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Protein inactivation in amorphous sucrose and trehalose matrices: effects of phase separation and crystallization
Biochimica et Biophysica Acta 1425 (1998) 235^244
Protein inactivation in amorphous sucrose and trehalose matrices: e¡ects of phase separation and crystallization Wendell Q. Sun*, Paul Davidson School of Biological Sciences, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore Received 15 April 1998; revised 11 June 1998; accepted 1 July 1998
Abstract Trehalose is the most effective carbohydrate in preserving the structure and function of biological systems during dehydration and subsequent storage. We have studied the kinetics of protein inactivation in amorphous glucose/sucrose (1:10, w/w) and glucose/trehalose (1:10, w/w) systems, and examined the relationship between protein preservation, phase separation and crystallization during dry storage. The glucose/trehalose system preserved glucose-6-phosphate dehydrogenase better than did the glucose/sucrose system with the same glass transition temperature (Tg ). The Williams-Landel-Ferry kinetic analysis indicated that the superiority of the glucose/trehalose system over the glucose/sucrose system was possibly associated with a low free volume and a low free volume expansion at temperatures above the Tg . Phase separation and crystallization during storage were studied using differential scanning calorimetry, and three separate domains were identified in stored samples (i.e., sugar crystals, glucose-rich and disaccharide-rich amorphous domains). Phase separation and crystallization were significantly retarded in the glucose/trehalose system. Our data suggest that the superior stability of the trehalose system is associated with several properties of the trehalose glass, including low free volume, restricted molecular mobility and the ability to resist phase separation and crystallization during storage. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Crystallization ; Desiccation tolerance; Glass transition; Phase separation; Protein preservation; Sucrose; Trehalose
1. Introduction The accumulation of disaccharides is associated with the desiccation-tolerant state of anhydrobiotic organisms (reviewed in [1^3]). The roles of carbohydrates in the stabilization of membranes, proteins, and cells upon dehydration have been extensively studied in the past 15 years. As a result, sucrose, Abbreviations: DSC, di¡erential scanning calorimetry; G6PDH, glucose-6-phosphate dehydrogenase; Tg , glass transition temperature; WLF, Williams-Landel-Ferry * Corresponding author. Fax: +65 779-2486; E-mail: [email protected]
lactose and more recently trehalose are widely used as excipients in stabilizing biomaterials by the pharmaceutical industry. Trehalose is the most e¡ective carbohydrate in conferring protection during dehydration and storage [4^9]. The e¤cacy order for membrane preservation is reported to be trehalose, followed by lactose, maltose, sucrose, and then glucose in a decreasing order [5,9]. Several studies have investigated possible mechanisms for the high e¤cacy of trehalose, but the properties that make trehalose superior to other carbohydrates are not fully understood. Green and Angell [10] suggested that the high e¤cacy of trehalose was connected with its glass-form-
0304-4165 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 8 ) 0 0 0 7 6 - 2
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ing characteristics. On the basis of mole percent glucose rings, trehalose had the highest glass transition temperature (Tg ) among tested sugars, followed by maltose, sucrose and glucose. This order of the Tg was that of the e¤cacy order of carbohydrates in biopreservation [10]. The high Tg of the trehalose/ water system was related to the use of trehalose as a desiccation protectant by certain anhydrobiotic organisms. Intracellular processes in trehalose/water solutions can be brought to a halt by a viscous slowdown (i.e., suspending life), while the water content remains quite high relative to other saccharide/water systems of the same Tg [11]. In seeds, which commonly accumulate sucrose and oligosaccharides, the formation of intracellular glasses is associated with the survival of desiccation and the maintenance of seed viability during storage [12,13]. It was noted, however, that glass formation alone was not su¤cient for the preservation of membranes [14,15] and proteins [16,17] during freeze-drying and storage, and for the survival of seeds upon desiccation [18]. Carbohydrates such as maltodextrin (MD) or poly(vinyl)pyrrolidone (PVP), with Tg values much higher than trehalose, are less e¡ective in stabilizing membranes and proteins [14,19^21]. Carpenter and Crowe [22] demonstrated that a direct interaction (hydrogen-bonding) between sugars and enzymes was required for the stabilization of protein structures during drying. The poor ability of MD and PVP in membrane and protein preservation is due to the lack of such hydrogen-bonding [14,23]. Trehalose is an e¡ective hydrogen-bond donor and acceptor, and has the highest potential among disaccharides to form hydrogen bonds with biomolecules [24]. However, this property may not necessarily explain why trehalose o¡ers better protection than other disaccharides. There is no clear correlation between the ability of biopreservation and the number and type of hydroxyl groups of carbohydrates, and not all carbohydrates that have the potential to form hydrogen bonds would preserve biomaterials [4,23]. The e¤cacy order in biopreservation is far di¡erent from the order of hydrogen-bonding potential for various carbohydrates. It is believed that both the glassy state and hydrogen-bonding are required for the preservation of membranes, proteins and cells [3,14^16]. Roser [6] proposed that trehalose might have un-
known properties over other disaccharides. Carbohydrate glasses are non-equilibrium, thermodynamically unstable solutions, and their physical state will change during storage. Unexpected changes in the physical state would have signi¢cant impact on the preservation of biomaterials [25,26]. Aldous et al. [27] recently o¡ered a novel interpretation of the trehalose superiority. They suggested that carbohydrates that could form hydrate crystals might better protect biomaterials during storage. Crowe et al. [28] compared the e¡ect of crystallization on the Tg of sucrose and trehalose glasses when rehydrated with water vapor. The formation of trehalose dihydrate crystals sequestered water, and therefore enhance membrane preservation by preventing the Tg depression. A large depression of the Tg in the sucrose glass was correlated with poor membrane preservation during rehydration [28]. There may be other factors contributing to the superiority of trehalose in biopreservation. Compared to trehalose, ra¤nose has comparable Tg values at low water contents [29,30]; ra¤nose is more e¡ective in hydrogen-bonding with biomolecules [24]; and ra¤nose is an even better water scavenger because it forms pentahydrate crystals when crystallized [29]. One might expect ra¤nose to be more e¡ective than trehalose in biopreservation. Yet ra¤nose was observed to be much less protective than trehalose for membranes and proteins during dehydration and storage [4,9]. Sastry and Agmon [31] recently reported that trehalose prevents myoglobin collapse through the retention of a few vital internal water molecules and the preservation of protein internal mobility. Their work suggested that molecular interactions among water, sugar and protein played an important role in protein structure preservation. The stability of biological systems in the glassy matrix cannot be understood fully unless the dynamic interactions between various participating components are examined. In the present study, we have examined the kinetics of protein inactivation in glucose/sucrose and glucose/trehalose systems with similar Tg values. The incorporation of glucose as a minor component into the glassy systems has allowed us to investigate the relationship between phase separation, crystallization and protein preservation during storage. The superior stability of the trehalose system in biopreservation
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may be associated with restricted molecular mobility and the ability to resist phase separation and crystallization of trehalose glass. 2. Materials and methods Dry protein/carbohydrate samples were prepared with glucose, sucrose, trehalose, and glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) (all from Sigma, St. Louis, MO, USA). G6PDH was delivered as a suspension in Tris-HCl bu¡er (50 mM, pH 7.5) with 3.2 M (NH4 )2 SO4 and 1 mM MgCl2 . Salts were removed using a 10-DG desalting column (cut-o¡, 6 kDa) (Bio-Rad, Hercules, CA, USA) with Tris-HCl bu¡er (50 mM, pH 7.5). Aqueous protein/carbohydrate solutions were made in 10 g/l glucose and 100 g/l sucrose or trehalose. Aliquots (20 Wl) were pipetted to 1.5-ml Eppendorf tubes, frozen with liquid nitrogen, and then freeze-dried for 18 h under vapor pressure of 100 mTorr. A dry carbohydrate/protein sample contained 0.2 mg glucose, 2.0 mg sucrose or trehalose, and 0.01 unit G6PDH. Residual moisture was V0.06 and 0.08 g H2 O/g dry mass in glucose/sucrose and glucose/trehalose samples, respectively. Freeze-dried samples were stored at temperatures ranging from 33³C to 93³C for different periods, and then rehydrated for enzyme activity measurements. Triplicate samples were used for each measurement. To study phase separation and crystallization, additional sets of samples were prepared with glucose, sucrose and trehalose at the composition pro¢le mentioned above. Freeze-dried samples were stored at 44³C and 60³C. Glass transition and state changes were determined by di¡erential scanning calorimetry (DSC-131, Setaram, France). Samples were sealed in 30-Wl aluminum crucibles, cooled with liquid nitrogen to 3120³C, and then warmed at 10³C/min to 130³C for glucose/trehalose samples and to 210³C for glucose/sucrose samples, with helium as inert purge gas. DSC thermograms of stored samples were compared with those of control samples kept at 320³C. The fraction of crystallization in a sample was calculated from the melting enthalpy of crystals, as compared to the melting enthalpy of pure crystalline carbohydrates. At least three samples were scanned for each determination.
