Efficient Refolding of Aggregation-prone Citrate Synthase by Polyol Osmolytes
2005; Elsevier BV; Volume: 280; Issue: 16 Linguagem: Inglês
10.1074/jbc.m410947200
ISSN1083-351X
AutoresRajesh Mishra, Robert Seckler, Rajiv Bhat,
Tópico(s)Protein Structure and Dynamics
ResumoEfficient refolding of proteins and prevention of their aggregation during folding are of vital importance in recombinant protein production and in finding cures for several diseases. We have used citrate synthase (CS) as a model to understand the mechanism of aggregation during refolding and its prevention using several known structure-stabilizing cosolvent additives of the polyol series. Interestingly, no parallel correlation between the folding effect and the general stabilizing effect exerted by polyols was observed. Although increasing concentrations of polyols increased protein stability in general, the refolding yields for CS decreased at higher polyol concentrations, with erythritol reducing the folding yields at all concentrations tested. Among the various polyols used, glycerol was the most effective in enhancing the CS refolding yield, and a complete recovery of enzymatic activity was obtained at 7 m glycerol and 10 μg/ml protein, a result superior to the action of the molecular chaperones GroEL and GroES in vitro. A good correlation between the refolding yields and the suppression of protein aggregation by glycerol was observed, with no aggregation detected at 7 m. The polyols prevented the aggregation of CS depending on the number of hydroxyl groups in them. Stopped-flow fluorescence kinetics experiments suggested that polyols, including glycerol, act very early in the refolding process, as no fast and slow phases were detectable. The results conclusively demonstrate that both the thermodynamic and kinetic aspects are critical in the folding process and that all structure-stabilizing molecules need not always help in productive folding to the native state. These findings are important for the rational design of small molecules for efficient refolding of various aggregation-prone proteins of commercial and medical relevance. Efficient refolding of proteins and prevention of their aggregation during folding are of vital importance in recombinant protein production and in finding cures for several diseases. We have used citrate synthase (CS) as a model to understand the mechanism of aggregation during refolding and its prevention using several known structure-stabilizing cosolvent additives of the polyol series. Interestingly, no parallel correlation between the folding effect and the general stabilizing effect exerted by polyols was observed. Although increasing concentrations of polyols increased protein stability in general, the refolding yields for CS decreased at higher polyol concentrations, with erythritol reducing the folding yields at all concentrations tested. Among the various polyols used, glycerol was the most effective in enhancing the CS refolding yield, and a complete recovery of enzymatic activity was obtained at 7 m glycerol and 10 μg/ml protein, a result superior to the action of the molecular chaperones GroEL and GroES in vitro. A good correlation between the refolding yields and the suppression of protein aggregation by glycerol was observed, with no aggregation detected at 7 m. The polyols prevented the aggregation of CS depending on the number of hydroxyl groups in them. Stopped-flow fluorescence kinetics experiments suggested that polyols, including glycerol, act very early in the refolding process, as no fast and slow phases were detectable. The results conclusively demonstrate that both the thermodynamic and kinetic aspects are critical in the folding process and that all structure-stabilizing molecules need not always help in productive folding to the native state. These findings are important for the rational design of small molecules for efficient refolding of various aggregation-prone proteins of commercial and medical relevance. Aggregation of proteins during folding, both in vitro and in vivo, is known to lead to low native protein yields as well as the onset of several age-related diseases (1Dobson C.M. Nature. 