ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 360, No. 1, December 1, pp. 10 –14, 1998 Article No. BB980906
Stabilization against Thermal Inactivation Promoted by Sugars on Enzyme Structure and Function: Why Is Trehalose More Effective Than Other Sugars? Mauro Sola-Penna*,1 and Jose´ Roberto Meyer-Fernandes† *Departamento de Fa´rmacos, Faculdade de Farma´cia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, 21944-910, Brasil; and †Departamento de Bioquı´mica Me´dica, Instituto de Cieˆncias Biome´dicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, 21941-590, Brasil
Received March 23, 1998, and in revised form August 10, 1998
Trehalose has been described to act as the best stabilizer of structure and function of several macromolecules. Although other sugars also stabilize macromolecules, none of them are as effective as trehalose. The extraordinary effect of trehalose has been attributed to several of its properties such as making hydrogen bonds with membranes or the ability to modify the solvation layer of proteins. However, the explanations always result in a question: Why is trehalose more effective than other sugars? Here, we show that trehalose has a larger hydrated volume than other related sugars. According to our results, trehalose occupies at least 2.5 times larger volume than sucrose, maltose, glucose, and fructose. We correlate this property with the ability to protect the structure and function of enzymes against thermal inactivation. When the concentrations of all sugars were corrected by the percentage of the occupied volume, they presented the same effectiveness. Our results suggest that because of this larger hydrated volume, trehalose can substitute more water molecules in the solution, and this property is very close to its effectiveness. Finally, these data drive us to conclude that the higher size exclusion effect is responsible for the difference in efficiency of protection against thermal inactivation of enzymes. © 1998 Academic Press Key Words: trehalose; osmolyte; thermal inactivation; enzyme; protection.
Trehalose is a disaccharide of glucose synthesized by several organisms that are able to survive heat shock
and other stress conditions (1– 6). Trehalose is considered to have an important role in survival of these organisms, stabilizing membranes and proteins in the face of stress (3–12). When baker’s yeast is submitted to a heat shock, it accumulates high concentrations of trehalose (6). Similar effects happen when yeasts are dried. Under this condition, trehalose can reach 35% of the dry weight, conferring yeast the ability to survive desiccation (1, 2). Trehalose has been described to modulate enzyme activity (8 –13). Modulation can be achieved in several ways. It was also shown that trehalose is capable of decreasing the Km for Pi of the sarcoplasmic reticulum calcium pump (13), of uncoupling the plasma membrane (Ca21 1 Mg21)ATPase (11), and of inhibiting the activity of yeast cytosolic pyrophosphatase2 (9, 10), yeast glucose 6-phosphate dehydrogenase, and yeast plasma membrane proton pump ATPase activity (12). The effectiveness of trehalose in uncoupling or inhibiting enzymes is greater than that achieved using other sugars such as maltose, sucrose, glucose, or fructose (9, 11, 12). Trehalose is more effective than other sugars in protecting yeast pyrophosphatase against thermal inactivation (9), and pyrophosphatase and glucose 6-phosphate dehydrogenase against inactivation promoted by guanidinium chloride (12). Modulation and protection of enzymes by trehalose and other sugars can be explained by their ability to preferentially solubilize in the bulk water, being excluded from the solvation layer of proteins (14). This phenomenon leads to a decrease in the solvation layer 2
1
To whom correspondence should be addressed. E-mail:
[email protected]. 10
Abbreviations used: glucose 6-phosphate dehydrogenase (EC 1.1.1.49); Pi, inorganic pyrophosphatase (EC 3.6.1.1); Tris, tris[hydroxymethyl]aminomethane. 0003-9861/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