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3. Results 3.1. Physical state of freeze-dried samples Fig. 1 shows DSC thermograms of freeze-dried G6PDH/glucose/sucrose and G6PDH/glucose/trehalose samples. Glass transitions were observed between 12³C and 35³C, and the mid-point temperatures of the transitions were taken to be the Tg . The Tg values of two systems were the same, being 23.5 þ 1.3³C (mean þ S.E.) and 22.4 þ 1.8³C for glucose/sucrose and glucose/trehalose systems, respectively. 3.2. G6PDH preservation during freeze-drying and storage Residual G6PDH activity in samples was assayed immediately after freeze-drying to determine e¡ects of glucose/sucrose and glucose/trehalose systems on G6PDH protection. The recovery of G6PDH activity was at 87.0 þ 2.0% (mean þ S.E.) for the glucose/sucrose system and 87.8 þ 2.0% for the glucose/trehalose system. Prepared samples permitted a fair comparison between glucose/sucrose and glucose/ trehalose systems for G6PDH preservation during subsequent storage. Fig. 2 shows the loss of G6PDH activity during storage. The loss of G6PDH activity in the glucose/ sucrose system followed the ¢rst-order reaction ki-
Fig. 1. DSC thermograms showing glass transition temperatures of freeze-dried protein/carbohydrate samples. Scan rate, 10³C/ min. DSC thermograms were normalized to a sample mass of 20 mg.
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various temperatures. The kinetics of G6PDH inactivation in the glucose/trehalose system deviated from the ¢rst-order reaction kinetics. The plot of log (% activity retention) against time showed signi¢cant curvature (Fig. 2B). The rate constant of G6PDH inactivation was a function of storage time, and decreased steadily as storage time increased. This kinetic behavior of G6PDH inactivation was similar at all temperatures in the glucose/ trehalose system. As an approximation, the rate constant of G6PDH inactivation in the glucose/trehalose system was calculated according to the ¢rst-order reaction kinetics, using the ¢rst few data points in the linear range, as indicated by the dashed lines in Fig. 2B. Temperature dependence of G6PDH inactivation was compared between glucose/sucrose and glucose/ trehalose systems. Inactivation rate constants were signi¢cantly lower in the glucose/trehalose system than in the glucose/sucrose system (Fig. 3), despite the similar Tg values in both systems (Fig. 1). Data of G6PDH inactivation was analyzed, using the Williams-Landel-Ferry (WLF) equation [32], kg 3C1 T3Tg R Log Log k Rg C2 T3Tg Fig. 2. Representative ¢rst-order plots of G6PDH inactivation during storage at constant temperatures. (A) The glucose/sucrose system. Slopes are rate constants of G6PDH inactivation. (B) The glucose/trehalose system. G6PDH inactivation deviated from the ¢rst-order kinetics. Rate constants of G6PDH inactivation in the glucose/trehalose system were calculated using the ¢rst few data points in the linear region (dashed lines). Each point is the mean of three independent measurements.
netics, and the plots of log (% activity retention) against storage time were straight lines (Fig. 2A). The slope of the ¢rst-order plot gave the rate constant (k) of G6PDH inactivation during storage at
where k and kg are rate constants; and R and Rg are viscosities at temperatures T and Tg , respectively. C1 and C2 are WLF equation constants and can be derived from experimental data. Experimental data ¢tted the WLF equation well, and derived WLF equation constants were presented in Table 1. Derived Tg values for both systems were almost identical to those determined by DSC. However, other equation constants were signi¢cantly di¡erent between glucose/sucrose and glucose/trehalose systems. The kg value for the glucose/sucrose system was three times more than that for the glucose/trehalose system,
Table 1 WLF equation constants of G6PDH inactivation in glucose/sucrose and glucose/trehalose systems during storagea Equation constants 31
kg (min ) C1 C2 Derived Tg (³C) Measured Tg (³C) a
1
Glucose/sucrose (1:10, w/w) 36
Glucose/trehalose (1:10, w/w) 5.14U1037 19.05 þ 2.18 183.6 þ 5.4 24.2 þ 2.2 22.4 þ 1.8
2.37U10 14.55 þ 1.51 141.2 þ 5.8 23.3 þ 2.2 23.5 þ 1.3
Values are mean þ S.E.
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Fig. 3. Temperature dependence of G6PDH inactivation in glucose/sucrose and glucose/trehalose systems. Solid curves were drawn according to the ¢tted WLF equation. Derived WLF equation constants are shown in Table 1. Data (R) for glucose/ trehalose samples represent instantaneous rate constants during storage at 60³C. Numbers below data points shows hours of storage.
whereas C1 and C2 of the glucose/sucrose system were smaller than those of the glucose/trehalose system. It was interesting that G6PDH inactivation in the glucose/trehalose system slowed down during storage (Fig. 2B). This time-dependent phenomenon was analyzed further with a time-dependent kinetic equaTable 2 Values of the time-dependent parameter (n) and apparent rate constant (k*) for G6PDH inactivation in the glucose/trehalose system at di¡erent temperatures Temperature (³C)
n valuea
33 37 44 50 55 60 65.5 75.5 85.5 90.5 Average
0.42 þ 0.10 0.55 þ 0.09 0.39 þ 0.05 0.71 þ 0.04 0.66 þ 0.06 0.52 þ 0.03 0.54 þ 0.05 0.41 þ 0.04 0.51 þ 0.03 0.61 þ 0.08 0.53 þ 0.04
a
k* (h30:53 )b (0.065 þ 0.029)? 0.007 þ 0.004 0.015 þ 0.002 0.038 þ 0.004 0.064 þ 0.003 0.089 þ 0.012 0.171 þ 0.011 0.420 þ 0.049 1.103 þ 0.059 1.535 þ 0.107
The values of n for each storage temperature were derived from Eq. 2. Data are means þ S.E. b Values of k* were derived with n being ¢xed at 0.53. Data are means þ S.E.
