pH-responsive polymer-assisted refolding of urea- and organic solvent-denatured {alpha}-chymotrypsin

I. Roy and M.N. Gupta1

Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India

1 To whom correspondence should be addressed. e-mail: mn_gupta{at}hotmail.com


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A pH-responsive polymer Eudragit S-100 has been found to assist in correct folding of {alpha}-chymotrypsin denatured with 8 M urea and 100 mM dithiothreitol at pH 8.2. The complete activity could be regained within 10 min during refolding. Both native and refolded enzymes showed emission of intrinsic fluorescence with {lambda}max of 342 nm. Gel electrophoresis showed that the presence of Eudragit S-100 led to dissociation of multimers followed by the appearance of a band at the monomer position. The unfolding (by 8 M urea) and folding (assisted by the polymer) also led to complete renaturation of {alpha}-chymotrypsin initially denatured by 90% dioxane. The implications of the data in recovery of enzyme activity from inclusion bodies and the interesting possibility in the in vivo context of reversing protein aggregation in amyloid-based diseases have been discussed.

Keywords: {alpha}-chymotrypsin/chemical denaturation/inclusion bodies/pH-responsive polymers/protein folding


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The correct folding of a protein in vivo often requires molecular chaperonins (Jaenicke and Seckler, 1999Go). If misfolding occurs, it leads to formation of protein aggregates. Such aggregates, in vivo, have been identified as the molecular basis of several human diseases such as Alzheimier’s disease, Creutzfeld–Jakobs disease and bovine spongiform encephalopathy (Carrell and Lomas, 1997Go). In bioprocessing (in vitro conditions), incorrect folding limits yields of recombinant proteins since over-expression (especially in Escherichia coli) generally forms insoluble inclusion bodies (Betts et al., 1999Go). Thus, understanding of protein refolding under various conditions is relevant to both in vivo and in vitro contexts.

The recovery of biological activity in the case of inclusion bodies is generally carried out by solubilizing these dense intracellular aggregates by unfolding protein molecules using denaturants such as 8 M urea or 6 M guanidine hydrochloride. It is for this reason that many attempts have been made to optimize the folding of denatured proteins. Such efforts include the use of water-soluble polymers (Middelberg, 2002Go). The pioneering work of Wang and coworkers (Cleland et al., 1992Go) indicated that polyethylene glycol, a water-soluble polymer, facilitates correct folding via hydrophobic interaction with initial folding intermediates that are prone to aggregation. In this respect, such polymers act similar to molecular chaperonins (Jaenicke and Seckler, 1999Go).

We have been working with a commercially available enteric polymer Eudragit S-100 for its application in bioseparation and biocatalysis (Sardar et al., 2000Go; Sharma and Gupta, 2002Go). This non-toxic methylmethacrylate polymer is a reversibly soluble polymer, which precipitates at slightly acidic pH (~4.8) and redissolves around pH 6.0. (The exact transition point for solubility depends upon medium properties such as ionic strength.) This pH-responsive behavior is the result of the presence of both free carboxyl groups and hydrophobic moieties. At lower pH, carboxyl groups are protonated and hydrophobic interactions dominate; this leads to precipitation of the polymer. In view of the presence of hydrophobic moieties, this polymer was thought to be a promising candidate for facilitating protein folding. In this work, this polymer is evaluated for facilitating correct folding of urea-denatured {alpha}-chymotrypsin. It is shown that this may be one of the best additives for assisting correct folding of proteins in vitro.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Enzyme assay

{alpha}-Chymotrypsin was assayed using benzoyl tyrosine ethyl ester as the substrate (Walsh and Wilcox, 1970Go). The enzyme unit is defined as the amount of enzyme which hydrolyzes 1 µmol of the ester per minute at pH 7.8 and at 25°C, under specified conditions.

