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• W2131692566 abstract "Trehalose, a naturally occurring osmolyte, is known to be an exceptional stabilizer of proteins and helps retain the activity of enzymes in solution as well as in the freeze-dried state. To understand the mechanism of action of trehalose in detail, we have conducted a thorough investigation of its effect on the thermal stability in aqueous solutions of five well characterized proteins differing in their various physico-chemical properties. Among them, RNase A has been used as a model enzyme to investigate the effect of trehalose on the retention of enzymatic activity upon incubation at high temperatures. 2 m trehalose was observed to raise the transition temperature, T m of RNase A by as much as 18 °C and Gibbs free energy by 4.8 kcal mol–1 at pH 2.5. There is a decrease in the heat capacity of protein denaturation (ΔC p) in trehalose solutions for all the studied proteins. An increase in the ΔG and a decrease in the ΔC p values for all the proteins points toward a general mechanism of stabilization due to the elevation and broadening of the stability curve (ΔG versus T). A direct correlation of the surface tension of trehalose solutions and the thermal stability of various proteins has been observed. Wyman linkage analysis indicates that at 1.5 m concentration 4–7 molecules of trehalose are excluded from the vicinity of protein molecules upon denaturation. We further show that an increase in the stability of proteins in the presence of trehalose depends upon the length of the polypeptide chain. The pH dependence data suggest that even though the charge status of a protein contributes significantly, trehalose can be expected to work as a universal stabilizer of protein conformation due to its exceptional effect on the structure and properties of solvent water compared with other sugars and polyols. Trehalose, a naturally occurring osmolyte, is known to be an exceptional stabilizer of proteins and helps retain the activity of enzymes in solution as well as in the freeze-dried state. To understand the mechanism of action of trehalose in detail, we have conducted a thorough investigation of its effect on the thermal stability in aqueous solutions of five well characterized proteins differing in their various physico-chemical properties. Among them, RNase A has been used as a model enzyme to investigate the effect of trehalose on the retention of enzymatic activity upon incubation at high temperatures. 2 m trehalose was observed to raise the transition temperature, T m of RNase A by as much as 18 °C and Gibbs free energy by 4.8 kcal mol–1 at pH 2.5. There is a decrease in the heat capacity of protein denaturation (ΔC p) in trehalose solutions for all the studied proteins. An increase in the ΔG and a decrease in the ΔC p values for all the proteins points toward a general mechanism of stabilization due to the elevation and broadening of the stability curve (ΔG versus T). A direct correlation of the surface tension of trehalose solutions and the thermal stability of various proteins has been observed. Wyman linkage analysis indicates that at 1.5 m concentration 4–7 molecules of trehalose are excluded from the vicinity of protein molecules upon denaturation. We further show that an increase in the stability of proteins in the presence of trehalose depends upon the length of the polypeptide chain. The pH dependence data suggest that even though the charge status of a protein contributes significantly, trehalose can be expected to work as a universal stabilizer of protein conformation due to its exceptional effect on the structure and properties of solvent water compared with other sugars and polyols. Sugars have been known to protect proteins against loss of activity (1Colaco C. Sen S. Thangavelu M. Pinder S. Roser B. Biotechnology. 