modification of the method of Ristow and Wetlaufer (10). The pH of the regeneration solution was first reduced to 5.5 with glacial acetic acid. Samples were removed for activity measurements. Unreacted thiols were then blocked by the addition of a lo-fold molar excess of N-ethylmaleimide (1 x lo-' M) after urea was added to a final concentration of 4 M to facilitate complete alkylation. The alkylation was allowed to proceed for 30 min, after which the pH was lowered to 2.0 by the addition of concentrated HCl. The alkylated, partially oxidized protein was isolated from this solution.

Product
The product was concentrated from the alkylation solution by ultrafiltration with an Amicon PM 10 membrane. The concentrated protein was then separated from regeneration and blocking reagents by gel filtration on a column (2.5 x 100 cm) of Sephadex G-25 (coarse) equilibrated and eluted with 0.10 N acetic acid. This protein was lyophilized and stored dry at O-5".

3148
Folding Pathway of Reduced Lysozyme The partly regenerated protein was dissolved in 5% formic acid to a concentration of 10 mg/ml. Pepsin was added at an enzyme to substrate ratio of 1:lOO by weight. After 18 hours a second equal aliquot of pepsin was added. The digestion was stopped after 24 hours by freezing and lyophilization.

Ziyptic Digest
The partially regenerated thiol-blocked protein or its peptic digest was dissolved at 10 mg/ml in 0.10 N acetic acid that was also 2.9 M in Ultra-pure urea (Schwarz/Mann). The pH of this mixture was adjusted to 6.0 with ammonium hydroxide, and diphenylcarbamyl chloride-treated trypsin was then added at an enzyme to substrate ratio of 1:lOO by weight. After 18 hours of digestion at 25", another equal aliquot of trypsin was added. The digestion was stopped after 30 hours by acidification to pH 3.0.

Gel Filtration
The enzymically digested regenerated material was initially fractionated by gel filtration on a column (2.5 x 150 cm) of Sephadex G-25 (fine) equilibrated and eluted with 0.10 N acetic acid. The absorbance of the effluent was monitored at 280 nm, and disulfides and alkylated cysteines were detected by the method of Anderson and Wetlaufer (11). The fractions containing disulfide and/or alkylated cysteine were lyophilized.
The dry peptides were stored at O-5" until further purification by ion exchange chromatography.

Ion Exchange Chromatography
CM-cellulose-Chromatography was carried out on a column (0.9 x 25 cm) of Whatman CM-cellulose CM32, using the method of Maron et al. (12). The column was equilibrated with 0.10 N ammonium acetate, pH 3.0, and the peptide samples were applied to the column in the same buffer. Two linear 500.ml ammonium acetate gradients were used to elute the column: a pH gradient of 0.10 N ammonium acetate from pH 3.0 to pH 6.0, followed by a concentration gradient of pH 6.0 ammonium acetate from 0.10 N to 1.0 N. The column effluent was continuously monitored at 280 nm with an ISCO dual beam monitor. Aminex 5OW-X4 and SP-Sephadex' C-25-Chromatography with Aminex 5OW-X4 and SP-Sephadex C-25 was carried out in columns (0.9 x 60 cm) using the method of Schroeder (13). The ion exchanger was washed and equilibrated with 0.10 N pyridine acetate, pH 2.7. The chromatogram was developed with a lOOO-ml pyridine acetate gradient from 0.10 N, pH 2.7, to 2.0 N, pH 5.0, and the effluent was automatically monitored using the ninhydrin reaction following alkaline hydrolysis of the peptides (14). Disulfides and alkylated cysteines were located in the column chromatographic separations by the method of Anderson and Wetlaufer (11). Purity of peptides was established by cellulose thin layer chromatography in the following solvent systems: I-butanol:acetic acid:water (4:1:5); 1-butanol:acetic acid:pyridine:water (30:6:20:24); and i-propanol:88% formic acid:water (20:1:5).
Peptide Mapping-Thin layer peptide mapping was carried out on digests (pepsin followed by trypsin) of regenerated material according to the method of Burns and Turner (15). Electrophoresis was for 2 hours at 400 V in acetic acid:88% formic acid:water (17:5:280). Chromatography was carried out using 1.butanol:acetic acid:water (4:1:5 upper layer).
Peptides were detected with ninhydrin, and cystines were detected by the method of Karush et al. (16).