Fig. 4. (A) Correlation between the initial rate constant (ki ) and apparent rate constant (k*) of G6PDH inactivation in the glucose/trehalose system. (B) Temperature dependence of k* for the glucose/trehalose system during storage. Solid curves were drawn according to the ¢tted WLF equation.
tion. The percent G6PDH inactivation (Aloss ) at time t was described by Aloss 13exp3k tn
2
where k* was the apparent rate constant, and n was the time-dependent parameter. Unlike inactivation rate constants calculated from the ¢rst few data points in the linear range (Fig. 2B), k* also took into consideration the factors that led to the increase of G6PDH stability during storage. Eq. 2 described very well experimental data at all storage temperatures (R2 s 0.96) except for 33³C. The values of n ranged from 0.39 to 0.71, with an average of 0.53 þ 0.04 (mean þ S.E.) (Table 2). The apparent rate constants, k*, were therefore derived for all tem-
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Fig. 5. DSC thermograms of glucose/sucrose samples after storage at 60³C. Arrows indicate glass transitions. Shaded areas represent devitri¢cation. Scan rate, 10³C/min. DSC thermograms were normalized to a sample mass of 20 mg.
peratures with n being ¢xed at 0.53. The k* was highly correlated with the initial rate constant (ki ) calculated from the data points in the linear range (Fig. 4A). The k* was also Tg -dependent, and followed WLF kinetics (Fig. 4B). The derived Tg value was 23.3 þ 1.5³C (mean þ S.E.), once again consistent with DSC measurements. Values of C1 and C2 were calculated to be 11.0 þ 0.8 and 162.2 þ 4.3, respectively.
because no similar change was observed in samples that had been stored at 320³C since freeze-drying. Additional sets of samples were prepared to study phase separation and solute crystallization during storage. Residual moisture was approximately 0.07 and 0.10 g H2 O/g dry mass for glucose/sucrose and glucose/trehalose samples. Fig. 5 shows DSC thermograms of glucose/sucrose samples after storage at 60³C. The control sample exhibited three thermal events during the heating scan between 3120³C and 220³C. The glass transition was observed at 11.5 þ 0.9³C. Devitri¢cation occurred between 60³C and 120³C, followed by the melting of sucrose crystals between 135³C and 180³C. Two major changes were observed during storage. One was phase separation in the amorphous matrix, as shown by two glass transitions in stored samples. The Tg of the major glassy domain increased rapidly during the ¢rst 6 h, and apparently remained at 60³C after 12 h of storage. The Tg of the minor domain declined to below 350³C, but increased slightly after 6 h (Fig. 6). Another major change during storage was crystallization. The devitri¢cation peak gradually became undetectable after 6 h, but the melting peak maintained approximately at the same size for all stored samples, indicating crystallization during storage. However, sucrose crystallization during storage could not be studied quantitatively with DSC, since
3.3. Phase separation and crystallization during storage Physical state of amorphous carbohydrate matrices changes during storage. In a preliminary experiment, we examined freeze-dried samples with DSC after storage at 44³C for 45 days. The glass transition of glucose/sucrose samples became undetectable, indicating the loss of the amorphous state. The glass transition in glucose/trehalose samples still remained at 23.9 þ 1.4³C (mean þ S.E.), although a small melting peak of trehalose dihydrate crystals was observed, indicating that a small fraction of trehalose crystallized. The data showed that the crystallization of sucrose and trehalose occurred during storage,
Fig. 6. Glass transition temperatures of glucose/sucrose samples after storage at 60³C. Data are means þ S.E. of at least three independent measurements. Bars are not shown when smaller than symbols. The shaded area represents the expected Tg for the local glucose domain separated after sucrose crystallization.
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devitri¢cation occurred in some samples during DSC measurements. Fig. 7 shows DSC thermograms of glucose/trehalose samples after storage. The control sample had the Tg at 12.3 þ 1.5³C, and a small melting peak at V95³C. Unlike glucose/sucrose samples, no apparent devitri¢cation was observed in glucose/trehalose samples during DSC measurements. Phase separation was also observed during storage, but was signi¢cantly delayed. The di¡erence in the Tg between two glassy domains were relatively small in comparison to glucose/sucrose samples (Fig. 8A). The Tg of the major glassy domain gradually increased to 22³C during storage, whereas the Tg of the minor domain declined to below 320³C. The endothermic peak at 95³C was the melting of trehalose dihydrate. The dehydration of trehalose dihydrate occurs at V130³C (data not shown). The peak size increased as storage time increased. The small amount of trehalose dihydrate in control samples was probably formed during DSC measurement. The fraction of trehalose dihydrate crystals in samples is shown in Fig. 8B. As storage time increased, the rate of trehalose dihydrate formation slowed down signi¢cantly, exhibiting a trend similar to G6PDH inactivation in the glucose/trehalose system. Even after 42 h of storage at 60³C, only 30% trehalose crystallized, showing
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Fig. 8. (A) Glass transition temperatures of glucose/trehalose samples after storage at 60³C. The shade area shows the expected Tg for the local glucose domain after trehalose crystallization. (B) Fraction of trehalose dihydrate in samples after storage at 60³C. Data are mean þ S.E. of at least three independent measurements. Bars are not shown when smaller than the symbols.
that the glucose/trehalose system was more stable than the glucose/sucrose system at the same Tg . 4. Discussion
Fig. 7. DSC thermograms of glucose/trehalose samples after storage at 60³C. Arrows indicate glass transitions. Scan rate, 10³C/min. DSC thermograms were normalized to a sample mass of 20 mg.
The high Tg value was previously suggested to be the cause of trehalose superiority over other carbohydrates in biopreservation [10]. Sucrose was commonly used in comparative studies. We checked recent reports [17,28,33,34], and found that the Tg of trehalose systems were typically 20^40³C higher than that of sucrose systems. This di¡erence in the Tg alone corresponds to an increase of 2^3 orders of magnitude in the stability of biomaterials in the glassy matrix [35,36], and a decrease of 4^6 orders
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of magnitude in the rates of relaxation processes according to WLF kinetics [35,37]. To verify whether better preservation by the trehalose glass was due to the high Tg or other special properties, the comparison between trehalose and other carbohydrates should be made at the same Tg value. In the present study, glucose/sucrose and glucose/trehalose systems were prepared to have the same Tg values (Fig. 1). Our data showed that the glucose/trehalose system conferred better protein preservation during storage at temperatures above the Tg (Fig. 3). The superior stability of the trehalose system may be attributed to a number of dynamic properties of the trehalose glass. Derived WLF equation constants, kg , C1 and C2 , are directly related to dynamic properties of amorphous systems [32]. C1 is proportional to the inverse of free volume at Tg , and C2 is proportional to the ratio of free volume at Tg to the thermal expansion of free volume above the Tg [37]. Free volume in an amorphous system is geometrically interpreted as a continuous network of `lakes and channels', which permit the di¡usive motion of its molecules [38]. Such a continuous network of `lakes and channels' disintegrates when the free volume is reduced to below a critical level, corresponding to the glass transition. When samples are stored at temperatures above the Tg , free volume and total volume of samples increase. Free volume expansion re-establishes the continuous network of `lakes and channels', thus providing su¤cient room for molecular motion. The stability of protein in carbohydrate glasses was closely related to molecular mobility [39], and therefore, WLF constants are of signi¢cance for protein preservation. Values of C1 and C2 for the glucose/trehalose system were signi¢cantly greater than those of the glucose/sucrose system (Table 1), suggesting that the glucose/trehalose system had a smaller free volume at the Tg and also a smaller free volume expansion coe¤cient at temperatures above the Tg than did the glucose/sucrose system. This implied that the di¡usive molecular motion in the glucose/trehalose system should be more restricted than that in the glucose/sucrose system. The analysis rationalized the observation that the kg for the glucose/trehalose system was less than that of glucose/trehalose system (Table 1). Phase separation and crystallization would occur as a result of increased molecular mobility. When an