1992; 10: 1007-1011Crossref PubMed Scopus (261) Google Scholar, 2Carninci P. Nishiyama Y. Westover A. Itoh M. Nagaoka S. Sasaki N. Okazaki Y. Muramatsu M. Hayashizaki Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 520-524Crossref PubMed Scopus (203) Google Scholar), chemical (3Taylor L.S. York P. Williams A.C. Edwards H.G.M. Mehta V. Jackson G.S. Badcoe I.G. Clarke A.R. Biochim. Biophys. Acta. 1995; 1253: 39-46Crossref PubMed Scopus (44) Google Scholar, 4Sola-Penna M. Ferreira-Pereira A. Lemos A.P. Meyer-Fernandes J.R. Eur. J. Biochem. 1997; 248: 24-29Crossref PubMed Scopus (66) Google Scholar), and thermal denaturation (5Back J.F. Oakenfull D. Smith M.B. Biochemistry. 1979; 18: 5191-5196Crossref PubMed Scopus (760) Google Scholar, 6Lee J.C. Timasheff S.N. Biochemistry. 1981; 256: 7193-7201Google Scholar, 7Arakawa T. Timasheff S.N. Biochemistry. 1982; 21: 6536-6544Crossref PubMed Scopus (985) Google Scholar, 8Lin T.-Y. Timasheff S.N. Protein Sci. 1996; 5: 372-381Crossref PubMed Scopus (230) Google Scholar, 9Xie G. Timasheff S.N. Biophys. Chem. 1997; 64: 25-43Crossref PubMed Scopus (348) Google Scholar). Among several sugars, α,α-trehalose (α-d-glucopyranosyl(1→1)-α-d-glucopyranoside) has been known to be a superior stabilizer in providing protection to biological materials against dehydration and desiccation (10Sampedro J.G. Guerra G. Pardo J.P. Uribe S. Cryobiology. 1998; 37: 131-138Crossref PubMed Scopus (43) Google Scholar, 11Sun W.Q. Davidson P. Biochim. Biophys. Acta. 1998; 1425: 235-244Crossref PubMed Scopus (128) Google Scholar). It is a compatible osmolyte that gets accumulated in organisms under stress conditions (12Somero G.N. Am. J. Physiol. 1986; 251: R197-R213Crossref PubMed Google Scholar, 13Singer M.A. Lindquist S. Trends Biotechnol. 1998; 16: 460-468Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). Because of this unique property, tremendous interest has been generated in understanding the molecular basis of stress management through induction of trehalose biosynthesis (13Singer M.A. Lindquist S. Trends Biotechnol. 1998; 16: 460-468Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 14Nwaka S. Holzer H. Prog. Nucleic Acids Res. Mol. Biol. 1998; 58: 197-237Crossref PubMed Scopus (156) Google Scholar). Trehalose has also been found to be very effective in the stabilization of labile proteins during lyophilization (15Carpenter J.F. Prestrelski S.J. Arakawa T. Arch. Biochem. Biophys. 1993; 303: 456-464Crossref PubMed Scopus (255) Google Scholar, 16Kreilgaard L. Frokjaer S. Flink J.M. Randolph T.W. Carpenter J.F. Arch. Biochem. Biophys. 1998; 360: 121-134Crossref PubMed Scopus (97) Google Scholar) and exposure to high temperatures in solution (2Carninci P. Nishiyama Y. Westover A. Itoh M. Nagaoka S. Sasaki N. Okazaki Y. Muramatsu M. Hayashizaki Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 520-524Crossref PubMed Scopus (203) Google Scholar, 8Lin T.-Y. Timasheff S.N. Protein Sci. 1996; 5: 372-381Crossref PubMed Scopus (230) Google Scholar, 9Xie G. Timasheff S.N. Biophys. Chem. 1997; 64: 25-43Crossref PubMed Scopus (348) Google Scholar). Sugars in general protect proteins against dehydration by hydrogen bonding to the dried protein by serving as water substitute (15Carpenter J.F. Prestrelski S.J. Arakawa T. Arch. Biochem. Biophys. 1993; 303: 456-464Crossref PubMed Scopus (255) Google Scholar, 17Carpenter J.F. Crowe J.H. Biochemistry. 