2003; 426: 884-890Crossref PubMed Scopus (3895) Google Scholar). Hence, there is a growing interest in developing strategies to prevent protein aggregation to enhance protein refolding yields and to design drugs for diseases involving protein aggregation. Several attempts have been made in this direction with successes as well as failures (2De Bernardez-Clark E. Schwarz E. Rudolph R. Methods Enzymol. 1999; 309: 217-236Crossref PubMed Scopus (224) Google Scholar, 3Mason J.M. Kokkoni N. Stott K. Doig A.J. Curr. Opin. Struct. Biol. 2003; 13: 526-532Crossref PubMed Scopus (119) Google Scholar, 4Hammarström P. Wiseman R.L. Powers E.T. Kelly J.W. Science. 2003; 299: 713-716Crossref PubMed Scopus (463) Google Scholar). In this study, we used citrate synthase (CS), 1The abbreviations used are: CS, citrate synthase; GdmCl, guanidinium chloride. a non-disulfide-bonded dimeric protein (∼100 kDa) highly prone to aggregation during in vitro refolding (5West S.M. Kelly S.M. Price N.C. Biochim. Biophys. Acta. 1990; 1037: 332-336Crossref PubMed Scopus (34) Google Scholar, 6Buchner J. Schmidt M. Fuchs M. Jaenicke R. Rudolph R. Schmid F.X. Kiefhaber T. Biochemistry. 1991; 30: 1586-1591Crossref PubMed Scopus (439) Google Scholar, 7Zhi W. Landry S.J. Gierasch L.M. Srere P.A. Protein Sci. 1992; 1: 522-529Crossref PubMed Scopus (98) Google Scholar, 8Jakob U. Lilie H. Meyer I. Buchner J. J. Biol. Chem. 1995; 270: 7288-7294Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 9Rozema D. Gellman S.H. J. Am. Chem. Soc. 1995; 117: 2373-2374Crossref Scopus (230) Google Scholar, 10Buchner J. Grallert H. Jakob U. 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Using the molecular chaperones GroEL and GroES, up to 80% refolding of CS could be obtained at 10 μg/ml protein, whereas unassisted refolding was only 5% (7Zhi W. Landry S.J. Gierasch L.M. Srere P.A. Protein Sci. 1992; 1: 522-529Crossref PubMed Scopus (98) Google Scholar). Inspired by the GroEL/ES-assisted two-step folding mechanism, Rozema and Gellman (9Rozema D. Gellman S.H. J. Am. Chem. Soc. 1995; 117: 2373-2374Crossref Scopus (230) Google Scholar) proposed an artificial chaperone-assisted refolding system in which a detergent was used to prevent aggregation, and cyclodextrin was added to remove the detergent, leading to the native structure formation. Using this strategy, a 65% refolding yield of CS was observed (21Daugherty D.L. Rozema D. Hanson P.E. Gellman S.H. J. Biol. Chem. 1998; 273: 33961-33971Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Recently, a similar two-step strategy has also been applied using cycloamylose, and complete recovery of the refolded protein has been claimed (12Machida S. Ogawa S. Xiaohua S. Takaha T. Fujii K. Hayashi K. FEBS Lett. 2000; 486: 131-135Crossref PubMed Scopus (173) Google Scholar). Despite its successful use in improving the refolding yield, the two-step artificial chaperone strategy is complicated, requiring the removal of the accessory agents before the enzyme can be effectively used. Another useful strategy that has been applied to improve the refolding yield of proteins is to use small molecular mass additives in the refolding buffer. The advantage of using such a strategy over the artificial chaperone strategy is that it is convenient and cost-effective, and the additives need not be removed from the refolding buffers. One can also select compounds that are simple in structure and that are biocompatible, thereby offering a possibility to be used in vivo to correct protein misfolding and to prevent aggregation responsible for a growing number of human and animal diseases (22Tatzelt J. Prusiner S.B. Welch W.J. EMBO J. 1996; 15: 6363-6373Crossref PubMed Scopus (271) Google Scholar, 23Sato S. Ward C.L. Krouse M.E. Wine J.J. Kopito R.R. J. Biol. Chem. 1996; 271: 635-638Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, 24Tamarappoo B.K. Yang B. Verkman A.S. J. Biol. Chem. 1999; 274: 34825-34831Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Efforts are being made to enable the design of such compounds, referred to as "neutral crowders," employing molecular dynamics simulations of protein associations in their presence (25Baynes B.M. Trout B.L. Biophys. J. 2004; 87: 1631-1639Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Low concentrations of denaturants such as arginine hydrochloride (2De Bernardez-Clark E. Schwarz E. Rudolph R. Methods Enzymol. 