aqueous glass is held at an elevated temperature, the system becomes increasingly unstable, being vulnerable to phase separation and subsequent crystallization. Preliminary storage experiment showed that the glucose/trehalose system was much more stable than the glucose/sucrose system at a high temperature. After 45-day storage at 44³C (T3Tg = V20³C), glucose/sucrose samples had lost their amorphous state completely, whereas only a small fraction ( 6 4%) of the glucose/trehalose material crystallized and the rest remained in the amorphous state with the Tg unchanged (23.9 þ 1.4³C). Crystallization was observed to destabilize enzymes, resulting in the rapid loss of enzyme activity [19^21]. The ability to resist phase separation and crystallization was probably associated with the restricted molecular motion in the glucose/trehalose system, and certainly contributed to its superiority over the glucose/sucrose system in conferring protection during storage. Crowe et al. [28] reported that the Tg of trehalose glass did not change signi¢cantly after exposure to elevated humidity because of the formation of trehalose dihydrate. In our experiment, samples were not humidi¢ed during storage, and the greater stability of the glucose/trehalose system can not be attributed to the sequestration of water. The formation of trehalose dihydrate ( 6 4%) in this case would have had little e¡ect on the rest of amorphous domain. Di¡erences in phase separation and crystallization between glucose/sucrose and glucose/trehalose systems were further studied using samples with lower Tg (V11^ 13³C) at 60³C. Three separate domains were identi¢ed in stored samples, representing carbohydrate crystal, glucose-rich and disaccharide-rich domain. Phase separation occurred rapidly in the glucose/sucrose system. The Tg of the sucrose-rich domain increased to about 60³C after 12 h, probably due to the dehydration at the high storage temperature (Fig. 6). In contrast, phase separation in the glucose/trehalose system was signi¢cantly delayed, and the Tg of the trehalose-rich domain increased by only 10³C (Fig. 8). Further dehydration during storage appeared to be more retarded in the glucose/trehalose system than in the glucose/sucrose system, showing greater restriction on the di¡usion of water molecules in the glucose/trehalose system. This suggestion is consistent with the results from our kinetic analysis mentioned above (i.e. the trehalose/glucose glass had a
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smaller free volume and a smaller free volume expansion coe¤cient at temperatures above the Tg than did the glucose/sucrose glass). Phase separation and crystallization of one component in a glassy product would lead to a dramatic change in the composition of the remaining amorphous phase. In the glucose/sucrose system, crystallization would result in a high local water content. The initial moisture in glucose/sucrose samples was about 0.07 g H2 O/g dry mass. If sucrose had crystallized, the local moisture of the remaining glucose domain would have increased to 0.6^0.7 g H2 O/g dry mass, and the Tg could have decreased to below 380³C. The observed Tg of the glucose-rich domain after sucrose crystallization was 20^30³C higher than the expected Tg , possibly due to partial dehydration during storage (water vapor goes to the head spaces of storage tubes) (Fig. 6). However, in the glucose/ trehalose glassy system, the local water content changed di¡erently because of the formation of trehalose dihydrate crystals. When trehalose crystallized, the local water content of the remaining glucose domain would have decreased to 0.07 g H2 O/g dry mass, corresponding to a Tg slightly below 320³C. The observed Tg of the remaining glucoserich domain was the same as the expected Tg (Fig. 8). The observed physical state changes in both systems could more or less be accounted for quantitatively according to system composition pro¢les. An interesting observation was that the rate constant of protein inactivation in the glucose/trehalose system was time-dependent (Fig. 2). The time-dependent parameter was 0.53 for the glucose/trehalose system, relative to the unity for the glucose/sucrose system, where protein inactivation simply followed the ¢rst order kinetics. The non-uniform distribution of residual water could give rise to a biphasic stability phenomenon, where the rate of loss of the stability was high during the initial period, and the product became more stable during the later stages [25]. However, the time-dependent phenomenon observed in the glucose/trehalose system was unlikely due to the non-uniform distribution of residual water in freeze-dried samples. DSC studies showed a single glass transition domain for both glucose/sucrose and glucose/trehalose samples before storage experiments (Fig. 1). The retardation of protein inactivation was likely due to the slight increase in the Tg
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during storage. An increase in the Tg would theoretically a¡ect protein stability as much as a decrease in storage temperature to the same extent. The data of protein inactivation during storage at 60³C were ¢tted with a third-order polynomial equation (R2 = 0.99). The instantaneous rate constants of protein inactivation were calculated by taking the ¢rst derivative of the ¢tted equation, and were superimposed onto Fig. 3 (triangle). The Tg dependence of the instantaneous rate constants was identical to the temperature dependence of initial rate constants at di¡erent temperatures, showing that the change of inactivation rate during storage was associated with the gradual increase in the Tg . A similar time dependence of crystallization was observed in the glucose/trehalose systems (Fig. 8). The gradual decrease in the rate of trehalose crystallization was also related to the increase of Tg during storage. In the present work, we have examined the kinetics of protein inactivation in two amorphous carbohydrate systems with similar Tg values. The superior stability of the glucose/trehalose system over the glucose/sucrose system was associated with a number of closely related dynamic properties of the glucose/trehalose glass, including low free volume, restricted molecular mobility of water and solutes, and the ability to resist phase separation and crystallization during storage. References [1] A.C. Leopold, in: G.J. Alscher, J.R. Cumming (Eds.), Stress Responses in Plants: Adaptation and Acclimation Mechanisms, Wiley-Liss, New York, 1990, pp. 37^56. [2] J.H. Crowe, L.M. Crowe, in: G.N. Somero, C.B. Osmond, C.L. Bolis (Eds.), Water and Life, Springer-Verlag, Berlin, 1992, pp. 87^103. [3] W.Q. Sun, A.C. Leopold, Comp. Biochem. Physiol. 117A (1997) 327^333. [4] J.H. Crowe, L.M. Crowe, S.A. Jackson, Arch. Biochem. Biophys. 220 (1983) 477^484. [5] R. Mouradian, C. Womersley, L.M. Crowe, J.H. Crowe, Biochim. Biophys. Acta 778 (1984) 615^617. [6] B. Roser, BioPharm 4 (1991) 47^53. [7] S.B. Leslie, E. Israeli, B. Lighthart, J.H. Crowe, L.M. Crowe, Appl. Environ. Microbiol. 61 (1995) 3592^3597. [8] M. Uritani, M. Takai, K. Yoshinaga, J. Biochem. 117 (1995) 774^779. [9] L.M. Crowe, R. Mouradian, J.H. Crowe, S.A. Jackson, C. Womersley, Biochim. Biophys. Acta 769 (1984) 141^150.
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[10] J.L. Green, C.A. Angell, J. Phys. Chem. 93 (1989) 2880^ 2882. [11] S.P. Ding, J. Fan, J.L. Green, Q. Lu, E. Sanchez, C.A. Angell, J. Therm. Anal. 47 (1996) 1391^1405. [12] W.Q. Sun, A.C. Leopold, Ann. Bot. 74 (1994) 601^604. [13] W.Q. Sun, Ann. Bot. 79 (1997) 291^291. [14] J.H. Crowe, S.B. Leslie, L.M. Crowe, Cryobiology 31 (1994) 355^366. [15] W.Q. Sun, A.C. Leopold, L.M. Crowe, J.H. Crowe, Biophys. J. 70 (1996) 1769^1776. [16] B.S. Chang, R.M. Beauvais, A. Dong, J.F. Carpenter, Arch. Biochem. Biophys. 331 (1996) 249^258. [17] S.P. Duddu, P.R. Dal Monte, Pharmaceut. Res. 14 (1997) 591^595. [18] W.Q. Sun, A.C. Leopold, Physiol. Plant. 90 (1994) 621^628. [19] C. Schebor, M.P. Buera, J. Chirife, J. Food Eng. 30 (1996) 269^282. [20] S. Cardona, C. Schebor, M.P. Buera, M. Karel, J. Chirife, J. Food Sci. 62 (1997) 105^109. [21] M.F. Mazzobre, M.P. Buera, J. Chirife, Food Sci. Technol. 30 (1997) 324^329. [22] J.F. Carpenter, J.H. Crowe, Biochemistry 28 (1989) 3916^ 3922. [23] T. Arakawa, S.J. Prestrelski, W.C. Kenney, J.F. Carpenter, Adv. Drug Deliv. Rev. 10 (1993) 1^28. [24] S.H. Ga¡ney, E. Haslam, T.H. Lilley, T.R. Ward, J. Chem. Soc. Faraday Trans. 84 (1988) 2545^2552.
