Refolding of Taiwan Cobra Neurotoxin: Intramolecular Cross-Link Affects Its Refolding Reaction

Archives of Biochemistry and Biophysics Vol. 387, No. 2, March 15, pp. 289 –296, 2001 doi:10.1006/abbi.2000.2236, available online at http://www.idealibrary.com on

Refolding of Taiwan Cobra Neurotoxin: Intramolecular Cross-Link Affects Its Refolding Reaction Long-sen Chang,* ,1 Shinne-ren Lin,† and Chen-chung Yang‡ *Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan; †School of Chemistry, Kaohsiung Medical University, Kaohsiung 807, Taiwan; ‡Department of Life Sciences, National Tsing Hua University, Hsinchu 300, Taiwan

Received August 25, 2000, and in revised form November 10, 2000; published online February 19, 2001

In order to explore the effect of intramolecular cross-linking in the folding reaction of cobrotoxin from Naja naja atra (Taiwan cobra) venom, the toxin molecule was modified with glutaraldehyde (GA). The monomeric GA-modified cobrotoxin (mGA-cobrotoxin) was separated from the dimeric and trimeric derivatives using gel filtration. The results of electrophoretic and chromatographic analyses revealed that mGA-cobrotoxin comprised two modified derivatives, which contained modified Lys residues at positions 26 and 27 and at positions 26, 27, and 47, respectively. Moreover, an intramolecular cross-linking of loops II and III by Lys residues was noted with the monomeric derivative containing three modified Lys residues. In sharp contrast to cobrotoxin observations, the folding rate of mGA-cobrotoxin decreased in the presence of GSH/ GSSG, but notably increased in the absence of thiol compounds. Particularly, the accelerated effect of GSH/GSSG on the refolding reaction was affected by the presence of the intramolecular cross-link. Comparative analyses on cobrotoxin and mGA-cobrotoxin CD spectra revealed that modification with the GA reagent caused a change in the gross conformation of cobrotoxin. Fluorescence measurement revealed that the stability of the microenvironment around the single Trp-29 in mGA-cobrotoxin and unfolded mGA-cobrotoxin was appreciably higher than in cobrotoxin and unfolded toxin. Moreover, the ordered structure formation around Trp-29 in refolded mGA-cobrotoxin was faster than in refolded cobrotoxin as evidenced by fluorescence quenching studies. Taken together, these results suggest that the structural flexibility of unfolded cobrotoxin should be favorable for the thiol catalyst to exert its action in the refolding reaction

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and that the cobrotoxin refold kinetics may change after modification with GA. © 2001 Academic Press Key Words: cobrotoxin; refolding; intramolecular cross-link; folding kinetic.

Cobrotoxin is a neurotoxic protein isolated from the venom of the Taiwan cobra (Naja naja atra) (1, 2). It is a small, basic protein consisting of a single polypeptide chain of 62 amino acids, cross-linked by four disulfide bonds (2). The four disulfide linkages are Cys3-Cys24, Cys17-Cys41, Cys43-Cys54, and Cys55-Cys60, respectively. As shown in Fig. 1, the established tertiary cobrotoxin structure shows a three-loop structure (3). Recently, we found that the disulfide bonds at the C-terminal region of the cobrotoxin exhibit a tendency to exchange with one another (4). Two cobrotoxin isomers (cobrotoxin II and cobrotoxin III) have been isolated from the cobrotoxin refolding mixtures and in Taiwan cobra venom as well (4). The disulfide linkages at the C-terminus of cobrotoxin II and cobrotoxin III are identified as Cys43–Cys55 and Cys54 –Cys60, and Cys43–Cys60 and Cys54 –Cys55, respectively. Moreover, the isomerization reaction is irreversible and decelerated by thiol compounds including GSSG, GSH, cystamine, and cysteamine in a pseudo-first-order kinetic. Chemical modification studies clearly indicate that disulfide isomerization of cobrotoxin is, in part, driven by the positively charged Lys residues at positions 26, 27, and 47 of the toxin molecule and that the thiol compounds are coordinated with the negatively charged cobrotoxin groups to exert their inhibitory action (5). Mutagenesis studies on cobrotoxin further supported this observation (6). Although disulfide isomers were always found with the intermediates along the folding pathway, the disulfide rearrangement caused the resulting product to 289