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of the enzyme, reducing its flexibility, and finally, the enzyme becomes more stable, but less active (9). The hypothesis above does not explain why trehalose is more efficient than other sugars in inhibiting and protecting enzymes. Here we present evidence that the higher efficiency of trehalose is a consequence of its larger hydrated volume. We believe that due to the larger size of trehalose molecule, it is more excluded from the hydration shell, and as a consequence, less trehalose is necessary to decrease the solvation layer of proteins and, thus, to stabilize and modulate enzyme activity. MATERIALS AND METHODS Materials. Yeast inorganic pyrophosphatase (EC 3.6.1.1) and yeast glucose 6-phosphate dehydrogenase (EC 1.1.1.49) were purchased from Sigma Chemical Co. (St. Louis, MO), and exhibited high purity (99.5%). Trehalose, glucose, fructose, sucrose, maltose, tetrasodium pyrophosphate, glucose 6-phosphate, NADP1, and Hepes were also purchased from Sigma Chemical Co. Other reagents were of the highest purity available. The capillary viscometer was from Cannon Instrument Co. (State College, PA). Viscosity measurements. Relative viscosity of solutions of sugars at different concentrations was measured using a Cannon–Manning semi-micro-type capillary viscometer No. 75 A882, using water as standard (viscosity 5 1.00). All measurements were performed at 25°C, and in triplicate. Determination of pyrophosphatase activity. Pyrophosphatase activity was determined by measuring the total Pi released at the end of incubation. The Pi concentration was determined as described by Lowry and Lopez (15). The enzyme activity assay was performed at 25°C in a medium containing 100 mM Hepes–KOH (pH 7.5), 10 mM MgCl2, 2 mM tetrasodium pyrophosphate, and 0.8 mg of purified enzyme per milliliter of reaction medium. Reaction was quenched after 1 min by addition of 2 vol of 20% (mass/vol) trichloroacetic acid. Determination of glucose 6-phosphate dehydrogenase activity. Yeast glucose 6-phosphate dehydrogenase activity was determined following the reduction of NADP1 by measurement of light absorption at 340 nm in a spectrophotometer. Experiments were performed at 25°C in a medium containing 100 mM Hepes–KOH (pH 7.5), 10 mM MgCl2, 1 mM glucose 6-phosphate, 0.1 mM NADP1, and 1 mg of purified enzyme per milliliter of reaction medium. Reactions were followed for 1 min and the amount of NADPH formed was calculated using its molar extinction coefficient (6.22 3 106 cm2/mol). Fluorescence measurements. Steady-state fluorescence measurements were performed on an Hitachi F4500 spectrofluorimeter. Protein concentration was fixed at 10 mg/ml in 100 mM Hepes–KOH (pH 7.5) at 25°C for yeast pyrophosphatase and 100 mg/ml for glucose 6-phosphate dehydrogenase. Appropriate reference spectra were subtracted from the data to correct for background interferences which were always less than 5% of the fluorescence signal. The excitation wavelength was set at 280 nm and the emission was measured at 330 nm.
RESULTS
Our first result was based on the observation that the amount of water used for the preparation of 1.5 M trehalose solution used in experiments was smaller than the amount used for the preparation of other sugar solutions. In a 1.5 M solution, trehalose itself occupies 37.5% of the volume of the solution. However,
FIG. 1. Relative viscosity of sugars. Viscosity measurements of different sugar concentrations were performed at 25°C, using a Cannon–Manning semimicro-capillary viscometer. Time (in seconds) of efflux of the sample was divided by time of efflux of water, and relative viscosity was obtained. (F) Trehalose, (h) sucrose, (■) maltose, (‚) glucose, or (Œ) fructose. Values are the mean of 3 independent measurements and standard errors were always less than 5%.
in a 1.5 M solution, sucrose occupies 13% and maltose occupies 14%. The monosaccharides glucose and fructose, in a 3 M solution, occupy 12.5 and 13%, respectively. These data suggest that trehalose presents a larger hydrated volume than the other sugars mentioned here. To demonstrate this hypothesis, we measured the viscosity of several concentrations of trehalose, sucrose, maltose, glucose, and fructose. Because the disaccharides used have almost the same mass and formula, they might have similar molecular volumes. Under this point of view, differences in the viscosity of the media should be due to differences in the hydrated volume of these sugars. This hypothesis is based on the fact that solutions of compounds with the same molecular formula are expected to present similar viscosity. Possible differences in this parameter can be due to a difference in the ability to form hydrogen bond with the solvent. In this case, a higher viscosity is a consequence of a higher hydrated volume. As can be seen in Fig. 1, the specific viscosity of trehalose solutions are nearly 2.5-fold higher than all of the correspondent concentrations of sucrose and maltose. Once glucose, fructose, and glycerol are smaller molecules, the viscosities do not represent comparable differences in hydrated volume of these molecules.