1999; 309: 217-236Crossref PubMed Scopus (224) Google Scholar, 26Buchner J. Rudolph R. Bio/Technology. 1991; 9: 157-162Crossref PubMed Scopus (404) Google Scholar, 27Arora D. Khanna N. J. Biotechnol. 1996; 52: 127-133Crossref PubMed Scopus (133) Google Scholar), polyethylene glycols (28Cleland J.L. Wang D.I. Bio/Technology. 1990; 8: 1274-1278Crossref PubMed Scopus (127) Google Scholar, 29Cleland J.L. Wang D.I. Biochemistry. 1990; 29: 11072-11078Crossref PubMed Scopus (152) Google Scholar, 30Wetlaufer D.B. Xie Y. Protein Sci. 1995; 4: 1535-1543Crossref PubMed Scopus (143) Google Scholar), detergents (31Tandon S. Horowitz P.M. J. Biol. Chem. 1987; 262: 4486-4491Abstract Full Text PDF PubMed Google Scholar, 32Krause M. Rudolph R. Schwarz E. FEBS Lett. 2002; 532: 253-255Crossref PubMed Scopus (19) Google Scholar), and polyols and sugars (33Valax P. Georgiou G. Am. Chem. Soc. Symp. Ser. 1991; 470: 97-109Google Scholar, 34Gorovits B.M. McGee W.A. Horowitz P.M. Biochim. Biophys. Acta. 1998; 1382: 120-128Crossref PubMed Scopus (31) Google Scholar, 35Yu Z. Li B. Protein Pept. Lett. 2003; 10: 199-211Crossref PubMed Scopus (11) Google Scholar) have been used to enhance the refolding yield of several proteins. Despite successful use of these molecules in enhancing protein refolding yields, their mechanism of action for the refolding process is far from clear. In this study, we used a series of polyol osmolytes with increasing numbers of hydroxyl groups in an attempt to enhance the refolding yield of CS as well as to investigate their mechanism of action given that their mechanism of action for enhancing protein stability is well understood (36Gekko K. Timasheff S.N. Biochemistry. 1981; 20: 4667-4676Crossref PubMed Scopus (1128) Google Scholar, 37Gekko K. Timasheff S.N. Biochemistry. 1981; 20: 4677-4686Crossref PubMed Scopus (446) Google Scholar, 38Gekko K. Morikawa T. J. Biochem. (Tokyo). 1981; 90: 39-50Crossref PubMed Scopus (139) Google Scholar, 39Gekko K. Morikawa T. J. Biochem. (Tokyo). 1981; 90: 51-60Crossref PubMed Scopus (91) Google Scholar, 40Gupta V. Bhat R. Geisow M.J. Epton R. Perspectives on Protein Engineering and Complementary Technologies. Mayflower Worldwide Ltd., Birmingham, United Kingdom1995: 209-212Google Scholar, 41Kaushik J.K. Bhat R. J. Phys. Chem. B. 1998; 102: 7058-7066Crossref Scopus (212) Google Scholar). Because glycerol was found to be the most effective among the polyols used in enhancing the refolding yield, extended studies were carried out with glycerol to understand its mechanism of action in detail. Understanding the mechanism of cosolvent-mediated folding of proteins and the prevention of their aggregation would be helpful not only in the rational design of additives for enhancing protein refolding yields, but also in the prevention of aggregation of proteins involved in several conformational diseases. Materials—Acetyl coenzyme A, CS (porcine heart), dithioerythritol, 5,5′-dithiobis(2-nitrobenzoic acid), erythritol, oxalacetate, sorbitol, Tris, and xylitol were purchased from Sigma. Ethylene glycol (Qualigens India Ltd.), glycerol (Roth Chemicals), and guanidinium chloride (GdmCl; Sigma and ICN Biomedicals) were of the highest purity grade and were used as such. For protein concentration determination and enzyme assay, Hitachi U2000, Specord (Analytik Jena AG), and Cary spectrophotometers were used. For maintaining constant temperature, a Haake Technik GmbH water bath was used. Refolding Protocol—The concentration of native CS was determined by measuring the absorbance at 280 nm using ϵ280 = 1.78 for a 1 mg/ml solution in a 1-cm path length cuvette (42Singh M. Brooks G.C. Srere P.A. J. Biol. Chem. 1970; 245: 4636-4640Abstract Full Text PDF PubMed Google Scholar), and the enzyme was used without further purification. CS (1 mg/ml) was denatured in 0.1 m Tris-HCl containing 6.0 m GdmCl and 20 mm dithioerythritol for at least 1 h at room temperature (6Buchner J. Schmidt M. Fuchs M. Jaenicke R. Rudolph R. Schmid F.X. Kiefhaber T. Biochemistry. 