Unfolding and refolding of {alpha}-chymotrypsin

{alpha}-Chymotrypsin (from bovine pancreas, 3x crystallized from 4x crystallized chymotrypsinogen, Cat. No. C-4129; Sigma Chemical Co., St Louis, MO, USA) was used without further purification. The stock solution of Eudragit S-100 (Rohm Pharma GmbH, Weiterstadt, Germany) was prepared as described earlier (Sardar et al., 2000Go). Eudragit S-100 is a copolymer of methacrylic acid and methyl methacrylate (in a molar ratio of 1:2), with molecular weight in the range of 200 000–250 000 (Product Sheet; Rohm Pharma GmbH). {alpha}-Chymotrypsin (10 mg) was dissolved in 0.05 M Tris–HCl buffer pH 8.2, containing 8 M urea (molecular biology grade; Sisco Research Laboratories, Mumbai, India) and 100 mM dithiothreitol (DTT) and incubated at 25°C for 4 h. A 0.1 ml aliquot of this unfolded enzyme was diluted with 0.1 ml of Eudragit S-100 (stock solution of 5%) and the final volume was made up to 1 ml with 0.05 M Tris–HCl buffer, pH 8.2. The diluted sample was then assayed for enzyme activity.

Native gel electrophoresis of {alpha}-chymotrypsin (Betts et al., 1999)

Gels were cast at room temperature. The stacking gel was 4% acrylamide/bisacrylamide, whereas the resolving gel was 10% acrylamide/bisacrylamide. The concern that stacking of aggregation intermediates will promote further aggregation in the gel was taken care of by comparing the distribution of multimers in the same sample separated by electrophoresis with and without a stacking gel. Gels and running buffer were equilibrated at 4°C before loading chilled samples. Sample loading and electrophoresis were performed in a cold room (4–5°C). The samples, collected at different time intervals, were stored on ice. The efficiency of cold quenching was determined by assaying the samples at various time intervals after chilling. The gel was stained with Coomassie Blue G-250 at room temperature for 45 min and then destained.

Inactivation of {alpha}-chymotrypsin by organic solvent

{alpha}-Chymotrypsin (10 mg) was suspended in 1 ml of 90% dioxane (in 0.05 M Tris–HCl, pH 7.8) and shaken at 25°C. After 1 h, the residual enzyme activity was measured in the following ways (Soler et al., 1997Go). (i) Aliquots were withdrawn at different time intervals and directly assayed in the presence of the organic co-solvent. (ii) Aliquots of the suspended enzyme were directly assayed using the standard protocol, in the absence of organic co-solvent. (iii) Aliquots were withdrawn and washed thoroughly with the assay buffer and incubated in the same for 24 h; they were then assayed as usual. (iv) Aliquots were withdrawn and washed thoroughly with the assay buffer. The samples were then incubated in 0.05 M Tris–HCl buffer, pH 8.2, containing 8 M urea and 100 mM DTT for 2 h at 25°C. The denatured samples were then renatured with 0.5% Eudragit S-100, as described earlier.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Role of Eudragit S-100 during unfolding and refolding

Figure 1 shows that the presence of Eudragit S-100 reduces the unfolding of {alpha}-chymotrypsin in the presence of various concentrations of urea. Unfolding of {alpha}-chymotrypsin leads to increase in emission intensity of intrinsic fluorescence (Desie et al., 1986Go), hence fluorescence emission is considered a sensitive tool to monitor the extent of unfolding. Figure 2 shows the effect of the presence of polymer during the refolding of urea-denatured enzyme. The presence of polymer not only led to a higher regain of activity but the refolding rate was also much faster. In the absence of the polymer, only 31% of the original activity could be regained after 15 h. No further regain of activity was observed beyond this time. On the other hand, the presence of the polymer led to a swift regain of 100% activity within 10 min.



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Fig. 1. Fluorescence analysis of unfolding of {alpha}-chymotrypsin in the presence of 8 M urea. The emission spectra (at {lambda} = 340 nm) of {alpha}-chymotrypsin were monitored with excitation ({lambda}max) at 282 nm. The relative fluorescence (F/F0) refers to the fluorescence of denatured enzyme (filled circle) (at a particular urea concentration) to that of the free enzyme (empty circle).