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SOLA-PENNA AND MEYER-FERNANDES
FIG. 2. Time course of thermal inactivation of enzymes at 50°C, in the presence of carbohydrates. Pyrophosphatase at 80 mg/ml (A) or glucose 6-phosphate dehydrogenase at 1 mg/ml (B) was incubated for the times indicated on abscissa in the absence (E) or in the presence of 1.5 M trehalose (F), 1.5 M sucrose (h), 1.5 M maltose (■), 3 M glucose (‚), 3 M fructose (Œ), 37.5% (v/v) glycerol (ƒ), or 0.5 M trehalose (). After preincubation enzymes were 100-fold diluted in the appropriate reaction medium described under Materials and Methods and assayed for activity. The activities (100% on the ordinate) were 1.02 6 0.05 mmol z mg21 z min21 (mean 6 standard error) for pyrophosphatase (A) and 3.7 6 0.01 mmol z mg21 z min21 for glucose 6-phosphate dehydrogenase (B) (mean 6 standard error of 4 independent experiments). Standard errors were always less than 5% of the absolute values. The curves were fitted by nonlinear regression (using the software Sigmaplot 3.03 from Jandel Scientific, USA) to the equation v 5 V0 z e2kt, where V0 is the initial rate of hydrolysis without preincubation; k is the decay constant; and t is the preincubation time.
We have previously demonstrated that trehalose was much more efficient than other sugars in protecting yeast pyrophosphatase against inactivation at 50°C (9). Now, based on the difference in the hydrated volume, we postulate that the extraordinary stability of pyrophosphatase in the presence of trehalose is due to the lower concentration of water in the trehalose solution. To test this hypothesis we should equalize the water concentration in all sugar solutions. According to this theory, a solution of sucrose, maltose, glucose, or fructose in which those molecules occupy 37.5% of the volume of solution should confer the same stability to the enzyme that a 1.5 M trehalose solution does. To achieve this condition sucrose, maltose, glucose, and fructose should be prepared at 4.3, 4.0, 9.0, and 8.7 M, respectively. However, in our experiments, those sugars are not soluble at such high concentrations. Another strategy was used to solve this problem. We used glycerol which is also a stabilizer and can be easily prepared at 37.5% (v/v). Figure 2 shows the protection promoted by sugars and glycerol on yeast pyrophosphatase (Fig. 2A) and on yeast glucose 6-phosphate dehydrogenase (Fig. 2B). It can be seen that the protection conferred by trehalose (filled circle), on both enzymes, is much higher than protection promoted by other sugars (squares and up triangles). On the other hand, 37.5% glycerol (4.4 M) (empty down triangles) conferred the same protection as 1.5 M trehalose on
both enzymes. Additionally, the protection promoted by 0.5 M trehalose (filled down triangles) on both enzymes was equal to that promoted by sucrose (empty squares) and maltose (filled squares) at 1.5 M, and glucose (empty up triangles) and fructose (filled up triangles) at 3.0 M (Fig. 2). Trehalose at 0.5 M occupies the same volume as sucrose and maltose at 1.5 M and glucose and fructose at 3 M. The effect of temperature on the tertiary structure of pyrophosphatase and glucose 6-phosphate dehydrogenase was also tested (Fig. 3). Exposure of both enzymes at 50°C promoted a significant reduction on the intensity of the intrinsic fluorescence at 330 nm (Fig. 3, empty circles). This reduction on intrinsic fluorescence intensity, which reflects an unfolding of enzyme tertiary structure (16), is dependent on the time of exposure. The presence of 1.5 M trehalose in the medium prevented the unfolding of both enzymes (Fig. 3, filled circles). The presence of 1.5 M sucrose or maltose, and 3 M glucose or fructose, promoted only a slight protection of the unfolding induced by exposure of enzymes to 50°C (Fig. 3, squares and up triangles). Glycerol, at a concentration of 37.5% (v/v) protected both enzymes to the same extent as 1.5 M trehalose (37.5 %) (compare Fig. 3, filled circles and down triangles). Using 0.5 M trehalose (where trehalose occupies the same volume as 1.5 M sucrose and maltose, and 3 M glucose and
STABILIZATION OF ENZYMES BY TREHALOSE
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FIG. 3. Time course of thermal denaturation of enzymes at 50°C, in the presence of carbohydrates. Pyrophosphatase at 1 mg/ml (A) or glucose 6-phosphate dehydrogenase at 10 mg/ml (B) was incubated for the times indicated on abscissa in the absence (E) or in the presence of 1.5 M trehalose (F), 1.5 M sucrose (h), 1.5 M maltose (■), 3 M glucose (‚), 3 M fructose (Œ), or 37.5% (v/v) glycerol (ƒ. After preincubation, enzymes were diluted in the appropriated medium described under Materials and Methods and assayed for intrinsic fluorescence determination.
