Protective Effect of Sorbitol on Enzymes Exposed to Microsecond

Oct 15, 2008 - Lysozyme (R-helix dominant structure) and pepsin were exposed to microsecond pulsed electric field (PEF) at 3.5 × 106 V/m. The respons...
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J. Phys. Chem. B 2008, 112, 14018–14025

Protective Effect of Sorbitol on Enzymes Exposed to Microsecond Pulsed Electric Field Wei Zhao and Ruijin Yang* State Key Laboratory of Food Science and Technology and School of Food Science and Technology, Jiangnan UniVersity No. 1800 Lihu Road, Wuxi 214122, China ReceiVed: July 15, 2008; ReVised Manuscript ReceiVed: August 25, 2008

Lysozyme (R-helix dominant structure) and pepsin (β-sheet dominant structure) were exposed to microsecond pulsed electric field (PEF) at 3.5 × 106 V/m. The response of enzymes to the stress of PEF was investigated in this study. Unfolding of enzyme structures and disruption of secondary and three-dimensional structures occurred when the exposed PEF dosage exceeds a critical value, which caused the decrease in activity. In this work, sorbitol was found to be effective to stabilize the conformations and activities of enzymes against electric field. The protective effect increased with the increase of concentration of sorbitol. Introduction With the development of high electric voltage transmission and distribution systems and wide application of power electronic equipments, the electric field in our living environment gradually increases. In the recent years, the biological effects of electric field and its applications have been widely studied including the thermal and nonthermal effects. Research on the nonthermal effects of pulsed electric field is a new field of bioelectromagnetics. Recent studies have shown that the “nonthermal” effect of pulsed electric field may cause changes in protein conformations, which in turn can change protein activity, causing harmful effects.1-3 On the other hand, electrostatics has been utilized by human beings in medicine and molecular biology such as electroporation3,4 and electrofusion.5-7 These techniques are based on breakdown or disruption of the cell membrane by applying a pulsed electric field, and the mechanism of electrical breakdown or disruption can be explained relatively easily by electromechanical compression. In recent years, commercial exploitation of high intensity pulsed electric field (PEF)8 as an alternative to traditional thermal pasteurization or sterilization of bioactive materials and foods has undergone substantial developments. The mechanism of PEF inactivation of microorganisms has been attributed to cell membrane permeabilization and damage when the applied electric field exceeds a certain critical value.9 Several researchers also extracted recombinant enzymes10 or DNA11 from microorganisms or tissue with PEF. Previous studies have been demonstrated that PEF can change the conformations and activities of proteins including enzymes. Budi et al.12 had performed molecular dynamics simulations on insulin chain-B under the influence of both static and oscillating electric fields, ranging from 107 to 109 V/m. They found that both variants had an effect on the normal behavior of the protein. Budi et al.1 observed that the intrinsic flexibility of insulin was constrained by lower-frequency oscillating fields or static field, thus potentially restricting access to the protein’s active state. It has been demonstrated that unfolding of lysozyme structure was induced by PEF, accompanied by the cleavage of disulfide bonds and self-association aggregation when the applied PEF dosage was higher than a critical level. The * To whom correspondence should be addressed. Fax/Phone: 86 510 85919150. E-mail: [email protected].