[25] F. Franks, Trends Biotechnol. 12 (1994) 114^117. [26] W.Q. Sun, Cryo-Letters 18 (1997) 99^106. [27] B.J. Aldous, A.D. Au¡ret, F. Franks, Cryo-Letters 16 (1995) 181^186. [28] L.M. Crowe, D.S. Reid, J.H. Crowe, Biophys. J. 71 (1996) 2087^2093. [29] A. Saleki-Gerhardt, J.G. Stowell, S.R. Byrn, G. Zogra¢, J. Pharmaceut. Sci. 84 (1995) 318^323. [30] W.F. Wolkers, H. Oldenhof, M. Alberda, F.A. Hoekstra, Biochim. Biophys. Acta 1379 (1998) 83^96. [31] G.M. Sastry, N. Agmon, Biochemistry 36 (1997) 7097^7108. [32] M.J. Williams, R.F. Landel, J.D. Ferry, J. Am. Chem. Soc. 77 (1955) 3701^3707. [33] K.L. Koster, C.L. Sommervold, Y.P. Lei, J. Therm. Anal. 47 (1996) 1581^1596. [34] J. O'Brien, J. Food Sci. 61 (1996) 679^682. [35] W.Q. Sun, Cryo-Letters 19 (1998) 66^70. [36] W.Q. Sun, Cryo-Letters 19 (1998) 105^114. [37] L. Slade, A. Levine, Crit. Rev. Food Sci. Nutr. 30 (1991) 115^360. [38] D.P. Miller, J.J. de Pablo, H. Corti, Pharmaceut. Res. 14 (1997) 578^590. [39] S. Yoshioka, Y. Aso, S. Kojima, Pharmaceut. Res. 14 (1997) 736^741.
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Protein inactivation in amorphous sucrose and trehalose matrices: e¡ects of phase separation and crystallization Wendell Q. Sun*, Paul Davidson School of Biological Sciences, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore Received 15 April 1998; revised 11 June 1998; accepted 1 July 1998
Abstract Trehalose is the most effective carbohydrate in preserving the structure and function of biological systems during dehydration and subsequent storage. We have studied the kinetics of protein inactivation in amorphous glucose/sucrose (1:10, w/w) and glucose/trehalose (1:10, w/w) systems, and examined the relationship between protein preservation, phase separation and crystallization during dry storage. The glucose/trehalose system preserved glucose-6-phosphate dehydrogenase better than did the glucose/sucrose system with the same glass transition temperature (Tg ). The Williams-Landel-Ferry kinetic analysis indicated that the superiority of the glucose/trehalose system over the glucose/sucrose system was possibly associated with a low free volume and a low free volume expansion at temperatures above the Tg . Phase separation and crystallization during storage were studied using differential scanning calorimetry, and three separate domains were identified in stored samples (i.e., sugar crystals, glucose-rich and disaccharide-rich amorphous domains). Phase separation and crystallization were significantly retarded in the glucose/trehalose system. Our data suggest that the superior stability of the trehalose system is associated with several properties of the trehalose glass, including low free volume, restricted molecular mobility and the ability to resist phase separation and crystallization during storage. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Crystallization ; Desiccation tolerance; Glass transition; Phase separation; Protein preservation; Sucrose; Trehalose
1. Introduction The accumulation of disaccharides is associated with the desiccation-tolerant state of anhydrobiotic organisms (reviewed in [1^3]). The roles of carbohydrates in the stabilization of membranes, proteins, and cells upon dehydration have been extensively studied in the past 15 years. As a result, sucrose, Abbreviations: DSC, di¡erential scanning calorimetry; G6PDH, glucose-6-phosphate dehydrogenase; Tg , glass transition temperature; WLF, Williams-Landel-Ferry * Corresponding author. Fax: +65 779-2486; E-mail: [email protected]
lactose and more recently trehalose are widely used as excipients in stabilizing biomaterials by the pharmaceutical industry. Trehalose is the most e¡ective carbohydrate in conferring protection during dehydration and storage [4^9]. The e¤cacy order for membrane preservation is reported to be trehalose, followed by lactose, maltose, sucrose, and then glucose in a decreasing order [5,9]. Several studies have investigated possible mechanisms for the high e¤cacy of trehalose, but the properties that make trehalose superior to other carbohydrates are not fully understood. Green and Angell [10] suggested that the high e¤cacy of trehalose was connected with its glass-form-
0304-4165 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 8 ) 0 0 0 7 6 - 2
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ing characteristics. On the basis of mole percent glucose rings, trehalose had the highest glass transition temperature (Tg ) among tested sugars, followed by maltose, sucrose and glucose. This order of the Tg was that of the e¤cacy order of carbohydrates in biopreservation [10]. The high Tg of the trehalose/ water system was related to the use of trehalose as a desiccation protectant by certain anhydrobiotic organisms. Intracellular processes in trehalose/water solutions can be brought to a halt by a viscous slowdown (i.e., suspending life), while the water content remains quite high relative to other saccharide/water systems of the same Tg [11]. In seeds, which commonly accumulate sucrose and oligosaccharides, the formation of intracellular glasses is associated with the survival of desiccation and the maintenance of seed viability during storage [12,13]. It was noted, however, that glass formation alone was not su¤cient for the preservation of membranes [14,15] and proteins [16,17] during freeze-drying and storage, and for the survival of seeds upon desiccation [18]. Carbohydrates such as maltodextrin (MD) or poly(vinyl)pyrrolidone (PVP), with Tg values much higher than trehalose, are less e¡ective in stabilizing membranes and proteins [14,19^21]. Carpenter and Crowe [22] demonstrated that a direct interaction (hydrogen-bonding) between sugars and enzymes was required for the stabilization of protein structures during drying. The poor ability of MD and PVP in membrane and protein preservation is due to the lack of such hydrogen-bonding [14,23]. Trehalose is an e¡ective hydrogen-bond donor and acceptor, and has the highest potential among disaccharides to form hydrogen bonds with biomolecules [24]. However, this property may not necessarily explain why trehalose o¡ers better protection than other disaccharides. There is no clear correlation between the ability of biopreservation and the number and type of hydroxyl groups of carbohydrates, and not all carbohydrates that have the potential to form hydrogen bonds would preserve biomaterials [4,23]. The e¤cacy order in biopreservation is far di¡erent from the order of hydrogen-bonding potential for various carbohydrates. It is believed that both the glassy state and hydrogen-bonding are required for the preservation of membranes, proteins and cells [3,14^16]. Roser [6] proposed that trehalose might have un-