 



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Fig. 2. Effect of time on the renaturation of {alpha}-chymotrypsin. {alpha}-Chymotrypsin (10 mg) was dissolved in 1 ml of 0.05 M Tris–HCl buffer, pH 8.2 containing 8 M urea and 100 mM DTT and incubated for 4 h at 25°C. After this, the solution was diluted 10-fold with 0.5 ml of Eudragit S-100 (0.1 ml of 5% stock solution) and the final volume was made up to 1 ml with 0.05 M Tris–HCl buffer, pH 8.2 (closed circles). The activity was determined at various time intervals (a) 0–20 min and (b) 0–24 h. A control was run where the dilution was done with 0.05 M Tris–HCl buffer, pH 8.2 (open circles). The starting enzyme activity has been taken as 100%.

 
The molar ratio of the polymer to enzyme was a critical parameter in influencing the polymer-assisted refolding (Figure 3). Even within 5 min of refolding time, as high as 71% activity could be regained with a polymer:enzyme ratio of 2. With 1 h and higher refolding time, 100% activity could be regained even with polymer:enzyme ratio of 1. At smaller ratios (of polymer:enzyme), there was a trade-off between this ratio and the time required to regain a specific level of enzyme activity. For example, when this ratio was 0.4, 50% activity was regained within 5 min; to regain the same level of activity at a molar ratio of 0.2, ~6 h were required. The optimum values, along with the appropriate control, are summarized in Table I.



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Fig. 3. Effect of varying polymer to enzyme ratio on the amount of activity regained. The unfolding of {alpha}-chymotrypsin was carried out as described in the caption to Figure 2. Different aliquots of Eudragit S-100 (stock solution of 5%) were added to the unfolded enzyme, the total volume made up to 1 ml with 0.05 M Tris–HCl, pH 8.2 and the regained activity measured after 5 min (circles), 1 (squares), 4 (triangles) and 6 h (crosses). The starting enzyme activity has been taken as 100%.

 

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Table I. Protein folding in the presence and absence of Eudragit S-100
 
The intrinsic fluorescence emission was also used to confirm that the refolded molecule with 100% regain in activity was structurally similar to the native {alpha}-chymotrypsin (Table II). Urea denaturation led to an increase in quantum yield as well as shift of {lambda}max (emission) to a higher wavelength. Both features are known to be associated with the protein folding process (Cleland and Wang, 1990Go). The refolded molecule, in the presence of Eudragit S-100, had fluorescence emission characteristics very close to the native enzyme.


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Table II. Emission spectra of {alpha}-chymotrypsin
 
It is well established that second and higher order aggregation processes interfere with the correct refolding of the proteins (Jaenicke and Seckler, 1999Go). Thus, the protein concentration during refolding is considered a critical parameter. Figure 4 shows the effect of the initial protein concentration with which refolding (by diluting it 10 times with Eudragit S-100 solution) was initiated. Up to 12 mg ml–1 (of protein concentration), the refolding process gave 100% regain of activity. Beyond this initial protein concentration, activity regain decreased rapidly. However, even at 20 mg ml–1, as high as 73% activity could be regained.



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Fig. 4. Effect of initial protein concentration on the amount of activity regained. Different amounts of {alpha}-chymotrypsin (1.8–18 mg) were weighed and denatured with 8 M urea, as described before. The samples were then diluted with 0.5% Eudragit S-100 and checked for enzyme activity. The starting enzyme activity has been taken as 100%.

 
Native gel electrophoresis for detection of aggregates during unfolding and refolding

The work of Betts et al. (Betts et al., 1999Go) has established that native gel electrophoresis constitutes one of the simplest and perhaps the best method for detection of aggregation intermediates during unfolding and refolding of proteins. Figure 5 shows that urea-denatured {alpha}-chymotrypsin, 5 min after being placed in the refolding buffer, consisted of dimers and multimers (lane A). Even after 1 h, higher molecular weight multimers, with a considerable extent of aggregation, could still be observed (lane B). However, if the refolding buffer contained Eudragit S-100, within 5 min, a considerable number of higher molecular weight multimers disappeared (lane C). After 1 h (in the presence of Eudragit S-100; lane D), only monomeric {alpha}-chymotrypsin could be observed at a position similar to the position of native {alpha}-chymotrypsin; lane E). Comparison of lanes C and D indicate that with time, protein–protein interactions are replaced by polymer–protein interactions. Thus, the latter are favored thermodynamically and lead to correct refolding in the presence of the polymer.