irreversible inactivation of lysozyme induced by PEF at 3.5 × 106 V/m was correlated to the loss of R-helix in secondary structure. Compared with thermal inactivation, lysozyme exposed to PEF did not form a molten globule.13 Yang et al.14 reported that PEF-induced inactivation of pepsin (β-sheet dominant structure) was correlated with the alteration of the secondary structure. Zhong et al.15 observed that the relative R-helix content of horseradish peroxidase (HRP) under PEF at 2.2 × 106 V/m compared to the untreated HRP (R-helix dominant structure) was 64.86%. From the point of view of human health and better application of electroporation and electrofusion, especially the sterilization of bioactive materials and extraction of enzymes from microorganisms by PEF, the effect of electric field on the behavior of proteins is unfavorable. Therefore, how to protect proteins against the stress of electric field remains a problem. It is known that the native conformation of many proteins and enzymes under dehydration, freezing, and thermal stresses can be stabilized by polyhydric molecules such as sorbitol. The stabilization mechanisms are still not fully understood at present. However, the stabilization mechanisms under different physical stresses are different.16-18 The protein thermoprotective mechanism with sorbitol has been discussed at length. The formation of a protein-sorbitol complex was suggested indicating the involvement of a direct interaction between sorbitol and protein in protein thermal stabilization.19 A recent study also demonstrated that sorbitol can be bound to protein molecular primarily by electrostatic force.20 It strengthens the bridge interaction between polypeptide chains to stabilize the secondary structures by replacing the water molecules.21,22 On the other hand, some researchers concluded that sorbitol stabilizes a protein against heat denaturation and does not involve any direct interaction with it, merely the exclusion of other solutes from the hydration shells close to the macromolecule.23 Several other mechanisms have also been proposed to be the dominant ones: (1) influence of sorbitol on strength of proteinsprotein interactions to modulate the attractive and repulsive forces between proteins; (2) effects of sorbitol to the structure of water; (3) preferential hydration; and (4) hydrophobic destabilization and hydrophilic stabilization.16,24,25 The stress from electric field is very different from others and exerts different action on proteins and organisms, but there is very limited literature on the stabilization methods of protein under electric field. In this study, lysozyme

10.1021/jp8062367 CCC: $40.75  2008 American Chemical Society Published on Web 10/15/2008

Effect of Sorbitol on Enzymes Exposed to Microsecond PEF (R-helix dominant structure) and pepsin (β-sheet dominant structure) were selected as model enzymes to investigate the enzyme behavior under pulsed electric field stress and the protective effect of sorbitol on enzyme conformations and activities. Experimental Section Chemicals and Materials. Hen egg-white lysozyme was purchased from Amresco Inc. (Solon, OH). Crystallized, pepsin powder, D-sorbitol, and freezedried Micrococcus lysodeikticus powder (M-3770) were purchased from Sigma Co. (St. Louis, MO). All other chemicals used were of reagent-grade. Preparation of Lysozyme and Pepsin Solutions. Lysozyme powder was dissolved in sodium phosphate buffer (10 mM, pH 6.2) with an electrical conductivity of 0.06 S/m at 25 °C. Pepsin powder was dissolved in water-phosphate-acetate mixture prepared by adding glacial acetic acid to 20 mM potassium phosphate to pH 3.8. Electrical conductivity of the media was adjusted to 0.15 S/m at 25 °C using sodium chloride. The concentrations of lysozyme and pepsin solutions were 7 and 500 µM. Different amounts of sorbitol were added into the lysozyme and pepsin solutions. Microsecond PEF Device. A bench-scale continuous PEF system (OSU-4 L, The Ohio State University, Columbus, OH) was used to treat the enzyme solutions. A schematic diagram is shown in Figure 1a. Six cofield flow tubular chambers (Figure 1b) with a 2.92 mm electrode gap and a 2.3 mm inner diameter were grouped in three pairs, and each pair was connected with stainless steel tubing with a 2.3 mm inner diameter. The enzyme solution was treated by PEF with alternating positive and negative pulses when bipolar pulses were applied. A model 9310 trigger generator (Quantum Composer Inc., Bozeman, MT) was used to control frequency and pulse duration time and delay time between opposite polarity. Signals of voltage, current, frequency, and waveform were monitored by a two channel 1 GS/s (60 MHz bandwidth) digital real-time oscilloscope (model TDS 210, Tektronix Inc., Wilsonville, OR). Figure 1c shows a typical set of voltage and current waveforms used in this study. A cooling coil with a 2.3 mm inner diameter was connected to each pair of chambers and submerged in a water bath (model 1016, Fisher Scientific Inc., Pittsburgh, PA) to regulate the temperature of enzyme solutions before and after PEF treatment. Type K thermocouples (Fisher Scientific, Pittsburgh, PA) were attached to the surface of the stainless steel coils near the inlet and outlet of each pair of PEF chambers. The temperatures of the inlet (T1, T3, and T5) and the outlet (T2, T4, and T6) of each pair of chambers were monitored during PEF treatment by dual channel digital thermocouple readers (Fisher Scientific, Pittsburgh, PA). The places where thermocouples were located were isolated from atmosphere by an insulation tape (Polyethylene Cloth, Bron, Phoenix, AR). The initial temperatures of enzyme solution in the sample bank (T0) and the sample temperature in the sample collecting bottle (T7) were measured by mercury thermometers. A micro gear pump (model 020000-010, Micropump, Inc., Vancouver, WA) maintained a continuous flow of enzyme solution. PEF treatment time (t) was calculated with the number of pulses received in a chamber (Np), which is obtained from residence time in a chamber (Tr) as follows