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tains an ordered state by chemical methods, the refold rate of cobrotoxin should markedly increase. Glutaraldehyde (GA) reacting via nonspecific cross-linking with lysine residues, in a few cases with tyrosine and histidine residues, forms an intra- or intermolecule crosslinked derivative (13). As shown in Fig. 1, the GAreactive residues in cobrotoxin are located near the loop II of cobrotoxin. Thus, the intramolecule crosslinked derivatives may have a propensity to maintain the ordered loop II structure. In this study, cobrotoxin was subjected to modification with GA and then unfolding and refolding of the GA-modified derivative was carried out. It was found that, in comparison with that observed with cobrotoxin, the refold rate of GA-cobrotoxin markedly decreased in redox buffer. On the contrary, the fold rate of GA-modified derivatives was faster than that of cobrotoxin in the absence of GSH/ GSSG. These results provide a novel view of the structural flexibility in how to affect the refold kinetics and the catalytic action of GSH/GSSG in the refolding reaction. FIG. 1. Schematic view of cobrotoxin structure. The model is based on the results of Yu et al. (3). Four disulfide linkages of cobrotoxin are Cys3–Cys24, Cys17–Cys41, Cys43–Cys54, and Cys55–Cys60. The arrows indicate GA-reacting residues.

have native disulfide linkages (7–9). Usually, the thiol compounds such as GSH/GSSG, by which the disulfide formation and reshuffling rates increase, are required for attaining the correct protein folding. The cobrotoxin refolding reaction was indeed accelerated by the addition of GSH/GSSG (4). Moreover, GSH/GSSG redox buffer probably decelerated the rate of disulfide isomerization, and thus the folded cobrotoxin became the predominant product in refolding reaction mixtures. Refold studies on several neurotoxins showed that cobrotoxin, Laticauda semifasciata erabutoxin a and Laticauda semifasciata erabutoxin b renatured slowly (10). This may reflect that different structural elements in neurotoxins are differentiately involved in controlling the formation of their native structures. Recent study suggested that the residues formed turn 2 (corresponding to the residues at positions 18 –23 of cobrotoxin) can account for the observed difference in the refold rate of snake neurotoxins (11). Refolding studies on cobrotoxin have revealed that the ordered structure formation around the single Trp-29 took place prior to the formation of its tertiary structure (4). The cobrotoxin refolding kinetics showed that the late stage(s) of the cobrotoxin refold pathway is characterized by change(s) in the local environment and optical asymmetry of Trp-29 indole ring (12). Thus, one may imagine that if the structure around the sole tryptophan residue of the unfolded cobrotoxin main-