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Fig. 5. Gel electrophoresis of {alpha}-chymotrypsin. Native gel electrophoresis (under non-reducing conditions) showing time course multimerization of {alpha}-chymotrypsin. Lane A, denatured enzyme in refolding buffer (5 min after dilution); lane B, denatured enzyme in refolding buffer (1 h after dilution); lane C, denatured enzyme in refolding buffer containing 0.5% Eudragit S-100 (5 min after dilution); lane D, denatured enzyme in refolding buffer containing 0.5% Eudragit S-100 (1 h after dilution); lane E, {alpha}-chymotrypsin. Unfolding and refolding of the enzyme were carried out as described in the Materials and methods.

 
Renaturation after inactivation by organic solvent

Soler et al. (Soler et al., 1997Go) have reported that {alpha}-chymotrypsin gets inactivated in the presence of 90% dioxane. Table III shows that placing {alpha}-chymotrypsin in 90% dioxane for 1 h at 25°C led to complete inactivation of the enzyme. Dilution with the aqueous assay buffer led to slow and only ~10% regain of enzyme activity. The organic solvent induced denaturation could be reversed after unfolding the inactivated enzyme by 8 M urea (Table III). After urea denaturation, whereas dilution with simple assay buffer led to only 5.5% regain of the activity after 1 h, dilution with Eudragit S-100 led to a fairly rapid regain of the activity. Approximately 78% activity could be regained within 5 min and after 1 h, 100% activity regain was observed.


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Table III. Denaturation of {alpha}-chymotrypsin by organic solvent
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Choice of urea as a denaturant

Solubilization of inclusion bodies is usually carried out in 8 M urea or 6 M guanidine hydrochloride at alkaline pH and in the presence of a reducing agent like ß-mercaptoethanol or dithiothreitol (Jaenicke and Rudolph, 1989Go). In the case of {alpha}-chymotrypsin, Hibbard and Tulinsky (Hibbard and Tulinsky, 1978Go) have shown the massive penetration of the interior of the enzyme by urea, ‘in contrast to guanidine hydrochloride which showed no significant changes in the protein interior’. In fact, urea bound within protein interiors is presumed to stabilize the partly folded protein chains. This ‘double-edged denaturation/activation of enzymes’ has been usefully employed for enhancing enzyme activity in organic solvents (Guo and Clark, 2001Go). Thus, for the purpose of unfolding/refolding proteins in inclusion bodies, urea appears to be a more suitable choice as it is expected to ‘stabilize’ the unfolded intermediates and simultaneously help unfolding the insoluble protein bodies sufficiently to correct any misfolded structures. As the aim of the present work was to use Eudragit S-100 in refolding denatured {alpha}-chymotrypsin, as a model system for possible future application to recovery of biological activity from inclusion bodies, 8 M urea was chosen as the denaturant.

Refolding of denatured {alpha}-chymotrypsin

In the case of recovery of active proteins from inclusion bodies, the unfolding step (which solubilizes the insoluble and misfolded protein moieties) is followed by the refolding step. The protein refolding is initiated by reduction in the concentration of denaturant. Two competing processes at this stage are the first order refolding and higher order aggregation. Thus, carrying out refolding at dilute protein concentration (<10–50 µg ml–1) reduces aggregation and leads to a varying extent of regain of protein activity. However, such low concentrations are impractical for obtaining refolded proteins in an economical way. This has been the motivation for the search for additives which facilitate correct refolding, even at a practical range of protein concentrations. As Middelberg (Middelberg, 2002Go) has pointed out, the search for conditions for efficient refolding of denatured proteins continues to be an ongoing effort. Recently, a temperature-sensitive polymer, poly(N-isopropylacrylamide), has been used for refolding of carbonic anhydrase B (Chen et al., 2003Go). Even with an initial protein concentration of 5 mg ml–1 (diluted 100 times with the refolding buffer), not more than 75% of activity could be regained. The time period was not clearly mentioned but appeared to be more than 1 h. The refolding yield decreased rapidly with increase in protein concentration; with an initial protein concentration of 15 mg ml–1, the reported refolding yield was 39%. It is also considered advisable to use denaturants at non-denaturing conditions at the refolding stage. The presence of these denaturants at much lower concentrations is presumed to prevent aggregation and hence promote correct folding (Clark et al., 1999Go). Thus, all refolding steps in this work were carried out in the presence of 0.8 M urea. In the present work, the use of the pH-responsive polymer as an additive in the refolding buffer led to a 100% regain of activity within 10 min, even when the initial protein concentration was 12.5 mg ml–1 (diluted 10 times with the refolding buffer). In fact, we have not come across a more efficient refolding additive in the literature. Our approach is fast, consists of a single step and leads to complete recovery of the enzyme activity.