Tr ) V/F where V is the volume of a chamber (ml) and F is the flow rate (ml/s);

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Np ) Tr × f where f is the pulse repetition rate (Hz);

t ) Np × Nc × Wp where Nc is the number of treatment chambers and Wp is the pulse width (µs). Lysozyme and pepsin solutions flowed through the PEF system to be exposed to the microsecond pulsed electric field. Lysozyme and Pepsin Activity Assay. Lysozyme activity was determined by the turbidimetric assay method as described by Shugar,26 measuring the decrease in absorbance at 450 nm of a M. lysodeikticus suspension versus time with a UV-vis spectrophotometer (UV1201, Beijing Ruili Instrument Co., Beijing, China). A fresh suspension of M. lysodeikticus (18 mg solid in 100 mL of phosphate buffer) was used as substrate. For each sample, 2.3 mL of substrate was placed in a cuvette held at 25 °C. At time 0, 0.3 mL of lysozyme sample, adequately diluted according to its expected activity, was added to give a total reaction volume of 2.6 mL and shaken quickly. Absorbance measurements were made in 0.5 s intervals, the decrease of absorbance versus time were plotted, and the activity of each sample was calculated (∆Abs450/min). One enzyme unit is equal to a decrease in turbidity of 0.001/min at 450 nm, the specific activity of per milliliter of lysozyme sample was calculated as follows

Units(U ) )

∆Abs450nm /min × 1000 0.3

The activity of pepsin was measured by the method of Anson27 with some modifications using hemoglobin as substrate. To prepare the 2.0% (w/v) hemoglobin substrate solution, 2.5 g of hemoglobin (Sigma-Aldrich Co., St. Louis, MO) was dissolved in 100 mL of distilled water and mixed vigorously, and then filtered with a glass wool filter. Before assay, 80 mL of filtrate was mixed with 20 mL of 300 mM HCl. Properly diluted enzyme solution (1.0 mL) was added to 5.0 mL of the above substrate solution which had been equilibrated in a 37 °C water bath for 20 min, mixed and incubated at 37 °C for exactly 10 min, and then 10 mL of 5.0% (W/V) trichloroacetic acid (TCA) (Sigma Co., St. Louis, MO) was added and mixed vigorously for 1 min by a vortex (Genie 2, Fisher Scientific). The resultant mixture was filtered with a Waterman no. 50 filter paper (Sigma Co., St. Louis, MO). The filtrate was transferred to a quartz cuvette (Fisher Scientific, Pittsburgh, PA) to record the absorbance at 280 nm by a UV-vis spectrophotometer (UV1201, Beijing Ruili Instrument Co., Beijing, China) at room temperature. The blank was prepared according to the above procedure, except that 1.0 mL of enzyme solution was replaced by 1.0 mL of distilled water. One unit was defined as a 0.001 increase of absorption unit at 280 nm per min by spectrometer. The relative residual activity (RRA) of lysozyme or pepsin was defined as a percentage of activity of the lysozyme or pepsin under PEF relative to that of the control. The control was kept in a 0 °C ice-water bath. Prior to activity assay, all samples were kept in a 0 °C ice-water bath. Circular Dichroism Analysis. Circular dichroism (CD) analysis was carried out with a CD spectropolarimeter (Jasco J-715, Jasco Corp., Tokyo, Japan) in the far- and near-UV regions at 25 °C. The former (200-250 nm) reflects the secondary structure, whereas the latter (260-320 nm) arises from the tertiary structure of the protein. Quartz CD cuvettes (Hellma, Muellheim, Baden, Germany) with 1 and 10 mm path lengths were used for the far- and near-UV measurements, respectively.