MATERIALS AND METHODS Cobrotoxin from Naja naja atra (Taiwan cobra) venom was isolated and purified as previously described (14). SynChropak RP-P column (4.6 mm ⫻ 25 cm) was obtained from SynChrom Inc. Reduced glutathione (GSH) and oxidized glutathione (GSSG) were purchased from Sigma Chemical Co., and glutaraldehyde (GA) was obtained from Fluka Chemie AG. Acetonitrile, sodium bicarbonate and trifluoroacetic acid (TFA) were purchased from E. Merck. All other reagents were of analytical grade. Modification of cobrotoxin with glutaraldehyde. The Cobrotoxin was modified with glutaraldehyde according to the method described by Onica et al. (15) with a slight modification. Cobrotoxin (50 mg) was dissolved in 5 ml of 0.1 M sodium phosphate buffer (pH 6.8) and then 0.5 ml of 0.25% glutaraldehyde was added dropwisely. The reaction was allowed to proceed at 30°C for 2 h and the mixtures were desalted by passage through a Sephadex G-25 column. The monomeric, dimeric and trimeric derivatives of the GA-modified cobrotoxin were further separated on a Sephadex G-50 column (2.2 ⫻ 137 cm). The homogeneity in the molecular weight of the GA-modified derivatives was identified using SDS–polyacrylamide gel electrophoresis. Identification of the GA-modified residues. To determine the position(s) of the GA-modified residues in the cobrotoxin sequence, the GA-modified derivatives were reduced and S-carboxymethylated (RCM) using the procedure described by Chang et al. (16), followed by hydrolysis with S. aureus V8 protease. RCM-protein was hydrolyzed with S. aureus V8 protease (protein:enzyme, 30:1, w/w) in 0.2 M ammonium bicarbonate buffer (pH 7.8) at 37°C for 3 h. The hydrolysates were then separated using HPLC on a SynChropak RP-P column (4.6 mm ⫻ 25 cm) equilibrated with 0.1% TFA and eluted with a linear gradient of 2.5–35% acetonitrile for 70 min. Reduction/refolding of cobrotoxin. Cobrotoxin and its modified derivatives were reduced and denatured in 0.1 M Tris buffer (pH 8.6) containing 6 M guanidine–HCl and 50 mM dithiotreitol. The reaction was allowed to proceed for 2 h. The sample was then desalted through a PD-10 column (Pharmacia Biotech Ltd.) equilibrated with 0.1 M acetic acid, and subjected to lyophilization. The sample (1 mg/ml) was dissolved in 50 mM sodium bicarbonate (pH 9.7) with or without the addition of 2 mM GSH/0.5 mM GSSG. The samples were

REFOLDING OF COBROTOXIN allowed to refold at 25°C. At appropriate intervals, a portion of each sample was withdrawn and immediately mixed with an equal volume of 4% TFA. The samples were further desalted through a PD-10 column equilibrated with 0.1 M acetic acid. After lyophilization, the samples were analyzed using native gel electrophoresis. In order to further identify the disulfide linkages of the refolded intermediates, the acid-trapped refolded intermediates were reacted with 4-N,N,-dimethylaminoazobenzene-4⬘-iodoacetamide (DABIA) as described by Chatrenet and Chang (17). The DABIA-reacting mixtures were then hydrolyzed with acid protease A. The proteins were dissolved in 1 ml of 5% acetic acid, and acid protease A was added to produce a protein:enzyme ratio of 30:1. The reaction was allowed to proceed for 24 h at 37°C. The hydrolysates were separated using HPLC on a SynChropak RP-P column (4.6 mm ⫻ 25 cm) equilibrated with 0.1% TFA and eluted with a linear gradient of 10 –35% acetonitrile for 70 min. The effluent was monitored at 345 nm to facilitate identification of the DABIA-labeled peptides. The DABIA-labeled peptide fractions were lyophilized for amino acid analysis. Circular dichroism. The CD spectra of cobrotoxin and it’s modified derivative were obtained on a Jasco J-720 spectropolarimeter at a concentration of 0.2 mg/ml in 0.1 M Tris–HCl (pH 8.0) with a cell pathlength of 0.1 cm. The CD spectra were measured from 250 to 200 nm and obtained using signals averaging five scans. Fluorescence measurement. Fluorescence spectra were recorded with a Hitachi model F-4500 spectrophotofluorometer equipped with a high-stability xenon lamp. The intrinsic fluorescence of the native protein and modified derivative were measured with an excitation wavelength at 295 nm in 0.1 M Tris–HCl (pH 8.0). The excitation and emission slit widths were 10 nm. An excitating wavelength at 295 nm was used to ensure that the light was absorbed almost entirely by the tryptophanyl group. Acrylamide quenching Trp fluorescence was studied essentially according to the method described by Chang et al. (4). An excitation wavelength at 295 nm was used to ensure selective excitation of Trp residue. The fluorescence intensity was monitored at the emission maximum, and quenched by the progressive addition of small aliquots of 5 M acrylamide to 1-cm fluorescence cuvettes. Data were corrected for dilution due to the addition of a titrant. Fluorescence quenching for the single Trp residue of cobrotoxin was analyzed according to Stern–Volmer equation: F o/F ⫽ 1 ⫹ K sv[Q], where F o is the fluorescence in the absence of quencher, F is the fluorescence at molar concentration [Q], and K sv is the Stern–Volmer quenching constant obtained from the slope of a plot of F o/F versus [Q]. To assess the ordered structure formation around the Trp, the changes in the K sv values were measured along with the refolding process of cobrotoxin and its modified derivatives. The degree of folded structure formation around Trp-29 was evaluated by the ratio of [K sv(t i) ⫺ K sv(t 120 )]/[K sv(t 0 ) ⫺ K sv(t 120 )]. K sv(t 0 ), K sv(t 120 ), and K sv(t i) represent the quenching constant for acrylamide after the refolding reaction was allowed to proceed for 0 min, 120 min, or indicated time. Previous studies (4) showed that the structure around Trp-29 in unfolded cobrotoxin was completely folded within 120 min. This was evidenced by the observation that the accessibility of the folded cobrotoxin for acrylamide was indistinguishable from native toxin. Native gel electrophoresis. Gel electrophoresis on a 7% polyacrylamide gel (pH 4.5) was carried out as described by Gabriel (18). The gel was stained with 0.05% Coomassie brilliant blue and destained with methanol/acetic acid/H 2O (3:1:6, v/v/v).