1989; 28: 3916-3922Crossref PubMed Scopus (661) Google Scholar). Several studies carried out by Timasheff and coworkers (9Xie G. Timasheff S.N. Biophys. Chem. 1997; 64: 25-43Crossref PubMed Scopus (348) Google Scholar, 18Xie G. Timasheff S.N. Protein Sci. 1997; 6: 211-221Crossref PubMed Scopus (239) Google Scholar) show that sugars and polyols stabilize the folded structure of proteins in solution as a result of greater preferential hydration of the unfolded state compared with the native state. The mechanism is fundamentally different from stabilization in the dried state and points toward the different origins of protein denaturation under different stress conditions (17Carpenter J.F. Crowe J.H. Biochemistry. 1989; 28: 3916-3922Crossref PubMed Scopus (661) Google Scholar). In solution, trehalose has been observed to stabilize RNase A by increasing the surface tension of the medium, which leads to the preferential hydration of the protein (8Lin T.-Y. Timasheff S.N. Protein Sci. 1996; 5: 372-381Crossref PubMed Scopus (230) Google Scholar, 9Xie G. Timasheff S.N. Biophys. Chem. 1997; 64: 25-43Crossref PubMed Scopus (348) Google Scholar). These studies have been carried out using a representative protein at a few selected conditions only. Different proteins are expected to interact with cosolvent molecules in varied ways depending on their physico-chemical properties. In general, trehalose has been observed to provide protection to different proteins to various extents and the efficacy of protection depends on the nature of the protein (2Carninci P. Nishiyama Y. Westover A. Itoh M. Nagaoka S. Sasaki N. Okazaki Y. Muramatsu M. Hayashizaki Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 520-524Crossref PubMed Scopus (203) Google Scholar, 4Sola-Penna M. Ferreira-Pereira A. Lemos A.P. Meyer-Fernandes J.R. Eur. J. Biochem. 1997; 248: 24-29Crossref PubMed Scopus (66) Google Scholar). Despite the availability of such data, the exact role of proteins and their physico-chemical properties in trehalose-mediated stability is still not clear. Studies done earlier by Gekko (19Gekko K. J. Biochem. (Tokyo). 1981; 90: 1633-1641Crossref PubMed Scopus (154) Google Scholar, 20Gekko K. J. Biochem. (Tokyo). 1981; 90: 1643-1652Crossref PubMed Scopus (53) Google Scholar), using polyol osmolytes and free energy of transfer studies, suggested that unfavorable interactions of the amino acid side chains with polyols dominate the stability effect, and peptide-polyol interactions contribute negligibly to the stability mediated by polyols. However, recently, Bolen and coworkers (21Liu Y. Bolen D.W. Biochemistry. 1995; 34: 12884-12891Crossref PubMed Scopus (426) Google Scholar, 22Qu Y. Bolen C.L. Bolen D.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9268-9273Crossref PubMed Scopus (272) Google Scholar, 23Bolen D.W. Baskakov I.V. J. Mol. Biol. 2001; 310: 955-963Crossref PubMed Scopus (559) Google Scholar), based on carefully conducted transfer studies of amino acids and model compounds, have shown that cumulative interactions between amino acid side chains and osmolytes (including sucrose) favor protein unfolding, whereas their overall stabilization is achieved due to unfavorable peptide-osmolyte interactions. The exact nature of interactions that govern the osmolyte-mediated stability of proteins is, therefore, not yet very clear. Overall, protein stability should depend upon a fine balance between favorable and unfavorable interactions of the native and the denatured protein states with the cosolvent molecules (24Timasheff S.N. Adv. Protein Chem. 1998; 51: 355-432Crossref PubMed Google Scholar). The stabilizing effect would, thus, depend on the nature of both the proteins as well as the cosolvent molecules and generalization of the effect may not be possible. To understand the mechanism of trehalose-mediated thermal stability of proteins in detail, we have studied its effect on the thermal stability of a set of five well characterized globular proteins, viz., ribonuclease A (RNase A), 1The abbreviations used are: RNase A, ribonuclease