1991; 30: 1586-1591Crossref PubMed Scopus (439) Google Scholar). Refolding experiments were carried out by diluting the denatured protein into the refolding buffer (0.1 m Tris-HCl, pH 8.1). In the case of cosolvent-assisted refolding, various cosolvents were added at different concentrations to the refolding buffer into which the denatured protein was mixed. Refolding was carried out by single shot dilution in a glass test tube, in which the denatured protein was diluted 1:100 in the refolding buffer by vigorous vortexing for 30–60 s. Extreme care had to be taken at this step because rapid mixing was essential for reproducibility and better refolding yields. Refolded samples were incubated at the desired temperatures until completion of the reaction. The refolding at 25 °C was complete in <2 h. CS activity assay was carried out to follow the formation of native CS molecules. CS catalyzes the following reaction: oxalacetate + acetyl coenzyme A → citrate + CoASH. The free–SH groups of CoASH react with 5,5′-dithiobis(2-nitrobenzoic acid) and form a mercaptide ion, 2-nitro-5-thiobenzoate–, which absorbs at 412 nm. The reaction mixture contained 100 mm Tris-HCl, 0.1 mm 5,5′-dithiobis(2-nitrobenzoic acid), 0.047 mm acetyl coenzyme A, and 0.023 mm oxalacetate. For the activity assay, 5 or 10 μl of enzyme was added to make a solution with a final concentration if 1.0 ml, and the absorbance was recorded for 180 s. For calculating the change in absorbance (ΔA/s), the initial linear portion was used (43Srere P.A. Brazil H. Gonen L. Acta Chem. Scand. 1963; 17: S129-S134Crossref Google Scholar). All activity assays were carried out at 25 °C. The change in absorbance of the native protein, which was treated in the same way as the refolded sample but without denaturing, was taken as 100% while expressing the relative refolding yields. The enzyme assays were carried out at least in triplicates and as many as five times. The average values are reported, and the error in the activity measurements was not more than ±5%. Aggregation Kinetics—Aggregation kinetics studies of CS were carried out by detecting light scattering of the aggregates formed during folding at 25 °C or after high temperature incubation at 45 °C using a Cary Varian Eclipse fluorometer and a 400-μl stoppered fluorescence cuvette with constant stirring. Excitation and emission wavelengths were set to 500 nm with a slit width of 2.5 nm each. For folding studies, the mixing of the denatured protein was carried out manually in the spectrometer cuvette with stirring, yielding a final protein concentration of 10 μg/ml. For aggregation studies at 45 °C, 10 μg/ml CS was also used. Refolding Kinetics—The refolding kinetics of CS in the absence and presence of polyols were monitored at 24 °C by fluorescence detection using a BioLogic SFM-300 stopped-flow module attached to a Jasco J-810 spectropolarimeter with an addition photomultiplier tube for fluorescence detection. The excitation wavelength for intrinsic tryptophan fluorescence was 280 nm, and an interference filter with a peak transmittance at 340 nm was used for emission detection. A mixing ratio of 1:9 denatured protein/folding buffer (0.1 m Tris-HCl, pH 8.1) was used for the refolding of the protein in a 30-μl cuvette. Data were acquired up to 1 s for the detection of any fast phase with a time interval of 0.5–2 ms as well as up to 3 min with an interval of 0.1 s. Measurements of refolding