RESULTS

Identification of the Residues Modified with Glutaraldehyde The cobrotoxin was modified with glutaraldehyde (GA) at pH 6.8 for 2 h. Three major GA-modified de-

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FIG. 2. Separation RCM-cobrotoxin and RCM-mGA-cobrotoxin peptide fragments after hydrolysis with S. aureus V8 protease. The chromatographic conditions are the same as those described under Materials and Methods. (A) cobrotoxin, (B) mGA-cobrotoxin. Inset: Native gel electrophoresis analysis of cobrotoxin and mGA-cobrotoxin. Lane 1, cobrotoxin; Lane 2, mGA-cobrotoxin.

rivatives, which represented monomeric, dimeric and trimeric GA-cobrotoxins, were separated by gel filtration on a Sephadex G-50 column (data not shown). Further studies on the monomeric GA-cobrotoxin (mGA-cobrotoxin) were carried out in this research. Although the mGA-cobrotoxin was homogeneous in molecular weight as evidenced by SDS–polyacrylamide gel electrophoresis analyses, the results of native gel electrophoresis showed two components appearing in this preparation (Inset of Fig. 2). The two components exhibited a slower electrophoretic mobility than cobrotoxin, indicating that the mGA-cobrotoxin was not contaminated with native toxin. Efforts were made to separate the two components by ion-exchange chromatography on a CM-52 or DE-52 column (Whatman international). The modified derivatives were applied on a CM-52 column (2 ⫻ 25 cm), equilibrated with 0.05 M ammonium acetate (pH 5.0), and eluted with a linear gradient of 350 ml of 0.05 M (pH 5.0) to 0.5 M ammonium acetate (pH 6.8) at the flow rate of 30 ml/h. Alternatively, the modified derivatives were applied on a DE-52 column (2 ⫻ 25 cm), equilibrated with 20 mM Tris–HCl (pH 8.0), and eluted with a linear gradient of 350 ml of 0 to 0.6 M NaCl in the same buffer at a flow rate of 30 ml/h. However, separation of the two components was not successfully achieved by both methods.

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FIG. 3. Electrophoresis analyses of cobrotoxin and mGA-cobrotoxin conversion into its isomers with time. Cobrotoxin (A), mGAcobrotoxin (B) and refolded mGA-cobrotoxin (C) were dissolved in 50 mM sodium bicarbonate (pH 9.7). At appropriate time intervals, the samples were withdrawn for electrophoresis analyses. (A) Lane 1, 0 days; Lane 2, 1 day; Lane 3, 2 days; Lane 4, 3 days; Lane 5, 4 days. (B) Lane 1, 0 days; Lane 2, 2 days; Lane 3, 4 days; Lane 4, 6 days. (C) Lane 1, 0 days; Lane 2, 4 days.