known properties over other disaccharides. Carbohydrate glasses are non-equilibrium, thermodynamically unstable solutions, and their physical state will change during storage. Unexpected changes in the physical state would have signi¢cant impact on the preservation of biomaterials [25,26]. Aldous et al. [27] recently o¡ered a novel interpretation of the trehalose superiority. They suggested that carbohydrates that could form hydrate crystals might better protect biomaterials during storage. Crowe et al. [28] compared the e¡ect of crystallization on the Tg of sucrose and trehalose glasses when rehydrated with water vapor. The formation of trehalose dihydrate crystals sequestered water, and therefore enhance membrane preservation by preventing the Tg depression. A large depression of the Tg in the sucrose glass was correlated with poor membrane preservation during rehydration [28]. There may be other factors contributing to the superiority of trehalose in biopreservation. Compared to trehalose, ra¤nose has comparable Tg values at low water contents [29,30]; ra¤nose is more e¡ective in hydrogen-bonding with biomolecules [24]; and ra¤nose is an even better water scavenger because it forms pentahydrate crystals when crystallized [29]. One might expect ra¤nose to be more e¡ective than trehalose in biopreservation. Yet ra¤nose was observed to be much less protective than trehalose for membranes and proteins during dehydration and storage [4,9]. Sastry and Agmon [31] recently reported that trehalose prevents myoglobin collapse through the retention of a few vital internal water molecules and the preservation of protein internal mobility. Their work suggested that molecular interactions among water, sugar and protein played an important role in protein structure preservation. The stability of biological systems in the glassy matrix cannot be understood fully unless the dynamic interactions between various participating components are examined. In the present study, we have examined the kinetics of protein inactivation in glucose/sucrose and glucose/trehalose systems with similar Tg values. The incorporation of glucose as a minor component into the glassy systems has allowed us to investigate the relationship between phase separation, crystallization and protein preservation during storage. The superior stability of the trehalose system in biopreservation
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may be associated with restricted molecular mobility and the ability to resist phase separation and crystallization of trehalose glass. 2. Materials and methods Dry protein/carbohydrate samples were prepared with glucose, sucrose, trehalose, and glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) (all from Sigma, St. Louis, MO, USA). G6PDH was delivered as a suspension in Tris-HCl bu¡er (50 mM, pH 7.5) with 3.2 M (NH4 )2 SO4 and 1 mM MgCl2 . Salts were removed using a 10-DG desalting column (cut-o¡, 6 kDa) (Bio-Rad, Hercules, CA, USA) with Tris-HCl bu¡er (50 mM, pH 7.5). Aqueous protein/carbohydrate solutions were made in 10 g/l glucose and 100 g/l sucrose or trehalose. Aliquots (20 Wl) were pipetted to 1.5-ml Eppendorf tubes, frozen with liquid nitrogen, and then freeze-dried for 18 h under vapor pressure of 100 mTorr. A dry carbohydrate/protein sample contained 0.2 mg glucose, 2.0 mg sucrose or trehalose, and 0.01 unit G6PDH. Residual moisture was V0.06 and 0.08 g H2 O/g dry mass in glucose/sucrose and glucose/trehalose samples, respectively. Freeze-dried samples were stored at temperatures ranging from 33³C to 93³C for different periods, and then rehydrated for enzyme activity measurements. Triplicate samples were used for each measurement. To study phase separation and crystallization, additional sets of samples were prepared with glucose, sucrose and trehalose at the composition pro¢le mentioned above. Freeze-dried samples were stored at 44³C and 60³C. Glass transition and state changes were determined by di¡erential scanning calorimetry (DSC-131, Setaram, France). Samples were sealed in 30-Wl aluminum crucibles, cooled with liquid nitrogen to 3120³C, and then warmed at 10³C/min to 130³C for glucose/trehalose samples and to 210³C for glucose/sucrose samples, with helium as inert purge gas. DSC thermograms of stored samples were compared with those of control samples kept at 320³C. The fraction of crystallization in a sample was calculated from the melting enthalpy of crystals, as compared to the melting enthalpy of pure crystalline carbohydrates. At least three samples were scanned for each determination.
237
3. Results 3.1. Physical state of freeze-dried samples Fig. 1 shows DSC thermograms of freeze-dried G6PDH/glucose/sucrose and G6PDH/glucose/trehalose samples. Glass transitions were observed between 12³C and 35³C, and the mid-point temperatures of the transitions were taken to be the Tg . The Tg values of two systems were the same, being 23.5 þ 1.3³C (mean þ S.E.) and 22.4 þ 1.8³C for glucose/sucrose and glucose/trehalose systems, respectively. 3.2. G6PDH preservation during freeze-drying and storage Residual G6PDH activity in samples was assayed immediately after freeze-drying to determine e¡ects of glucose/sucrose and glucose/trehalose systems on G6PDH protection. The recovery of G6PDH activity was at 87.0 þ 2.0% (mean þ S.E.) for the glucose/sucrose system and 87.8 þ 2.0% for the glucose/trehalose system. Prepared samples permitted a fair comparison between glucose/sucrose and glucose/ trehalose systems for G6PDH preservation during subsequent storage. Fig. 2 shows the loss of G6PDH activity during storage. The loss of G6PDH activity in the glucose/ sucrose system followed the ¢rst-order reaction ki-
Fig. 1. DSC thermograms showing glass transition temperatures of freeze-dried protein/carbohydrate samples. Scan rate, 10³C/ min. DSC thermograms were normalized to a sample mass of 20 mg.
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various temperatures. The kinetics of G6PDH inactivation in the glucose/trehalose system deviated from the ¢rst-order reaction kinetics. The plot of log (% activity retention) against time showed signi¢cant curvature (Fig. 2B). The rate constant of G6PDH inactivation was a function of storage time, and decreased steadily as storage time increased. This kinetic behavior of G6PDH inactivation was similar at all temperatures in the glucose/ trehalose system. As an approximation, the rate constant of G6PDH inactivation in the glucose/trehalose system was calculated according to the ¢rst-order reaction kinetics, using the ¢rst few data points in the linear range, as indicated by the dashed lines in Fig. 2B. Temperature dependence of G6PDH inactivation was compared between glucose/sucrose and glucose/ trehalose systems. Inactivation rate constants were signi¢cantly lower in the glucose/trehalose system than in the glucose/sucrose system (Fig. 3), despite the similar Tg values in both systems (Fig. 1). Data of G6PDH inactivation was analyzed, using the Williams-Landel-Ferry (WLF) equation [32], kg 3C1 T3Tg R Log Log k Rg C2 T3Tg Fig. 2. Representative ¢rst-order plots of G6PDH inactivation during storage at constant temperatures. (A) The glucose/sucrose system. Slopes are rate constants of G6PDH inactivation. (B) The glucose/trehalose system. G6PDH inactivation deviated from the ¢rst-order kinetics. Rate constants of G6PDH inactivation in the glucose/trehalose system were calculated using the ¢rst few data points in the linear region (dashed lines). Each point is the mean of three independent measurements.
netics, and the plots of log (% activity retention) against storage time were straight lines (Fig. 2A). The slope of the ¢rst-order plot gave the rate constant (k) of G6PDH inactivation during storage at
where k and kg are rate constants; and R and Rg are viscosities at temperatures T and Tg , respectively. C1 and C2 are WLF equation constants and can be derived from experimental data. Experimental data ¢tted the WLF equation well, and derived WLF equation constants were presented in Table 1. Derived Tg values for both systems were almost identical to those determined by DSC. However, other equation constants were signi¢cantly di¡erent between glucose/sucrose and glucose/trehalose systems. The kg value for the glucose/sucrose system was three times more than that for the glucose/trehalose system,
Table 1 WLF equation constants of G6PDH inactivation in glucose/sucrose and glucose/trehalose systems during storagea Equation constants 31
kg (min ) C1 C2 Derived Tg (³C) Measured Tg (³C) a
1
Glucose/sucrose (1:10, w/w) 36
Glucose/trehalose (1:10, w/w) 5.14U1037 19.05 þ 2.18 183.6 þ 5.4 24.2 þ 2.2 22.4 þ 1.8
2.37U10 14.55 þ 1.51 141.2 þ 5.8 23.3 þ 2.2 23.5 þ 1.3
Values are mean þ S.E.