A; ΔASA, change in accessible surface area; ΔC p, apparent heat capacity of denaturation; ΔH m, enthalpy of denaturation; ΔG 0, Gibbs energy; ΔΔG 0, free energy of stabilization; T m, midpoint of thermal denaturation; α-CTgen, α-chymotrypsinogen; cyt c, cytochrome c; Trp-Inh, Trypsin Inhibitor; GdmCl, guanidinium chloride; CD, circular dichroism; MOPS, 4-morpholinepropanesulfonic acid. lysozyme, cytochrome c (cyt c), α-chymotrypsinogen (α-CTgen), and trypsin-inhibitor (Trp-Inh) at varying cosolvent concentrations and pH values. These proteins vary in their molecular size, ranging from 12.3 kDa for cyt c to 25.7 kDa for α-CTgen, their hydrophobicities, and the net charges, with pI values in the range of 4.1 for Trp-Inh to 10.7 for lysozyme. The data have been analyzed in the light of the role of bulk properties of the solvent environment and the physico-chemical properties of proteins. Because trehalose in addition to imparting thermodynamic stability to proteins also helps in the retention of activity of enzymes during storage at high temperatures, we have carried out activity studies for RNase A at elevated temperatures and denaturing conditions to understand the stability-activity relationship of the enzyme in the presence of trehalose. Materials—RNase A (bovine pancreatic), lysozyme (hen egg white, HEW), α-CTgen (bovine pancreatic), cyt c (horse heart, type IV), Trp-Inh (HEW), trehalose, and 2′,3′-cCMP (NH4+ salt) were all from Sigma Chemical Co., St. Louis, MO. All the proteins were dialyzed against distilled de-ionized water and lyophilized followed by drying over P2O5. Glycine and sodium acetate were purchased from E. Merck, India. The pH of the solutions was adjusted on a Radiometer PHM84 research pH meter by adding HCl or NaOH solutions. 20 mm glycine HCl buffer at pH 2.5, 40 mm acetate buffer at pH 4.0, and 20 mm MOPS (Sigma Chemical Co.) buffers at pH 7.0 were used from their stock solutions. The solutions at pH 7.0 were made in the presence of 1.5 m GdmCl (Amresco, Solon, OH). Thermal Denaturation Experiments—For monitoring the unfolding of the tertiary structure, thermal denaturation experiments were carried out using a Cecil 599 UV-visible spectrophotometer to which a linear temperature programmer (CE-247, Cecil) was attached. The concentration of the protein solutions was ∼0.5 mg/ml, except for cyt c where 0.1 mg/ml protein was used. The protein solutions were loaded in a 0.5-ml masked and Teflon-stoppered quartz cuvette (Hellma, Germany). A temperature scan rate of 1 °C/min was used in all the experiments. The wavelengths for monitoring conformational changes related to the tertiary structure were 287 nm for RNase A, 293 nm for α-CTgen, 301 nm for lysozyme, 285 nm for Trp-Inh, and 394 nm for cyt c based on their difference spectra. The reversibility of the thermal transitions recorded for the proteins was ascertained by reheating the protein solutions. To investigate the unfolding of secondary structure elements, thermal denaturation was monitored by far-UV CD measurements using a Jasco J715 spectropolarimeter at selected pH conditions, one for each protein, at a scan rate of 1 °C/min. A wavelength of 222 nm was used to specifically probe the opening up of helical regions in the proteins. Analysis of Data—The evaluation of thermodynamic parameters from the thermal denaturation curves was based on the equilibrium constant K, for N ⇔ D conversion for a two-state reversible transition, where N represents the native state and D is the denatured state. The equilibrium constant was deduced from the equation, K=[unfolded]/[native]=(AN-AO)/(AO-AD)(Eq. 1) where A N is the absorbance of the pure native state in the transition zone after extrapolation from the pre-transition region, A D is the corresponding absorbance of the