The results of amino acid analyses of the mGA-cobrotoxin showed that only Lys residues were modified and all other amino acids remained essentially unchanged. It indicated that GA reagent exclusively reacted with cobrotoxin Lys residues. Chromatographic analyses on the V8-protease-digested hydrolysates of RCM-mGA-cobrotoxin and RCM-cobrotoxin were carried out as described under Materials and Methods. As shown in Fig. 2, four peptide fragments were separated from the RCM-cobrotoxin hydrolysates. The results of amino acid composition and sequence determinations show that the four peptides, a, b, c, and d are the segments at positions 52– 62, 1–21, 39 –51, and 22–38, respectively. Instead of peptides c and d, two new peptide fractions, e and f, appeared in the map of the RCM-mGA-cobrotoxin hydrolysates. It should be noted that peak c still appeared in the RCM-mGA-cobrotoxin hydrolysates, but its content notably decreased in comparison with peaks a and b. The results of amino acid composition and sequence determinations revealed that peak e represented the segment at positions 22–38 containing the modified residues Lys-26 and Lys-27. Alternatively, fraction f comprised two peptide segments at positions 22–38 and 39 –51, which modified the Lys residues at positions 26, 27, and 47. This indicated that the two peptide fragments 22–38 and 39 –51 were linked together through a cross-linking between Lys-26 (or Lys-27) with Lys-47. Based on these obser-

vations, it was deduced that the two mGA-cobrotoxin components visualized from the native gel (Inset of Fig. 2) were Lys-26 and -27 modified derivative and Lys-26, -27, and -47 modified derivative, respectively. Previous studies showed that accumulative lysine residue trinitrophenylation caused a stepwise decrease in the electrophoretic mobility of cobrotoxin with an increasing number of modified Lys residues (5). It is believed that in a native gel, Lys-26, -27, and -47 modified cobrotoxin should migrate at a slower rate than Lys-26 and -27 modified cobrotoxin. As shown in Fig. 3, mGA-cobrotoxin as well as the native toxin exhibited a tendency to convert into its disulfide isomers in alkali solution. Likewise, the disulfide isomers of mGA-cobrotoxin had a slower electrophoretic mobility, as those noted with cobrotoxin. However, the rate of disulfide isomerization for mGAcobrotoxin was slower than for cobrotoxin. This is in line with previous results showing that cobrotoxin disulfide isomerization is driven by the positively charged Lys residues (5, 6). Folding Reaction of mGA-Cobrotoxin Previous studies showed that two cobrotoxin isomers (cobrotoxin II and cobrotoxin III) have been produced along the refolding reaction of cobrotoxin (4). The disulfide isomers (cobrotoxin II and cobrotoxin III) had the same electrophoretic mobility which slower than that observed for cobrotoxin. As shown in Fig. 4A, after the refolding reaction was allowed to proceed for 3 h, the two bands visualized from the native gels repre-

FIG. 4. Electrophoresis analyses on the folded products of cobrotoxin and mGA-cobrotoxin in the presence of GSH/GSSG. The refolding buffer was 50 mM bicarbonate buffer (pH 9.7) containing 2 mM GSH/0.5 mM GSSG. (A) Lane 1, cobrotoxin; Lane 2, folded products at time 3 h; Lane 3, folded products at time 7 h. The two bands appearing in lanes 2 and 3 represent cobrotoxin and its disulfide isomers, respectively (4). (B) Lane 1, mGA-cobrotoxin; Lane 2, folded products at time 3 h; Lane 3, folded products at time 7 h. The lane 2 showed that the derivative containing modified Lys-26, -27, and -47 residues did not completely refolded within 3 h. This suggested that this mGA-cobrotoxin should fold more slowly in the presence of GSH/GSSG.