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Fig. 3. Temperature dependence of G6PDH inactivation in glucose/sucrose and glucose/trehalose systems. Solid curves were drawn according to the ¢tted WLF equation. Derived WLF equation constants are shown in Table 1. Data (R) for glucose/ trehalose samples represent instantaneous rate constants during storage at 60³C. Numbers below data points shows hours of storage.
whereas C1 and C2 of the glucose/sucrose system were smaller than those of the glucose/trehalose system. It was interesting that G6PDH inactivation in the glucose/trehalose system slowed down during storage (Fig. 2B). This time-dependent phenomenon was analyzed further with a time-dependent kinetic equaTable 2 Values of the time-dependent parameter (n) and apparent rate constant (k*) for G6PDH inactivation in the glucose/trehalose system at di¡erent temperatures Temperature (³C)
n valuea
33 37 44 50 55 60 65.5 75.5 85.5 90.5 Average
0.42 þ 0.10 0.55 þ 0.09 0.39 þ 0.05 0.71 þ 0.04 0.66 þ 0.06 0.52 þ 0.03 0.54 þ 0.05 0.41 þ 0.04 0.51 þ 0.03 0.61 þ 0.08 0.53 þ 0.04
a
k* (h30:53 )b (0.065 þ 0.029)? 0.007 þ 0.004 0.015 þ 0.002 0.038 þ 0.004 0.064 þ 0.003 0.089 þ 0.012 0.171 þ 0.011 0.420 þ 0.049 1.103 þ 0.059 1.535 þ 0.107
The values of n for each storage temperature were derived from Eq. 2. Data are means þ S.E. b Values of k* were derived with n being ¢xed at 0.53. Data are means þ S.E.
Fig. 4. (A) Correlation between the initial rate constant (ki ) and apparent rate constant (k*) of G6PDH inactivation in the glucose/trehalose system. (B) Temperature dependence of k* for the glucose/trehalose system during storage. Solid curves were drawn according to the ¢tted WLF equation.
tion. The percent G6PDH inactivation (Aloss ) at time t was described by Aloss 13exp3k tn
2
where k* was the apparent rate constant, and n was the time-dependent parameter. Unlike inactivation rate constants calculated from the ¢rst few data points in the linear range (Fig. 2B), k* also took into consideration the factors that led to the increase of G6PDH stability during storage. Eq. 2 described very well experimental data at all storage temperatures (R2 s 0.96) except for 33³C. The values of n ranged from 0.39 to 0.71, with an average of 0.53 þ 0.04 (mean þ S.E.) (Table 2). The apparent rate constants, k*, were therefore derived for all tem-
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Fig. 5. DSC thermograms of glucose/sucrose samples after storage at 60³C. Arrows indicate glass transitions. Shaded areas represent devitri¢cation. Scan rate, 10³C/min. DSC thermograms were normalized to a sample mass of 20 mg.
peratures with n being ¢xed at 0.53. The k* was highly correlated with the initial rate constant (ki ) calculated from the data points in the linear range (Fig. 4A). The k* was also Tg -dependent, and followed WLF kinetics (Fig. 4B). The derived Tg value was 23.3 þ 1.5³C (mean þ S.E.), once again consistent with DSC measurements. Values of C1 and C2 were calculated to be 11.0 þ 0.8 and 162.2 þ 4.3, respectively.
because no similar change was observed in samples that had been stored at 320³C since freeze-drying. Additional sets of samples were prepared to study phase separation and solute crystallization during storage. Residual moisture was approximately 0.07 and 0.10 g H2 O/g dry mass for glucose/sucrose and glucose/trehalose samples. Fig. 5 shows DSC thermograms of glucose/sucrose samples after storage at 60³C. The control sample exhibited three thermal events during the heating scan between 3120³C and 220³C. The glass transition was observed at 11.5 þ 0.9³C. Devitri¢cation occurred between 60³C and 120³C, followed by the melting of sucrose crystals between 135³C and 180³C. Two major changes were observed during storage. One was phase separation in the amorphous matrix, as shown by two glass transitions in stored samples. The Tg of the major glassy domain increased rapidly during the ¢rst 6 h, and apparently remained at 60³C after 12 h of storage. The Tg of the minor domain declined to below 350³C, but increased slightly after 6 h (Fig. 6). Another major change during storage was crystallization. The devitri¢cation peak gradually became undetectable after 6 h, but the melting peak maintained approximately at the same size for all stored samples, indicating crystallization during storage. However, sucrose crystallization during storage could not be studied quantitatively with DSC, since
3.3. Phase separation and crystallization during storage Physical state of amorphous carbohydrate matrices changes during storage. In a preliminary experiment, we examined freeze-dried samples with DSC after storage at 44³C for 45 days. The glass transition of glucose/sucrose samples became undetectable, indicating the loss of the amorphous state. The glass transition in glucose/trehalose samples still remained at 23.9 þ 1.4³C (mean þ S.E.), although a small melting peak of trehalose dihydrate crystals was observed, indicating that a small fraction of trehalose crystallized. The data showed that the crystallization of sucrose and trehalose occurred during storage,
Fig. 6. Glass transition temperatures of glucose/sucrose samples after storage at 60³C. Data are means þ S.E. of at least three independent measurements. Bars are not shown when smaller than symbols. The shaded area represents the expected Tg for the local glucose domain separated after sucrose crystallization.
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devitri¢cation occurred in some samples during DSC measurements. Fig. 7 shows DSC thermograms of glucose/trehalose samples after storage. The control sample had the Tg at 12.3 þ 1.5³C, and a small melting peak at V95³C. Unlike glucose/sucrose samples, no apparent devitri¢cation was observed in glucose/trehalose samples during DSC measurements. Phase separation was also observed during storage, but was signi¢cantly delayed. The di¡erence in the Tg between two glassy domains were relatively small in comparison to glucose/sucrose samples (Fig. 8A). The Tg of the major glassy domain gradually increased to 22³C during storage, whereas the Tg of the minor domain declined to below 320³C. The endothermic peak at 95³C was the melting of trehalose dihydrate. The dehydration of trehalose dihydrate occurs at V130³C (data not shown). The peak size increased as storage time increased. The small amount of trehalose dihydrate in control samples was probably formed during DSC measurement. The fraction of trehalose dihydrate crystals in samples is shown in Fig. 8B. As storage time increased, the rate of trehalose dihydrate formation slowed down signi¢cantly, exhibiting a trend similar to G6PDH inactivation in the glucose/trehalose system. Even after 42 h of storage at 60³C, only 30% trehalose crystallized, showing
241
Fig. 8. (A) Glass transition temperatures of glucose/trehalose samples after storage at 60³C. The shade area shows the expected Tg for the local glucose domain after trehalose crystallization. (B) Fraction of trehalose dihydrate in samples after storage at 60³C. Data are mean þ S.E. of at least three independent measurements. Bars are not shown when smaller than the symbols.
that the glucose/trehalose system was more stable than the glucose/sucrose system at the same Tg . 4. Discussion
Fig. 7. DSC thermograms of glucose/trehalose samples after storage at 60³C. Arrows indicate glass transitions. Scan rate, 10³C/min. DSC thermograms were normalized to a sample mass of 20 mg.