Renaturation of {alpha}-chymotrypsin inactivated by dioxane

The use of enzymes and other proteins in a predominantly non-aqueous environment has emerged as an important approach in organic synthesis and the resolution of enantiomers (Gupta, 2000Go). Thus, understanding their instability/inactivation in organic solvents is vital to enlarging the scope of non-aqueous enzymology. Soler et al. (Soler et al., 1997Go) showed that {alpha}-chymotrypsin, inactivated in 90% dioxane, did not show significant renaturation even after 24 h. However, unfolding–refolding gave complete recovery in the case of the immobilized preparation. The polymer-assisted refolding worked equally well in the current work even when the enzyme was free and not anchored to a stabilizing matrix. The activity regain was much faster and complete recovery within 1 h was observed.

Possible role of the polymer in folding

Eudragit S-100 is a stimulus-responsive or smart polymer (Roy and Gupta, 2003Go). It is a pH-responsive polymer, which is soluble at pH 8.2, the pH used during the refolding experiments. At this pH, it is an anionic polymer consisting of carboxylate moieties. It also has hydrophobic structural components in the form of esterified carboxylate groups. Cleland et al. (Cleland et al., 1992Go) concluded in their work that both polyethylene glycol and the chaperonin GroEL, bind to hydrophobic structures in the partially unfolded protein molecules and thus inhibit their aggregation without altering their rate of folding. The mode of increasing percent refolding (to correct structure) by Eudragit S-100 is likely to be similar. Chen et al. (Chen et al., 2003Go), while working with a temperature-responsive smart polymer, have also explained the role of such a polymer in assisting protein refolding in a similar way. However, unlike the results of Cleland et al. (Cleland et al., 1992Go) with polyethylene glycol, Eudragit S-100 in fact did alter the rate of refolding. Such an acceleration of refolding by anionic polymers has been reported by Rentzeperis et al. (Rentzeperis et al., 1999Go), who implicated electrostatic interactions in the rate acceleration of the polyanion-mediated refolding. It is likely that Eudragit S-100 achieves the acceleration of refolding in a similar way.

Potential in vitro and in vivo applications

As mentioned in the Introduction, water-soluble polymers (and chaperonins in vivo) are assumed to assist correct refolding by preventing aggregation by attachment to exposed hydrophobic portions of the polypeptide. We have not come across any study where the refolding additives are shown to dissociate first the preformed aggregates, and then push the folding of the monomeric protein along the right pathway. The native gel electrophoresis data (Figure 5) shown in this work indicate this new role of refolding additives. It is possible that other workers have not observed it because most of the refolding studies have been done with fairly dilute solutions. Thus, it is possible that upon extensive dilution, refolding additives merely had to assist refolding of monomers since, in these cases, dilution prevented aggregate formation. It may be intriguing to determine whether molecular chaperonins also play a similar role in vivo.

One of the key approaches in understanding protein folding has been to study refolding of unfolded proteins. Besides, recovery of activity from over-expressed proteins (in the form of inclusion bodies) and involvement of protein aggregation in the case of several human diseases (Sadana, 2000Go; Carrio and Villaverde, 2001Go) are two important areas wherein studying protein unfolding–refolding is relevant. The use of a non-toxic, pH-responsive polymer in the present work has the additional advantage that the polymer can be separated and reused merely by precipitation around pH 5.0. Most of the proteins, including {alpha}-chymotrypsin, are stable around this pH region. The possibility of refolding at fairly high protein concentration provides one with a practical method for renaturation.


    Acknowledgements
 
The partial support provided by the Council for Scientific and Industrial Research (CSIR) (Extramural Division) and Department of Science and Technology, both Government of India organizations, is gratefully acknowledged.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received June 17, 2003; revised September 26, 2003; accepted October 21, 2003





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