REFOLDING OF COBROTOXIN

FIG. 5. Electrophoresis analyses on cobrotoxin and mGA-cobrotoxin folding in the absence of GSH/GSSG. The refolding buffer was 50 mM bicarbonate buffer (pH 9.7) without GSH/GSSG. (A) Lane 1, cobrotoxin; Lane 2, reduced cobrotoxin; Lane 3, folded products at time 24 h; Lane 4, 2 mM GSH/0.5 mM GSSG was added into the folding mixtures at 24 h in bicarbonate buffer, and the folded products were subjected to analysis after 2 h; Lane 5, folded products at 48 h; Lane 6, 2 mM GSH/0.5 mM GSSG was added into the folding mixtures at 48 h in bicarbonate buffer, and the folded products were subjected to analysis after 2 h. (B) Lane 1, mGA-cobrotoxin; Lane 2, folded products at time 24 h; Lane 3, folded products at time 48 h; Lane 4, reduced mGA-cobrotoxin.

sented cobrotoxin and its disulfide isomers, respectively. There is no change in the ratio of cobrotoxin and its isomers after 3 h, suggesting the refold reaction went to completion within 3 h in redox buffer. However, unfolded mGA-cobrotoxin cannot be completely refolded during this period. In particular, the refold rate for the derivative containing modified Lys-26,-27, and -47 residues appreciably decreased (Fig. 4B). Alternatively, unfolded cobrotoxin folded slowly in the absence of thiol compounds. Folded toxin was not noted in the folding reaction mixtures even after the reaction was allowed to proceed for 48 h (Fig. 5A). Nevertheless, the folding reaction could be resumed following the addition of GSH/GSSG, but an increase in the amount of cobrotoxin II and cobrotoxin III isomers was observed with the resumed folding reaction. In the meantime, the refolding rate of mGA-cobrotoxin was faster than cobrotoxin in the absence of thiol compounds (Fig. 5). Unfolded mGA-cobrotoxin flowed primarily toward folded mGA-cobrotoxon within 24 h. The chromatographic profiles of acid protease-digested hydrolysates of refolded mGA-cobrotoxin and its parent protein were not significantly different (data not shown), supporting that the disulfide pairings of mGA-cobrotoxin and refolded mGA-cobrotoxin are the same. Refolded mGAcobrotoxin was further converted into its disulfide isomers with a slower electrophoretic mobility when it was dissolved in alkali buffer (lane 3 of Fig. 5B). The rate of disulfide isomerization of refolded mGA-cobrotoxin was not appreciably distinguishable from that of its parent protein (Fig. 1C). This observation again emphasized the view that refolded mGA-cobrotoxin had native disulfide linkages, and that the refolding reaction could indeed be attained in the absence of GSH/GSSG.

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As shown in Fig. 6, CD measurements reveal that cobrotoxin modified with GA caused a change in the gross cobrotoxin conformation. This probably arose the intramolecular cross-link partly affecting the backbone flexibility of cobrotoxin. Since the unordered polypeptide chain has a strong negative maximum around 200 nm (19), the observed increase in a positive ellipticity at 205 nm may suggest that the cobrotoxin structure shifted toward an ordered form resulting from modification with GA reagent. In fact, bovine trypsin modification with GA leads to a modified catalyst that is resistant to heat and denaturing agent (20). As shown in Fig. 7A, the intensity of cobrotoxin and mGA-cobrotoxin Trp fluoresence gradually decreased with increasing temperature from 25 to 70°C. However, the extent of decrease in Trp fluorescence was greater for cobrotoxin than for mGA-cobrotoxin, suggesting that the stability of the microenvironment around the single Trp-29 in mGA-cobrotoxin was higher than that in cobrotoxin. Moreover, the intensity of intrinisic fluorescence in mGA-cobrotoxin was affected less by pH increment (Fig. 7B). It appeared that the Trp-29 environment in mGA-cobrotoxin than in cobrotoxin was less distorted by alkali-induced denaturation. Likewise, studies on the changes in intrinsic fluorescence of RCM-cobrotoxin and RCM-mGA-cobrotoxin under thermal denaturing and alkali denaturing conditions also revealed that the stability of the microenvironment around Trp-29 in unfolded mGA-cobrotoxin was higher than that in unfolded cobrotoxin (data not shown). The Trp-29 of reduced cobrotoxin and reduced mGAcobrotoxin was more accessible for acrylamide than that of their parent proteins (data not shown). Thus the changes in the accessibility (K sv) for acrylamide along the refolding reaction provided a probe to moni-

FIG. 6. CD spectra of cobrotoxin and mGA-cobrotoxin. The symbols – – – and –䡠–䡠– represent cobrotoxin and mGA-cobrotoxin, respectively.