pure denatured state, and A O is the observed absorbance at a temperature in the transition zone. We used all the experimental data points obtained in a transition reaction to fit Equation 2, as follows, and described in detail elsewhere (25Kaushik J.K. Bhat R. J. Phys. Chem. B. 1998; 102: 7058-7066Crossref Scopus (208) Google Scholar, 26Kaushik J.K. Bhat R. Protein Sci. 1999; 8: 222-233Crossref PubMed Scopus (64) Google Scholar), AO=[AN+AD·exp·(-1/R(ΔHm(1/T-1/Tm)-ΔCp(Tm/T-1+1n(T/Tm)))][1+exp(-1/R(ΔHm(1/T-1/Tm)-ΔCp(Tm/T-1+1n(T/Tm)))](Eq. 2) where R is the gas constant, T m is the midpoint temperature of transition in kelvin, ΔH m is the enthalpy of protein denaturation calculated at the T m, and ΔC p is the apparent heat capacity of protein denaturation. For nonlinear least square analysis a minimum of 50 iterations or more using Marquardt-Levenberg routine as available in the Origin™ software (Microcal Inc., Northampton, MA) were performed until the fractional change in χ2 value was within the tolerance limit set to 5 × 10–4. All the parameters were floated freely to deduce their values simultaneously from thermal transition curves. RNase A Activity Assay—RNase A catalyzed hydrolysis of 2′,3′-cCMP was measured by the change in the absorbance at 286 nm (27Crook E.M. Mathias A.P. Robin B.R. Biochem. J. 1960; 74: 234-238Crossref PubMed Scopus (355) Google Scholar). Two sets of experiments were conducted in the presence of 1.5 m trehalose and 20 mm Tris, pH 7.0. In set 1, RNase A was incubated at high temperatures (66 °C and 60 °C, with 1 m GdmCl) for 13 h followed by cooling to room temperature and monitoring the activity by addition of 2′,3′-cCMP from the stock. In set 2, RNase was added to the reaction buffer and allowed to equilibrate at high temperatures (63 °C, 56 °C, and 52 °C, with 1 m GdmCl) at which the activity was monitored by the addition of 2′,3′-cyclic CMP. All the reactions were carried out in a 1.0-ml Teflon-stoppered quartz cuvette. The temperature of the cuvette was maintained by using a programmable thermoelectric cuvette holder. Thermal denaturation experiments were carried out for RNase A, lysozyme, cyt c, α-CTgen, and Trp-Inh in the absence and presence of 1–2 m trehalose at pH 2.5, 4.0, and 7.0 by absorbance measurements. The data have been presented in Fig. 1. The insets in Fig. 1 (A–E) show thermal denaturation of the helical structure of these proteins monitored at 222 nm by CD spectroscopy in the absence and presence of 1 m trehalose. The studies were not feasible at all the pH conditions for the proteins. cyt c is known to be partially denatured at pH 2.5 (28Dyson H.J. Beattie J.K. J. Biol. Chem. 1982; 257: 2267-2273Abstract Full Text PDF PubMed Google Scholar), and hence the thermal stability studies at this pH were not possible. α-CTgen undergoes aggregation at pH 4.0 and 7.0 at higher temperatures, thereby limiting thermodynamic analysis. In the presence of trehalose, lysozyme at pH 4.0 and 7.0 and cyt c at pH 7.0 undergoes partial aggregation even in the presence of 1.5 m GdmCl. The two-state transition analysis using the van't Hoff equation in these cases was carried out only to determine the approximate values of thermodynamic parameters. Santoro et al. (29Santoro M.M. Liu Y. Khan S.M.A. Hou L.-X. Bolen D.W. Biochemistry. 1992; 31: 5278-5283Crossref PubMed Scopus (355) Google Scholar) have observed irreversible aggregation of lysozyme in osmolyte solutions, and its inactivation at high temperatures in buffer has also been reported (30Zale S.E. Klibanov A.M. Biochemistry. 