RESULTS
In order for inferences based on the identification of disulfide intermediates in the folding of lysozyme to be meaningful, it is essential that the method used to stop the refolding be rapid and efficient and that it not alter the disulfides that have been formed. Reducing the pH of the regeneration mixture from 8.0 to 5.5 prevents shuffling involving enzymically active species for at least 2 hours, as evidenced by the time-stability of the enzymic activity of a sample quenched to pH 5.5 at any point during the course of the regeneration. Following acidification, ' The abbreviations used are: SP-Sephadex, sulfopropyl-Sephadex; GdmCl, guanidinium chloride; RNase A, ribonuclease A from the bovine pancreas. the unreacted thiols are rapidly blocked with N-ethylmaleimide.
Independent tests of the effectiveness of these blocking conditions were carried out. In the first control, native lysozyme was added to the regeneration mixture, and standard blocking and isolation operations were carried out. As can be seen in Table I, these conditions do not decrease the number of disulfide bonds (4.0) in native lysozyme. In the second control experiment, the pH of the regeneration mixture was reduced to 5.5 before the addition of reduced lysozyme. From this point, standard blocking and isolation procedures were carried out. In the second control no disulfides were formed. These results indicate that the methods used to stop the regeneration, block thiols, and isolate the product are effective both in preventing further disulfide formation and in preventing the destruction of existing disulfide bonds. The blocking and isolation procedures are thus valid for isolation of covalent intermediates in the folding process.
Figs. 1 and 2 compare the rate of disulfide formation with the rate of regain of enzymic activity. There is a rapid formation of two disulfide bonds followed by a slower formation of the remaining two disulfides in the molecule. Two first order kinetic processes are required to fit the disulfide rate data, one with a kinetic constant k = 0.6 min', and one with k = 0.08 min'. The initial rapid disulfide formation is completely lost when either 3.0 or 6.0 M GdmCl is included in the regeneration buffer. From Fig. 2 it is evident that no enzymic activity appears during the initial rapid formation of disulfide; significant activity is not observed until more than (on the average) two disulfide bonds are formed. Due to the existence of the lag period (confirmed in dozens of experiments) and the poor precision of the lysozyme assay, we have not attempted to fit the activity data to simple kinetic models.
Thin layer mapping on digests (pepsin followed by trypsin) of partially regenerated material was carried out in order to extend the observations of Ristow and Wetlaufer (10) to the glutathione regeneration system. Only six disulfide peptides could be observed in maps of material regenerated to an average of 0.8 disulfides/molecule.' No disulfide peptides were observed in maps of material regenerated to the same extent of disulfide formation in 3.0 M GdmCl.' These results extend the findings of Ristow and Wetlaufer to the glutathione system: only a few disulfides are formed early in the regeneration.

One-half-minute
Regeneration-To identify the first, rapidly formed disulfides, we regenerated a 40-mg batch of lysozyme for '/z min under optimal conditions as outlined above. This material was digested with trypsin, and the digest was initially fractionated on a Sephadex G-25 column. The gel filtration clearly separates the peptides containing disulfide and alkylated cysteine into three distinct major fractions, as shown in Fig. 3.
CM-cellulose chromatography of peptides in the largest gel filtration fraction (Fraction A) is shown in Fig. 4 Fig. 3. A column (0.9 x 60 cm) was eluted with a IOOO-ml pyridine nooled fractions. acetate eradient as described under "Materials and Methods." FIG. 4 (center).
CM-cellulose chromatography of Fraction A from  Conditions are the same as described in Fig. 3. -, absorbance at 280 nm; ---, absorbance due to disulfide and alkylated cysteine.

DISCUSSION
The mechanisms of disulfide formation in the glutathione thiol-disulfide regeneration system have been formally described by Bradshaw et al. (18) and Saxena and Wetlaufer (9). In such a system disulfide bonds are formed by thiol-disulfide exchange reactions. The rates of this reaction have been determined for exchanges between cystamine and glutathione under conditions similar to those used in this communication (19). If the exchange rates between glutathione and protein thiol are similar to the rates described for cystamine and glutathione, then complete equilibration of disulfide with all thiols on lysozyme is possible in less than 1 min (8). This means that there are four types of disulfides to be considered for lysozyme folding in the glutathione regeneration system: native protein disulfides, wrongly paired protein disulfides, mixed disulfides between protein and glutathione, and GSSG. Any proposed folding mechanism should account for all four classes of disulfides.
If the one stochiometric disulfide observed after ?&min regeneration were due to the presence of four native disulfides in one quarter of the molecules, we would expect to find approximately 25% of native enzymic activity. That is, if all-or-none folding were taking place, one would expect to