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were subjected to reactions with DABIA. The hydrolysates of acid protease digested DABIA-reacting mixtures were separated using HPLC (Fig. 9). The amino acid composition and sequence of each peptide fraction was determined. The positions of the cysteine residues inside the peptide fractions are shown in the inset of Fig. 9. The formation of disulfide linkages impeded the reaction of DABIA with Cys residues, thus the amount of DABIA-reacted Cys residues should decrease along with the folding reaction. As shown in Fig. 9, the amount of Cys-3 containing peptide did not decrease in a concerted way with other DABIA-reacting peptides. It is suggested that the disulfide pairing of Cys3–Cys24 should form in the late stage of the cobrotoxin refolding reaction. The same procedure was also carried out for analyzing the disulfide pairings of mGA-cobrotoxin refolded intermediates. Unfortunately, the DABIA-labeled mGA-cobrotoxin was precipitated in an acetic acid solution, thus hydrolyzing the derivative with an acid protease became impossible. DISCUSSION

FIG. 7. Effect of temperature and pH on cobrotoxin and mGAcobrotoxin Trp fluorescence. Sample cuvette contained 10 ␮M of cobrotoxin (䊐) or mGA-cobrotoxin (E). The excitation wavelength is 295 nm, and the fluorescence intensity is monitored at 345 nm. (A) Fo and F are intensities at 25°C and at indicated temperature from 25 to 70°C, respectively. All values are the means of triplicated determinations. (B) Fo and F are intensities at pH 8.0 and at indicated pH from 8.0 to 13.2, respectively. All values are the means of triplicated determinations.

tor the ordered structure formation around Trp-29. As illustrated in Fig. 8, the accessibility (K sv) of reduced cobrotoxin and reduced mGA-cobrotoxin for acrylamide gradually decreased with time. The susceptibility of the folded products for acrylamide was restored to the same degree as that noted for their parent proteins within 120 min. However, the decrease in the susceptibility of mGA-cobrotoxin for acrylamide was faster than that observed with cobrotoxin, reflecting that the ordered structure formation around mGAcobrotoxin Trp-29 should be faster than cobrotoxin Trp-29. This suggested the possibilities that the ordered structure of mGA-cobrotoxin Trp-29 was not distorted completely by reduction of disulfide bonds, and/or an alteration in the cobrotoxin refolding kinetic arose from modification with GA reagent. The Disulfide Pairings of Refolded Intermediates In order to determine the extent of participation of individual cysteines in disulfide pairing, the refolding intermediates of cobrotoxin obtained from redox buffer

The results from this study showed that a decrease in the amount of DABIA-reactive Cys-3 was not accompanied with a decrease in its inherent partner Cys-24. This allows to deduction that disulfide linkage(s) in refolded intermediates of cobrotoxin are not always native before the completion of the folding reaction. In order to achieve the correct pairing between Cys-3 and Cys-24, disulfide rearrangement of the refolding intermediates is expected to occur. The presence of disulfide isomers was also observed along with the refolding process of other proteins such as bovine pancreatic trypsin inhibitor, ribonuclease T, epidermal growth factor, and hirudin (7–9, 21–23). Finally, the disulfide

FIG. 8. The accessibility of Trp for acrylamide along with the folding process of cobrotoxin and mGA-cobrotoxin. The reduced cobrotoxin and reduced mGA-cobrotoxin were dissolved in 0.1 M Tris–1 M NaCl (pH 8.0). The refolding buffer contained 2 mM GSH and 0.5 mM GSSG. All values are the means of triplicate determinations.