1986; 25: 5432-5444Crossref PubMed Scopus (270) Google Scholar). RNase A in the presence of trehalose undergoes a completely reversible denaturation at pH 2.5 and 4.0. At high trehalose concentrations, however, RNase A solutions at pH 7.0 showed partial aggregation even in the presence of 0.5 m GdmCl and required the addition of 1–1.5 m GdmCl for reversibility in thermal denaturation. It must be pointed out that a two-state cooperative transition has been observed for all the proteins studied. Even though Trp-Inh possesses three tandem homologous domains, only a single cooperative transition has been observed. We have observed that multidomain and multimeric proteins usually undergo irreversible thermal denaturation 2D. P. Kumar and R. Bhat, unpublished data. and hence cannot be studied for the effect of trehalose by either spectroscopic or calorimetric techniques. Effect of Trehalose on the Thermodynamics of Protein Denaturation—Thermodynamic parameters for protein denaturation in trehalose solutions at different conditions, obtained from the data in Fig. 1, have been presented in Table I. ΔT m and ΔΔG 0 are the increments in the midpoint of transition, T m, and Gibbs free energy of stabilization, ΔG 0, respectively. ΔΔG 0 in the presence of trehalose has been calculated at the T m of the control, where ΔG 0 for control is zero. For all the proteins, both ΔT m and ΔΔG 0 increase linearly with an increase in the trehalose concentration. ΔH m and ΔS m were also observed to be increasing with trehalose concentration in the case of RNase A, α-CTgen, Trp-Inh, and cyt c at pH 4.0. However, these parameters show a decrease in the case of lysozyme at pH 4.0 and 7.0, and cyt c at pH 7.0, which could be due to partial irreversible aggregation of the proteins in the post denaturation region. Overall, barring these exceptions involving protein aggregation, trehalose was observed to gradually increase the ΔH m and ΔS m of protein denaturation. The experimental errors in ΔH m were in the range of ±1–4% for RNase and cyt c and ±5–7% for α-CTgen, lysozyme, and Trp-Inh. The error in T m measurements was within ±0.5 °C for all the proteins. The fitting errors were always within the experimental error limits. The CD data presented as insets in Fig. 1 have not been used in Table I. However, the transition temperatures and the various thermodynamic parameters obtained by CD measurements matched well with those evaluated by absorption spectroscopy.Table IThermodynamic parameters for several proteins in aqueous trehalose solutions at various pH valuesCosolventRNase Aα-CTgenLysozymeCytochrome cTmΔTmΔHmΔSmΔΔG 0TmΔTmΔHmΔSmΔΔG 0TmΔTmΔHmΔSmΔΔG 0TmΔTmΔHmΔSmΔΔG 0°C°Ckcal mol-1e.u. ae.u., entropy units (cal k -1 mol-1).kcal mol-1°C°Ckcal mol-1e.u.kcal mol-1°C°Ckcal mol-1e.u.kcal mol-1°C°Ckcal mol-1e.u.kcal mol-1pH 2.5Control38.381.02600.0044.996.33030.0061.198.02930.00Trehalose (1.0 M)46.68.386.02692.1350.35.498.53041.5966.55.494.52781.50(1.5 M)51.613.390.72793.4553.28.3100.43072.4370.69.597.02822.68(2.0 M)56.518.295.32894.4856.411.5109.73333.5674.713.6101.42923.96pH 4.0Control54.294.02870.0073.198.52840.0064.561.51820.00Trehalose (1.0 M)62.48.2102.93072.4279.96.895.12691.8371.26.763.11831.19(1.5 M)66.212.0104.53083.4983.710.694.02632.7978.714.267.01902.56(2.0 M)70.516.3107.13124.7181.917.468.91943.15pH 7.0bSolutions at pH 7.0 also contain 1.5 M GdmCl.Trypsin inhibitorControl46.092.02880.0059.056.51700.0058.594.92860.0048.038.41200.00Trehalose (1.0 M)51.55.596.52971.5865.46.458.21721.0866.98.490.02652.2251.43.434.01050.35(1.5 M)56.910.9103.03123.1974.515.660.51762.5972.914.491.02633.7956.68.635.81090.88(2.0 M)59.313.3106.93223.95>75>16.590.02584.26a e.u., entropy units (cal k -1 mol-1).b Solutions at pH 7.0 also contain 1.5 M GdmCl. Open table in a new tab The slopes of the curves (ΔH m versus T m) plotted in Fig. 2 represent the heat capacity of protein denaturation, ΔC p for RNase A obtained in the presence of trehalose and are 0.87 kcal mol–1 K–1 at pH 2.5 and 4.0, and 1.1 kcal mol–1 K–1 at pH 7.0, respectively. The values are considerably lower than the spectroscopically obtained ΔC p values of 2.07 kcal mol–1 K–1 and 2.2 kcal mol–1 K–1 evaluated by varying the T m of RNase A using GdmCl (26Kaushik J.K. Bhat R. Protein Sci. 1999; 8: 222-233Crossref PubMed Scopus (64) Google Scholar) and urea (31Pace C.N. Laurents D.V. Biochemistry. 