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Figure 1. Diagram of the (a) PEF processing unit, (b) PEF treatment chamber, and (c) bipolar square wave pulse pair. Trace 1 is the measured voltage with 5 × 103 V per division. Trace 2 is the measured current with 20 A per division. Time scale is 5 µs per division.

Effect of Sorbitol on Enzymes Exposed to Microsecond PEF

Figure 2. Far-UV CD spectra of (a) lysozyme and (b) pepsin in the presence or absencce of 3 mg/ml sorbitol under PEF at 3.5 × 106 V/m.

The final lysozyme concentration was 7 µM for near-UV CD experiments and was adujsted to 2.5 µM with phosphate buffer for far-UV CD analysis. The concentrations of the pepsin solutions were adjusted to 50 and 3 µM for near and far-UV CD experiments, respectively. Five scans were averaged to obtain one spectrum. The CD data were expressed in terms of molar ellipticity, (θ), in degree cm2/dmol. Results and Discussion Protective Effect of Sorbitol on Secondary Structures of Lysozyme and Pepsin Exposed to Microsecond Pulsed Electric Field. Generally, enzymes are globular proteins whose catalytic activity relies on the native configuration of their active site and the conformation of surrounding proteins. Figure 2 shows the effects of electric field stress on proteins secondary structures and the protective effect of sorbitol against PEF. Lysozyme is a R-helix dominant protein. The CD spectrum of native lysozyme in Figure 2a displays negative bands in a wavelength range shorter than 240 nm, which is characterized mainly by two negative bands at 208 and 222 nm. Theses negative bands are rationalized by the nfπ* transition in the peptide bond of R-helical structure.28 Pepsin is a β-sheet dominant acid proteinase. As illustrated in Figure 2b, native pepsin has a single minimum at 210-215 nm, which is typical for a very highly β-sheet rich protein. Under pulsed electric field, lysozyme and pepsin exhibited disruption to the secondary structures. Figure 2a shows that the intensity of the negative peak of 208 nm decreased when lysozyme exposed to pulsed electric field at 3.5 × 106 V/m for 1200 µs, indicating a loss of R-helical structure under electric field stress. Similar to the results of lysozyme, the minima of the CD spectrum of the pepsin (Figure 2b) under pulsed electric field at 3.5 × 106 V/m

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Figure 3. The change of secondary structure of (a) lysozyme and (b) pepsin in the presence or absence of various amounts of sorbitol under PEF at 3.5 × 106 V/m for various times. For lysozyme and pepsin, the transitions of secondary structure were monitored by the changes in ellipticity at 208 and 215 nm, respectively.

for 500 µs shifted to a lower wavelength region, and the intensity of the negative peak between 210 and 215 nm decreased. It indicates a gradual loss of β-sheet structure and the emergence of intensity characteristic of random coil regions of structure.29,30 These results indicate that no matter whether the enzyme is R-helix or β-sheet dominant structure, loss of the secondary structure occurred under electric field stress. The stability of a protein itself is mainly depending on the interactions of the amino acid side chains and secondary structure elements. It is noted from Figure 2a that the CD spectra of lysozyme in the presence and absence of 3 mg/ml sorbitol under PEF were remarkably different. Compared with the disruption of secondary structure induced by PEF, lysozyme with 3 mg/ml sorbitol has the same backbone secondary structure as the native protein under PEF at 3.5 × 106 V/m for 1200 µs. Similar results were also obtained when pepsin was exposed to PEF. This implies that sorbitol could effectively prevent the disruption of secondary structure of enzymes under PEF. Figure 3 shows the unfolding process of secondary structures of lysozyme and pepsin in the presence or absence of various amounts of sorbitol under PEF at 3.5 × 106 V/m for various times. The molecular ellipticity at 208 nm ([θ]208nm) is a standard measure of helical content of a protein and has been used to estimate the secondary structural change of the protein. The decrease in the intensity of the negative band at 208 nm represents the decrease in the content of R-helix and changes in the secondary structure of lysozyme.31 Acid proteinases have a characteristic minimum at 215 nm.32 The decrease in the

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Figure 4. Near-UV CD spectra of (a) lysozyme and (b) pepsin in the presence or absencce of 3 mg/ml sorbitol under PEF at 3.5 × 106 V/m.