REFOLDING OF COBROTOXIN

FIG. 9. A colorimetric cysteine peptide mapping method was employed to evaluate the extent of involvement of the eight cysteines in disulfide pairings of cobrotoxin. The refolding buffer was 50 mM bicarbonate buffer (pH 9.7) containing GSH/GSSG. The refolding intermediates at time 0 h (A), 1/2 h (B), and 1 h (C) were reacted with DABIA, and the DABIA proteins were hydrolyzed with acid protease A. The chromatographic conditions are the same as those described under Materials and Methods. The Cys residues of the peptides are indicated in the inset.

rearrangement produced native disulfide pairings in the resulting products. Previous studies have shown that the protein packing (disulfide formation) is promoted primarily by GSSG, whereas consolidation (disulfide reshuffling) requires GSH as a catalyst (21). The refold rate observed with cobrotoxin in the presence of GSH/GSSG is faster than that in the absence of GSH/GSSG, suggesting that the thiol compounds indeed accelerate the refolding reaction. In contrast to cobrotoxin, the refold rate of mGA-cobrotoxin decreased in the presence of thiol compounds, but increased in the absence of thiol compounds. Previous findings showed that the stability and folding/unfolding properties of Lys-modified Naja naja atra (Taiwan

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cobra) cardiotoxin III, which is structurally homologous to cobrotoxin, is indistinguishable from those of native toxin (24). Thus, it is likely that the changes in the folding kinetics of mGA-cobrotoxin may arise from the intramolecular cross-linking rather than Lys-modification. The observations that refolded mGA-cobrotoxin as its parent protein exhibited a disulfide isomerization reaction (Fig. 1C), emphasized that the pairings of disulfide bonds were essentially not affected by the changes in refolding kinetics. Noticeably, a change in the conformation of mGA-cobrotoxin, probably shifted toward an ordered one, was evidence by CD spectra and fluorescence measurement. Moreover, studies on the intrinsic fluorescence under the conditions of thermal denaturation and alkali denaturation suggest the idea that the ordered structure around Trp-29 in mGAcobrotoxin can not distort completely after its disulfide bonds were reduced. This suggestion was in part supported by the results of fluorescence quenching studies showing that Trp-29 of RCM-mGA-cobrotoxin was less accessible for acrylamide than RCM-cobrotoxin (data not shown). Previous studies showed that the refolding kinetics of cobrotoxin follow a framework model, and the complete secondary structure formation occurs prior to the clustering of the hydrophobic residues (12). In fact, oxidation of Tyr-25, Trp-29, and Tyr-35 with N-bromosuccinimide notably distorted the cobrotoxin refold reaction, and the reduced protein was minimally refolded in the presence of or the absence of GSH/GSSG (data not shown). Thus, it is not surprising to find that the preexistence of an ordered structure around Trp-29 may result in an alteration of the refold kinetics. The finding that the ordered structure around Trp-29 of mGA-cobrotoxin formed rapidly in comparison to cobrotoxin partly supported this proposition. Furthermore, intrinsic fluorescence measurement along with the refolding process showed that a decrease in the fluorescence intensity was noted when the unfolded mGA-cobrotoxin became a folded protein, but an increase in the fluorescence intensity was noted when reduced cobrotoxin was folded (data not shown). This again emphasizes the view that the refolding kinetics of mGA-cobrotoxin is different from that of cobrotoxin. Three mechanisms, including a framework model, nucleation model, and hydrophobic collapse model, have been widely employed to describe the protein folding routes (25). The hydrophobic collapse model hypothesized that a protein would collapse rapidly around its hydrophobic side chains and then rearrange from the restricted conformational space occupied by the intermediates. The ordered structure around Trp-29 may allow a hydrophobic collapse occur prior to the conformational rearrangement, and thus an accelerated rate in the mGA-cobrotoxin refold reaction is observed in the absence of GSH/GSSG. Whether the cobrotoxin

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refold kinetic shifts from a framework model to a hydrophobic collapse model after modification with GA remains to be studied. Interestingly, a sharp contrast effect by thiol compounds on the refolding of mGAcobrotoxin and cobrotoxin highly suggests the view that the structural flexibility of unfolded cobrotoxin is required for the thiol catalyst action. ACKNOWLEDGMENTS This work was supported by Grants NSC 89-2320-B110-008 (to L. S. Chang) and NSC89-2311-B007-035 (to C. C. Yang) from the National Science Council, Republic of China.

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