1989; 28: 2520-2525Crossref PubMed Scopus (199) Google Scholar), respectively, and calorimetric values of 1.74 kcal mol–1 K–1 in buffer and 2.16 kcal mol–1 K–1 in 1 m GdmCl reported by Liu and Sturtevant (32Liu Y. Sturtevant J.M. Biochemistry. 1996; 35: 3059-3062Crossref PubMed Scopus (58) Google Scholar). Previously, several other osmolytes like sarcosine (29Santoro M.M. Liu Y. Khan S.M.A. Hou L.-X. Bolen D.W. Biochemistry. 1992; 31: 5278-5283Crossref PubMed Scopus (355) Google Scholar) and polyols (25Kaushik J.K. Bhat R. J. Phys. Chem. B. 1998; 102: 7058-7066Crossref Scopus (208) Google Scholar) have also been observed to decrease the apparent heat capacity of denaturation of globular proteins. Neutral salts, including carboxylic acid salts (26Kaushik J.K. Bhat R. Protein Sci. 1999; 8: 222-233Crossref PubMed Scopus (64) Google Scholar), which increase the thermal stability of proteins, also lead to a decrease in the denaturation heat capacity of several proteins just like osmolytes. For proteins other than RNase A, e.g. for α-CTgen and lysozyme at pH 2.5, cyt c at pH 4.0, and Trp-Inh at pH 7.0, ΔH m also increases as a function of T m, though marginally (Table I), and results in much lower ΔC p values than the corresponding values reported for these proteins without trehalose. Solution Surface Tension and Protein Stability— Fig. 3 presents data showing the effect of the surface tension of trehalose solutions on the thermal stability, as monitored by T m of various proteins studied. Surface tension of aqueous trehalose solutions has been observed to increase linearly with the concentration resulting in a slope of 1.34 dyne cm–1 mol–1 at 20 °C (33Kita Y. Arakawa T. Lin T.-Y. Timasheff S.N. Biochemistry. 1994; 33: 15178-15189Crossref PubMed Scopus (275) Google Scholar). The data presented in Fig. 3 suggest a good correlation of the effect of the increased surface tension of trehalose solutions with the increase in the T m for all the proteins studied. Studies done by us earlier using a series of polyols (25Kaushik J.K. Bhat R. J. Phys. Chem. B. 1998; 102: 7058-7066Crossref Scopus (208) Google Scholar) and carboxylic salts (26Kaushik J.K. Bhat R. Protein Sci. 1999; 8: 222-233Crossref PubMed Scopus (64) Google Scholar) also indicate a strong correlation of the surface tension effect with the thermal stability of proteins, suggesting an important role of water and the solvent environment in the stability of proteins. Wyman Linkage and Interaction of Trehalose with Proteins— Trehalose stabilizes proteins by shifting the equilibrium constant in favor of the native state. To analyze the effect of trehalose on the denaturation reaction, the Wyman linkage equation (24Timasheff S.N. Adv. Protein Chem. 1998; 51: 355-432Crossref PubMed Google Scholar) given below (Equation 3) was used to determine the relative preferential interaction of trehalose with the two end states of the proteins, d(lnK)/d(lna)=(nD-nN)=Δn(Eq. 3) where K is the equilibrium constant for the conversion reaction N ⇔ D, a is the cosolvent activity, and n D and n N are the numbers of cosolvent molecules bound to the denatured and the native protein molecules, respectively. For approximation, a second order equation was fit to a ln-ln plot of the equilibrium constant versus the trehalose concentration (instead of activity) for various proteins (Fig. 4). The Wyman slope of the tangent, Δn at a point on the curve provides