The high Tg value was previously suggested to be the cause of trehalose superiority over other carbohydrates in biopreservation [10]. Sucrose was commonly used in comparative studies. We checked recent reports [17,28,33,34], and found that the Tg of trehalose systems were typically 20^40³C higher than that of sucrose systems. This di¡erence in the Tg alone corresponds to an increase of 2^3 orders of magnitude in the stability of biomaterials in the glassy matrix [35,36], and a decrease of 4^6 orders
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of magnitude in the rates of relaxation processes according to WLF kinetics [35,37]. To verify whether better preservation by the trehalose glass was due to the high Tg or other special properties, the comparison between trehalose and other carbohydrates should be made at the same Tg value. In the present study, glucose/sucrose and glucose/trehalose systems were prepared to have the same Tg values (Fig. 1). Our data showed that the glucose/trehalose system conferred better protein preservation during storage at temperatures above the Tg (Fig. 3). The superior stability of the trehalose system may be attributed to a number of dynamic properties of the trehalose glass. Derived WLF equation constants, kg , C1 and C2 , are directly related to dynamic properties of amorphous systems [32]. C1 is proportional to the inverse of free volume at Tg , and C2 is proportional to the ratio of free volume at Tg to the thermal expansion of free volume above the Tg [37]. Free volume in an amorphous system is geometrically interpreted as a continuous network of `lakes and channels', which permit the di¡usive motion of its molecules [38]. Such a continuous network of `lakes and channels' disintegrates when the free volume is reduced to below a critical level, corresponding to the glass transition. When samples are stored at temperatures above the Tg , free volume and total volume of samples increase. Free volume expansion re-establishes the continuous network of `lakes and channels', thus providing su¤cient room for molecular motion. The stability of protein in carbohydrate glasses was closely related to molecular mobility [39], and therefore, WLF constants are of signi¢cance for protein preservation. Values of C1 and C2 for the glucose/trehalose system were signi¢cantly greater than those of the glucose/sucrose system (Table 1), suggesting that the glucose/trehalose system had a smaller free volume at the Tg and also a smaller free volume expansion coe¤cient at temperatures above the Tg than did the glucose/sucrose system. This implied that the di¡usive molecular motion in the glucose/trehalose system should be more restricted than that in the glucose/sucrose system. The analysis rationalized the observation that the kg for the glucose/trehalose system was less than that of glucose/trehalose system (Table 1). Phase separation and crystallization would occur as a result of increased molecular mobility. When an
aqueous glass is held at an elevated temperature, the system becomes increasingly unstable, being vulnerable to phase separation and subsequent crystallization. Preliminary storage experiment showed that the glucose/trehalose system was much more stable than the glucose/sucrose system at a high temperature. After 45-day storage at 44³C (T3Tg = V20³C), glucose/sucrose samples had lost their amorphous state completely, whereas only a small fraction ( 6 4%) of the glucose/trehalose material crystallized and the rest remained in the amorphous state with the Tg unchanged (23.9 þ 1.4³C). Crystallization was observed to destabilize enzymes, resulting in the rapid loss of enzyme activity [19^21]. The ability to resist phase separation and crystallization was probably associated with the restricted molecular motion in the glucose/trehalose system, and certainly contributed to its superiority over the glucose/sucrose system in conferring protection during storage. Crowe et al. [28] reported that the Tg of trehalose glass did not change signi¢cantly after exposure to elevated humidity because of the formation of trehalose dihydrate. In our experiment, samples were not humidi¢ed during storage, and the greater stability of the glucose/trehalose system can not be attributed to the sequestration of water. The formation of trehalose dihydrate ( 6 4%) in this case would have had little e¡ect on the rest of amorphous domain. Di¡erences in phase separation and crystallization between glucose/sucrose and glucose/trehalose systems were further studied using samples with lower Tg (V11^ 13³C) at 60³C. Three separate domains were identi¢ed in stored samples, representing carbohydrate crystal, glucose-rich and disaccharide-rich domain. Phase separation occurred rapidly in the glucose/sucrose system. The Tg of the sucrose-rich domain increased to about 60³C after 12 h, probably due to the dehydration at the high storage temperature (Fig. 6). In contrast, phase separation in the glucose/trehalose system was signi¢cantly delayed, and the Tg of the trehalose-rich domain increased by only 10³C (Fig. 8). Further dehydration during storage appeared to be more retarded in the glucose/trehalose system than in the glucose/sucrose system, showing greater restriction on the di¡usion of water molecules in the glucose/trehalose system. This suggestion is consistent with the results from our kinetic analysis mentioned above (i.e. the trehalose/glucose glass had a
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smaller free volume and a smaller free volume expansion coe¤cient at temperatures above the Tg than did the glucose/sucrose glass). Phase separation and crystallization of one component in a glassy product would lead to a dramatic change in the composition of the remaining amorphous phase. In the glucose/sucrose system, crystallization would result in a high local water content. The initial moisture in glucose/sucrose samples was about 0.07 g H2 O/g dry mass. If sucrose had crystallized, the local moisture of the remaining glucose domain would have increased to 0.6^0.7 g H2 O/g dry mass, and the Tg could have decreased to below 380³C. The observed Tg of the glucose-rich domain after sucrose crystallization was 20^30³C higher than the expected Tg , possibly due to partial dehydration during storage (water vapor goes to the head spaces of storage tubes) (Fig. 6). However, in the glucose/ trehalose glassy system, the local water content changed di¡erently because of the formation of trehalose dihydrate crystals. When trehalose crystallized, the local water content of the remaining glucose domain would have decreased to 0.07 g H2 O/g dry mass, corresponding to a Tg slightly below 320³C. The observed Tg of the remaining glucoserich domain was the same as the expected Tg (Fig. 8). The observed physical state changes in both systems could more or less be accounted for quantitatively according to system composition pro¢les. An interesting observation was that the rate constant of protein inactivation in the glucose/trehalose system was time-dependent (Fig. 2). The time-dependent parameter was 0.53 for the glucose/trehalose system, relative to the unity for the glucose/sucrose system, where protein inactivation simply followed the ¢rst order kinetics. The non-uniform distribution of residual water could give rise to a biphasic stability phenomenon, where the rate of loss of the stability was high during the initial period, and the product became more stable during the later stages [25]. However, the time-dependent phenomenon observed in the glucose/trehalose system was unlikely due to the non-uniform distribution of residual water in freeze-dried samples. DSC studies showed a single glass transition domain for both glucose/sucrose and glucose/trehalose samples before storage experiments (Fig. 1). The retardation of protein inactivation was likely due to the slight increase in the Tg
243
during storage. An increase in the Tg would theoretically a¡ect protein stability as much as a decrease in storage temperature to the same extent. The data of protein inactivation during storage at 60³C were ¢tted with a third-order polynomial equation (R2 = 0.99). The instantaneous rate constants of protein inactivation were calculated by taking the ¢rst derivative of the ¢tted equation, and were superimposed onto Fig. 3 (triangle). The Tg dependence of the instantaneous rate constants was identical to the temperature dependence of initial rate constants at di¡erent temperatures, showing that the change of inactivation rate during storage was associated with the gradual increase in the Tg . A similar time dependence of crystallization was observed in the glucose/trehalose systems (Fig. 8). The gradual decrease in the rate of trehalose crystallization was also related to the increase of Tg during storage. In the present work, we have examined the kinetics of protein inactivation in two amorphous carbohydrate systems with similar Tg values. The superior stability of the glucose/trehalose system over the glucose/sucrose system was associated with a number of closely related dynamic properties of the glucose/trehalose glass, including low free volume, restricted molecular mobility of water and solutes, and the ability to resist phase separation and crystallization during storage. References [1] A.C. Leopold, in: G.J. Alscher, J.R. Cumming (Eds.), Stress Responses in Plants: Adaptation and Acclimation Mechanisms, Wiley-Liss, New York, 1990, pp. 37^56. [2] J.H. Crowe, L.M. Crowe, in: G.N. Somero, C.B. Osmond, C.L. Bolis (Eds.), Water and Life, Springer-Verlag, Berlin, 1992, pp. 87^103. [3] W.Q. Sun, A.C. Leopold, Comp. Biochem. Physiol. 117A (1997) 327^333. [4] J.H. Crowe, L.M. Crowe, S.A. Jackson, Arch. Biochem. Biophys. 220 (1983) 477^484. [5] R. Mouradian, C. Womersley, L.M. Crowe, J.H. Crowe, Biochim. Biophys. Acta 778 (1984) 615^617. [6] B. Roser, BioPharm 4 (1991) 47^53. [7] S.B. Leslie, E. Israeli, B. Lighthart, J.H. Crowe, L.M. Crowe, Appl. Environ. Microbiol. 61 (1995) 3592^3597. [8] M. Uritani, M. Takai, K. Yoshinaga, J. Biochem. 117 (1995) 774^779. [9] L.M. Crowe, R. Mouradian, J.H. Crowe, S.A. Jackson, C. Womersley, Biochim. Biophys. Acta 769 (1984) 141^150.
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