intensity of molar ellipticity at 215 nm means the loss of the second structure of pepsin, featuring a very high β-sheet content. As shown in Figure 3, when the exposed PEF time was longer than 100 µs at 3.5 × 106 V/m, the unfolding of secondary structure was observed for lysozyme. It indicates that to change the structure of lysozyme, PEF dosage should be higher than a critical level, which may be defined as critical dosage. This critical dosage increases as the concentrations of sorbitol increases. The secondary structure of lysozyme in the presence of 1 and 2 mg/ml sorbitol started to unfold under PEF at 3.5 × 106 V/m for 400 and 900 µs, respectively. No change of secondary structure of lysozyme in the presence of 3 mg/ml sorbitol was found under the tested condition. The protective effect of sorbitol on pepsin against PEF was also very pronounced. Pepsin without sorbitol started to unfold under 50 µs of PEF at 3.5 × 106 V/m. In the presence of 3 mg/ml sorbitol, the unfolding of sencondary structure was observed under PEF for 450 µs. This demonstrates that pepsin (β-sheet dominant structure) is more sensitive to electric field than lysozyme (Rhelix dominant structure).

Protective Effect of Sorbitol on Three-dimensional Structures of Lysozyme and Pepsin Exposed to Microsecond Pulsed Electric Field. The near-UV CD spectra of proteins reflect mainly the contribution of aromatic amino acids to protein tertiary structure and are very sensitive to structural perturbations. Figure 4 shows the effects of electric field stress on protein three-dimensional structures and the protective effect of sorbitol against PEF. As shown in Figure 4, the spectra of native lysozyme and pepsin were dominated by positive bands in the 280-300 nm range, which reflect significant tertiary structure of the protein.33 After exposure to PEF at 3.5 × 106 V/m for 1200 and 500 µs for lysozyme and pepsin, respectively, there were decreases in the amplitude of positive molar ellipticity in the range 280-300 nm, indicating that the tertiary structure became less-defined and less compact and the unfolding of the tertiary structure occurred. In contrast to the flat spectrum of lysozyme or pepsin without sorbitol, the near-UV CD spectra of lysozyme and pepsin in the presence of 3 mg/ml sorbitol under PEF were almost identical to that of native enzymes, suggesting that sorbitol can effectively preserve packing around

Effect of Sorbitol on Enzymes Exposed to Microsecond PEF

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Figure 5. The change of tertiary structure of (a) lysozyme and (b) pepsin in the presence or absencce of various amounts of sorbitol under PEF at 3.5 × 106 V/m for various times. The transitions of tertiary structure were monitored by the changes in ellipticity at 295 nm.

the aromatic amino acids under PEF. Figure 5 shows the change of tertiary structure of lysozyme and pepsin in the presence or absence of various amounts of sorbitol under PEF at 3.5 × 106 V/m for various times. From Figure 4 and 5, it is noteworthy that the secondary and tertiary structures of lysozyme and pepsin unfolded synchronously when PEF reached the critical dosage. It indicates that the molten-globule state did not exist in the structures unfolding of lysozyme and pepsin induced by PEF. A molten globule as a thermal stress response is an intermediate protein structure between native and denatured protein forms but is distinguished from native proteins by a nonrigid sidechain arrangement and nonfixed tertiary structures.34 This demonstrates that the behavior of enzymes under PEF is different from that of a thermal stress response. Similar to the results of Figure 3, the protective effect of sorbitol on tertiary structure of pepsin against PEF was also pronounced. Effect of Sorbitol on the Activities of Lysozyme and Pepsin Exposed to Microsecond Pulsed Electric Field. Changes in enzyme activity may occur due to the structural changes taking place in the enzyme induced by PEF. Figure 6 shows the inactivation kinetics of lysozyme and pepsin in the presence or