kinetics were carried out using an Applied Photophysics RX.2000 rapid kinetics setup based on a pneumatic ram drive system using a microvolume cuvette interfaced with a Varian Eclipse fluorometer. The mixing ratio for CS folding in this case was 1:10 using a 250-μl syringe for the protein solution and 2.5-ml syringe for the refolding buffer. The refolding protein concentration was 10 μg/ml. Experiments were carried out at 0.9, 2.7, 4.5, and 6.4 m glycerol in 0.1 m Tris, pH 8.1, as well as in 1 m each ethylene glycol, erythritol, xylitol, and sorbitol at 24 °C. The excitation and emission wavelengths were 280 and 338 nm with slit widths of 10 and 20 nm, respectively. The limitation of the instrumental setup allowed the first data point to be collected at 31 ms only for experiments with data collection up to 1 s. Both the fast phase (up to 1s) and the slow phase (up to 3 min) were acquired. The 338-nm wavelength was selected based on equilibrium measurements of the fluorescence spectra of the native and denatured protein solutions. Furthermore, pH jump experiments to monitor the dimer formation kinetics were carried out by mixing, at a 1:10 ratio, a 100 μg/ml CS solution in 50 mm phosphate buffer, pH 5.8, with a 50 mm phosphate buffer solution, pH 8, yielding a final protein concentration of 10 μg/ml at pH 8. Effect of Polyols—Table I shows the results of the effect of polyols, viz. ethylene glycol, glycerol, erythritol, xylitol, and sorbitol ranging from 0.5 to 9 m, depending on their solubility, on the CS refolding yield at a concentration of 10 μg/ml and 25 °C. The polyols vary in the number of–OH groups in them: ethylene glycol has two, glycerol three, erythritol four, xylitol five, and sorbitol six. Except erythritol, which decreased the refolding yield at all concentrations, all other polyols led to an initial increase in the refolding yield, followed by a decrease at higher concentrations. Erythritol is known to increase the thermal stability of proteins (44Back J.F. Oakenfull D. Smith M.B. Biochemistry. 1979; 18: 5191-5196Crossref PubMed Scopus (764) Google Scholar), but resulted in a decrease in the refolding yields for CS. In contrast, ethylene glycol, which is known as a destabilizer of protein conformation (39Gekko K. Morikawa T. J. Biochem. (Tokyo). 1981; 90: 51-60Crossref PubMed Scopus (91) Google Scholar), led to a gradual increase in the refolding yield of CS, with a maximum yield of 52% at 5 m, whereas the control yield was 36%. There was a sharp decline in the refolding yield after 5 m, with no refolding activity obtained at 9 m.Table IEffect of polyols on the refolding yield of CSConcRefolding yieldEGGlycerolErythritolXylitolSorbitol%0.0 m36373439350.5 m1137341.0 m3551760521.5 m1154382.0 m4956817313.0 m46675.0 m52717.0 m32839.0 m071 Open table in a new tab Glycerol also led to a gradual increase in the refolding yield, and a very high refolding yield of 83% was obtained at 7 m, followed by a decrease. It was not possible to use glycerol at concentrations higher than 9 m, as there were mixing problems due to the high viscosity of the solutions. Glycerol has long been known to stabilize proteins against chemical and thermal denaturation. Gekko and Timasheff (36Gekko K. Timasheff S.N. Biochemistry. 1981; 20: 4667-4676Crossref PubMed Scopus (1128) Google Scholar, 37Gekko K. Timasheff S.N. Biochemistry. 1981; 20: 4677-4686Crossref PubMed Scopus (446) Google Scholar) proposed that the solvophobic effect of glycerol is responsible for its stabilizing action. Glycerol has been used to increase the refolding yield of proteins (34Gorovits B.M. McGee W.A. Horowitz P.M. Biochim. Biophys. Acta. 1998; 1382: 120-128Crossref PubMed Scopus (31) Google Scholar, 45Jaspard E. Arch. Biochem. Biophys. 