the difference in the cosolvent molecules bound to the denatured and the native protein molecules. The Δn values obtained for various proteins at 1.5 m trehalose concentration vary from –7 to –4. The negative values indicate the preferential exclusion of trehalose from the hydration shell of the protein upon denaturation. The values of Δn extrapolated to 0.1–0.9 m trehalose agree well with those obtained by Xie and Timasheff (9Xie G. Timasheff S.N. Biophys. Chem. 1997; 64: 25-43Crossref PubMed Scopus (348) Google Scholar) for RNase A at similar concentrations Activity of RNase A in the Presence of Trehalose—RNase A was taken as a model enzyme to analyze the effect of trehalose on its bioactivity at high temperatures. The relative activity of RNase A in the presence of trehalose presented in Table II has been calculated by dividing the slopes of the linear zone of the corresponding activity plots by the slope of the data for control (buffer). The stabilization factor, f Nt/f Nc presented in Table II, wherein f Nt is the fraction of the protein in the native state in the trehalose solution and f Nc is the fraction of the native protein in the control buffer, was calculated from the thermal denaturation curves for RNase A in the respective solvent systems at the indicated temperatures. The data indicate a remarkable retention of activity of the enzyme in the presence of trehalose under various conditions of the experiment compared with control. Both the storage (set 1) as well as the operational (set 2) stabilities of the enzyme increased in the presence of trehalose as suggested by activity measurements under different conditions. Interestingly, greater relative retention of activity was observed in the presence of a mixture of 1.5 m trehalose and 1.5 m GdmCl compared with trehalose alone. We have obtained similar results earlier using lysozyme as a model enzyme (34Sangeeta Devi Y. Nair U.B. Bhat R. Prog. Biotechnol. 1998; 15: 263-268Google Scholar).Table IIRelative activities of RNase A in the presence of trehalose at different temperaturesCosolventRelative activitiesSet 1aActivities were measured at 25 °C after 13 h of incubation at the indicated temperatures.Set 2bActivities were measured at the indicated temperatures.66 °C56 °C63 °CTrehalose2.02 ± 0.11.47 ± 0.082.41 ± 0.35(9.64)cParentheses include the value of stabilization factor, f Nt/f Nc.(1.041)(2.82)60 °CaActivities were measured at 25 °C after 13 h of incubation at the indicated temperatures.52 °CbActivities were measured at the indicated temperatures.Trehalose + 1 M3.03 ± 0.13.44 ± 0.05GdmCl(12.3)(1.77)a Activities were measured at 25 °C after 13 h of incubation at the indicated temperatures.b Activities were measured at the indicated temperatures.c Parentheses include the value of stabilization factor, f Nt/f Nc. Open table in a new tab Exceptional Stabilization by Trehalose—Among the various osmolytes selected by nature to counteract deleterious environmental effects, trehalose seems to be exceptional among the compatible osmolytes of the sugar and the polyol series, because it increases the transition temperature (ΔT m) of proteins to a maximal extent. A comparatively high value of ΔT m of 18.2 °C (ΔG 0 ∼ 4.5 kcal mol–1) for RNase A at 2 m concentration of trehalose and pH 2.5 is indicative of this (Table I). This increase is much higher compared with any oth" @default.
• W2131692566 created "2016-06-24" @default.
• W2131692566 creator A5058646356 @default.
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• W2131692566 title "Why Is Trehalose an Exceptional Protein Stabilizer?" @default.
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• W2131692566 doi "https://doi.org/10.1074/jbc.m300815200" @default.
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