absence of various amounts of sorbitol under PEF at 3.5 × 106 V/m for various times. As seen in Figure 6, sorbitol prevented the inactivation of lysozyme and pepsin under PEF. The plots of RRA values versus exposed PEF time indicate that inactivation of enzymes induced by PEF followed zero order kinetics on linearity. The inactivation rates of lysozyme and pepsin were found to decrease with increasing amounts of sorbitol. In particular, no inactivation of lysozyme was observed in the presence of 3 mg/ml sorbitol under PEF at 3.5 × 106 V/m for 1200 µs. In the absence of sorbitol, the activity of lysozyme decreased by approximately 40% under 1200 µs of PEF at 3.5 × 106 V/m; however, the activity of pepsin decreased by more than 90% induced by 500 µs of PEF at the same electric field strength. This indicates that pepsin is much more sensitive to electric field, which is agreement with the results of structure analysis. From type K thermocouples attached to the surface of the stainless steel coils near the inlet and outlet of each pair of PEF chambers in Figure 1, the highest temperature was lower than 45 °C, so the effect of heat on these the activity and conformational changes could be ruled out.

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Figure 6. The inactivation kinetics of (a) lysozyme and (b) pepsin in the presence or absencce of various amounts of sorbitol under PEF at 3.5 × 106 V/m for various times. Experiments were triplicated and the mean ( standard deviation of the three data sets are presented.

Polyhydric compounds have been found to be effective stabilizers of the native conformation of globular proteins and biological assemblies against the stresses of dehydration,17 freezing,35 drying,19 and heating.36 However, the mechanisms involved in the protecting effect of polyols on various types of protein denaturation under these stresses mentioned above are not fully understood at present. Proposed mechanisms are as follows: (1) preservation of native conformation through preferential exclusion of solutes during freezing or through a direct interaction between polyols and polar residues in the protein surface during drying;19 (2) promotion of preferential hydration which is facilitated by an increased surface tension of water during freezing;35 (3) strengthening the bridge interaction between polypeptide chains to stabilize the protein structure by replacing the water molecular.17,36 Back et al.36 showed that the mechanism by which polyols stabilize proteins against heat denaturation is through their effect on the structure of water molecules; hydrophobic interactions between pairs of hydrophobic groups in protein are stronger in polyols solutions than in pure water. From the previous studies, it is known that the pH and electrical conductivity of the system have critical influence on the stability of enzymes under PEF.14 In this study the addition of sorbitol did not alter the pH and electrical conductivity of the system. This indicates that the different behaviors of

enzymes in the presence or absence of sorbitol under electric field are not caused by the change of pH or electrical conductivity of the enzyme system. Further research is needed to explain the mechanism of sorbitol stabilization of enzymes under electric field stress. Conclusions In this work, lysozyme (R-helix dominant structure) and pepsin (β-sheet dominant structure) were selected to investigate the enzyme behavior under pulsed electric field stress and the protective effect of sorbitol. The results indicate that unfolding of enzyme structures and disruption of secondary and threedimensional structures occurred when the exposed PEF dosage exceeds a critical value. Pepsin (β-sheet dominant structure) is more sensitive to electric field than lysozyme (R-helix dominant structure). From the transitions of secondary and tertiary structure under PEF, it seems reasonable to conclude that the molten-globule state did not exist in the protein denaturation of lysozyme and pepsin. The protective effect of sorbitol on the structures and activities of enzymes against PEF was pronounced, which increased with the increase of concentration. Acknowledgment. The authors gratefully acknowledge the financial support provided by National 863 Hi-Tech R&D Plan