2000; 375: 220-228Crossref PubMed Scopus (21) Google Scholar, 46Tieman B.C. Johnston M.F. Fisher M.T. J. Biol. Chem. 2001; 276: 44541-44550Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Glycerol has been used not only in in vitro studies, but also in in vivo studies, where it has been used to prevent the formation of the scrapie form (PrPsc) of the soluble prion protein (PrPc) (22Tatzelt J. Prusiner S.B. Welch W.J. EMBO J. 1996; 15: 6363-6373Crossref PubMed Scopus (271) Google Scholar). The refolding yield obtained by us for CS in glycerol is comparable with that using the GroEL/ES system, in which a similar yield has been obtained (7Zhi W. Landry S.J. Gierasch L.M. Srere P.A. Protein Sci. 1992; 1: 522-529Crossref PubMed Scopus (98) Google Scholar). Both xylitol and sorbitol resulted in maximum refolding yields of 60 and 52%, respectively, at 1 m, followed by a decrease as their concentrations increased. The results suggest that there is no parallel correlation between the ability of the polyols to stabilize proteins and their effect in enhancing the refolding yield of CS. Effect of Protein Concentration and Temperature—It has been reported previously that the refolding yield of CS depends on protein concentration (6Buchner J. Schmidt M. Fuchs M. Jaenicke R. Rudolph R. Schmid F.X. Kiefhaber T. Biochemistry. 1991; 30: 1586-1591Crossref PubMed Scopus (439) Google Scholar, 7Zhi W. Landry S.J. Gierasch L.M. Srere P.A. Protein Sci. 1992; 1: 522-529Crossref PubMed Scopus (98) Google Scholar). As the protein concentration increased, there was a drastic decrease in the spontaneous refolding yield of the enzyme, with negligible activity observed at a refolding concentration of 50 μg/ml (Table II). The addition of 7 m glycerol led to an increase in the refolding yield at 10 and 20 μg/ml almost to a similar extent but a smaller increase was observed relatively at 50 μg/ml. Refolding of proteins is also known to be highly temperature-dependent, with a tendency to form aggregates with increasing temperature, as a consequence of which, the refolding yield is reduced (47Fink A.L. Folding Des. 1998; 3: R9-R23Abstract Full Text Full Text PDF PubMed Scopus (1058) Google Scholar). This is quite obvious from the data on CS presented in Table III. The data on the effect of glycerol studied at various temperatures (10, 15, 25, 30, and 35 °C) reveal that glycerol helped to refold CS to a greater extent with increasing temperature, resulting in a 100% yield at 30 and 35 °C (Table III), at which the control activities were quite low. This result is much superior to the results obtained for CS refolding using molecular chaperones (7Zhi W. Landry S.J. Gierasch L.M. Srere P.A. Protein Sci. 1992; 1: 522-529Crossref PubMed Scopus (98) Google Scholar).Table IIEffect of protein concentration on the refolding yield of CS in 7.0 m glycerol at 25 °CCS concSpontaneous refoldingGlycerol-assisted refoldingμg/ml%%10408320217350340 Open table in a new tab Table IIIEffect of temperature on CS refolding in the absence and presence of 7 m glycerolTemperatureSpontaneous refoldingGlycerol-assisted refolding°C%%10475115504725408330291003511100 Open table in a new tab Addition of Glycerol at Different Time Intervals—To determine at which stage glycerol can affect the refolding of CS, glycerol was added at different time intervals during the folding of CS by manual mixing. The results show that there was almost a negligible effect of glycerol even as early as 15 s after refolding. No significant effect of glycerol on the CS refolding yield (activity) was observed when the addition was delayed further (data not shown). This suggests that the presence of glycerol, leading to an increase in the refolding yield, is necessary during the beginning or very early stages of refolding. Compensation for the Urea Effect by Glycerol—Studies on the compensation effect of glycerol on the refolding of CS in the presence of various concentrations of urea were carried out. Urea was used as a cosolvent additive in the concentration range of 0.5–2 m (Table IV). There was no effect of urea at 0.5 m, whereas a further increase in its concentration led to a decrease in the refolding yield, with no refolding observed at 2 m. It has been shown previously in the case of P22 tailspike protein folding that low concentrations of urea destabilize folding intermediates (48Fuchs A. Seiderer C. Seckler R. Biochemistry. 