Effect of Sorbitol on Enzymes Exposed to Microsecond PEF (2007AA100405) and National Key Project of Scientific and Technical Supporting Programs Funded by Ministry of Science and Technology of China During the 11th Five-year Plan (Nos. 2006BAD05A17 and 2006BAD05A02). This study was also supported by 111 Project-B07029, Program for Changjiang Scholars and Innovative Research Team in University and the Graduate Student Innovation Project (Jiangsu, China). References and Notes (1) Budi, A.; Legge, F. S.; Treutlein, H.; Yarovsky, I. J. Phys. Chem. B 2007, 111, 5748. (2) de Pomerai, D. I.; Smith, B.; Dawe, A.; North, K.; Smith, T.; Archer, D. B.; Duce, I. R.; Jones, D.; Candido, E. P. FEBS Lett. 2003, 543, 93. (3) Salford, L. G.; Brun, A. E.; Eberhardt, J. L.; Malmgren, L.; Persson, B. R. R. EnViron. Health Perspect. 2003, 111, 881. (4) Vassilopoulos, G.; Wang, P. R.; Russell, D. W. Nature 2003, 422, 901. (5) Heller, R.; Grasso, R. J. Biochim. Biophys. Acta 1990, 1024, 185. (6) Andreason, G. L.; Evans, G. A. BioTechniques 1988, 6, 650. (7) Zimmermann, U. Trends Biotechnol. 1983, 1, 149. (8) Loghavi, L.; Sastry, S. K.; Yousef, A. E. Biotechnol. Prog. 2008, 24, 148. (9) Sale, A. J. H.; Hamilton, W. A. Biochim. Biophys. Acta 1967, 148, 781. (10) Shiina, S.; Ohshima, T.; Sato, M. Biotechnol. Prog. 2004, 20, 1528. (11) Yin, Y. G.; Jin, Z. X.; Wang, C. L.; An, W. Z. Sep. Purif. Technol. 2007, 56, 127. (12) Budi, A.; Legge, F. S.; Treutlein, H.; Yarovsky, I. J. Phys. Chem. B 2005, 109, 22641. (13) Zhao, W.; Yang, R.; Lu, R.; Tang, Y.; Zhang, W. J. Agric. Food Chem. 2007, 55, 9850. (14) Yang, R.; Li, S. Q.; Zhang, Q. H. J. Agric. Food Chem. 2004, 52, 7400.

J. Phys. Chem. B, Vol. 112, No. 44, 2008 14025 (15) Zhong, K.; Hu, X. S.; Zhao, G. H.; Chen, F.; Liao, X. J. Food Chem. 2005, 92, 473. (16) Xie, G.; Timasheff, S. N. Protein Sci. 1997, 6, 211. (17) Yoo, B.; Lee, C. M. J. Agric. Food Chem. 1993, 41, 190. (18) McClements, D. J. Crit. ReV. Food Sci. 2002, 45, 417. (19) Crowe, J. H.; Carpenter, J. F.; Crow, L. M.; Anchordoguy, T. J. Cryobiology 1990, 27, 219. (20) Li, D.; Jian, X.; Yan, Z. Spectrosc. Spect. Anal. 2008, 28, 13121. (21) Usha, R.; Sundar Raman, S.; Subramanian, V.; Ramasami, T. Chem. Phys. Lett. 2006, 430, 391. (22) Gekko, K.; Koga, S. J. Biochem. 1983, 94, 199. (23) Timasheff, S. N.; Arakawa, T. Stabilization of protein structure by solvents. In Protein Structure: A Practical Approach; Creighton, T. E., Eds.; IRL Press: Oxford, 1989; p 331. (24) Record, M. T.; Zhang, W.; Anderson, C. F. AdV. Protein Chem. 1998, 51, 281. (25) Steffen, B. P.; Virpi, J.; Peter, F.; Reinhard, W.; Shona, P. J. Biotechnol. 2004, 114, 269. (26) Shugar, D. Biochim. Biophys. Acta 1952, 8, 302. (27) Anson, M. L. J. Gen. Physiol. 1938, 22, 79. (28) Neumann, E.; Katchalsky, A. Proc. Natl. Acad. Sci. U.S.A. 1972, 69, 993. (29) Sielecki, A. R.; Fedorov, A. A.; Boodhoo, A.; Andreeva, N. S.; James, M. N. J. Mol. Biol. 1990, 214, 143. (30) Favilla, R.; Parsoli, A.; Mazzini, A. Biophys. Chem. 1997, 67, 75. (31) Cowgill, R. W. Biochim. Biophys. Acta 1967, 140, 37. (32) Tello-Solı´s, S. R.; Hernandez-Arana, A. Biochem. J. 1995, 311, 969. (33) Kelly, S. M.; Price, N. C. Biochim. Biophys. Acta 1997, 1338, 161. (34) Ohgushi, M.; Wada, A. FEBS Lett. 1983, 164, 21. (35) Lee, J. C.; Timasheff, S. N. J. Biol. Chem. 1981, 256, 7193. (36) Back, J. F.; Oakenfull, D.; Smith, M. B. Biochemistry 1979, 18, 519.

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