1991; 30: 6598-6604Crossref PubMed Scopus (80) Google Scholar). It is possible that an intermediate in CS refolding may also be susceptible to low urea concentrations, which in turn decrease the refolding yield. To determine whether glycerol stabilizes folding intermediates and in turn increases the refolding yield, CS refolding was carried out at 7 m glycerol in the presence of various concentrations of urea (Table IV). We observed that 1.5 m urea almost completely inhibited the refolding of CS, but 1.5 m urea and 7 m glycerol led to a very high refolding yield. To inhibit the refolding completely, 3 m urea was required in the presence of 7 m glycerol.Table IVEffect of urea (Part A) and a mixture of urea and glycerol (Part B) on CS refolding yield at 25 °CRefolding yield%Part A. Urea0.0 m350.5 m381.0 m261.5 m42.0 m0Part B. SolventSpontaneous331.5 m urea + 7.0 m glycerol682.5 m urea + 7.0 m glycerol63.0 m urea + 7.0 m glycerol0 Open table in a new tab Denaturation Transition and Thermal Inactivation—Denaturation transition curves measured by activity assay of CS (Fig. 1) in varying concentrations of GdmCl showed that CS was very sensitive to denaturation by GdmCl. At a concentration as low as 0.5 m, it started losing activity rapidly. In the presence of 3 m glycerol, however, the denaturation transition curve was shifted toward higher GdmCl concentrations. These experiments suggest that glycerol helps in the stabilization of the native CS structure against GdmCl denaturation. Glycerol concentrations higher than 3 m were difficult to achieve due to mixing problems with GdmCl. To study the effect of glycerol on the thermal inactivation of CS, inactivation kinetics experiments with the enzyme were performed at 45 °C. This temperature was chosen because it has been reported that CS inactivates rapidly at this temperature, resulting in no activity after 20 min of incubation (18Grallert H. Rutkat K. Buchner J. J. Biol. Chem. 2000; 275: 20424-20430Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Fig. 2 shows that there was a complete loss of enzymatic activity after incubation at 45 °C for 25 min, whereas in the presence of 7.0 m glycerol, 20% of the enzymatic activity was retained. This further suggests that glycerol stabilizes the native structure of CS against the effect of high temperature. Refolding and Aggregation Kinetics—Fig. 3 presents data on the refolding kinetics in the absence and presence of 7 m glycerol determined by activity measurements. The addition of glycerol slowed down the kinetics of refolding as evidenced by the rate constants of 0.213 and 0.126 min–1 in 7 m glycerol compared with 0.635 and 0.148 min–1 in the control buffer at 10 and 35 °C, respectively. Data on the aggregation kinetics of CS during refolding by manual mixing in the presence and absence of 1 m polyols as monitored by fluorescence at 25 °C are shown in Fig. 4a. Polyols suppressed the aggregation of CS depending on the number of hydroxyl groups in them, which is in accordance with their effect on the thermal stability of proteins (39Gekko K. Morikawa T. J. Biochem. (Tokyo). 1981; 90: 51-60Crossref PubMed Scopus (91) Google Scholar, 44Back J.F. Oakenfull D. Smith M.B. Biochemistry. 1979; 18: 5191-5196Crossref PubMed Scopus (764) Google Scholar). Interestingly, erythritol, which led to a decrease in the refolding yield, resulted in suppression of aggregation, suggesting that factors other than aggregation of CS also contribute to the polyol-mediated refolding of the protein.Fig. 4a and b, aggregation kinetics of CS during refolding from the denatured state at 25 °C in the absence and presence of 1 m polyols and increasing concentrations of glycerol. The traces in a are as follows: trace 1, control; trace 2, ethylene glycol; trace 3, glycerol; trace 4, erythritol; trace 5, xy
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