fructose), the degree of protection obtained is comparable with those using the other sugars. The degree of protection promoted by trehalose on the thermal inactivation of pyrophosphatase and glucose 6-phosphate dehydrogenase can be reproduced in all range of concentrations using glycerol at a concentration that is equivalent to the volume occupied by trehalose (Fig. 4). Additionally, the effects of all sugars tested are equal, when concentrations used were corrected by the percentage of the occupied volume (Figs. 4A and 4B, insets). These data suggest that the effectiveness of sugars to stabilize enzymes depends on the size exclusion effect of each sugar. DISCUSSION
The higher efficiency of trehalose in stabilizing macromolecules and modulating enzyme function has been pointed out elsewhere (3, 5, 9 –12, 17, 18). As far as we can understand, no satisfactory explanation has been given until now. Explanations concerning the effects of trehalose on stabilization and modulation of enzymes are based on the ability of the disaccharide to make hydrogen bonds, and that this kind of interaction should stabilize enzymes (9, 11). Nevertheless, other carbohydrates have similar properties, and are not as effective as trehalose. In addition, Timasheff suggested that sugars, in general, do not interact directly with protein structure, preferentially solubilizing in bulk water in a phenomenon called preferential hydration of proteins (14).
Preferential hydration of proteins postulates that in a triphasic system consisting of water, protein, and a cosolvent—that can be a sugar—a stabilizer (cosolvent) is excluded from viccinal water that composes the solvation layer of protein (14). As a result, the protein becomes preferentially hydrated, but the radius of the solvation layer and the apparent volume of protein decreases, in a phenomenon that leads to a more stable protein conformation. The data presented here can be explained by correlating stabilization promoted by sugars on pyrophosphatase and glucose 6-phosphate dehydrogenase, with the preferential hydration phenomenon. Once trehalose occupies a larger volume in solution in comparison to other sugars, the size-exclusion effect will be more pronounced in the case of trehalose. As a consequence, preferential hydration of enzyme is attained at lower concentrations and therefore result in much strong stabilization effects. Actually, trehalose is more efficient as a stabilizer and as an inhibitor of several enzymes as was shown here and elsewhere (5, 9–12, 18). When glycerol was added in a concentration that occupies the same volume as trehalose, the degree of effectiveness of both was coincident. In addition, all sugars have similar effects if the concentrations are corrected by the volume occupied by each sugar. These data allow us to suggest that the extraordinary properties of trehalose are due to a large hydrated volume in comparison to other sugars, and consequently, a higher size exclusion effect.
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FIG. 4. Protection promoted by sugars and glycerol on enzyme activity against thermal inactivation. Pyrophosphatase at 80 mg/ml (A) or glucose 6-phosphate dehydrogenase at 1 mg/ml (B) was preincubated at 50°C for 20 and 10 min for pyrophosphatase and glucose 6-phosphate dehydrogenase, respectively, in the presence of the concentrations of trehalose (F) and glycerol (E) (A and B) and trehalose (F), sucrose (h), maltose (■), glucose (‚), or fructose (Œ) (insets to A and B) indicated on abscissa. After preincubation enzymes were 100-fold diluted in the appropriate reaction media described under Materials and Methods and assayed for activity. The activities (100% on the ordinate) were 0.98 6 0.04 mmol z mg21 z min21 (mean 6 standard error) for pyrophosphatase (A) and 3.7 6 0.1 mmol z mg21 z min21 for glucose 6-phosphate dehydrogenase (B) measured in control experiments without preincubation (mean 6 standard error of 4 independent experiments). Standard errors were always less than 5% of the absolute values. The curves were drawn free-hand.
ACKNOWLEDGMENTS We thank Dr. Gisela Maria Dellamora Ortiz (Faculdade de Farma´cia, UFRJ) for critical reading of the manuscript and Dr. Adalberto Vieyra (Departamento de Bioquı´mica Me´dica, ICB, UFRJ) for the use of spectrofluorimeter. This was supported by grants from Fundac¸a˜o Universita´ria Jose´ Bonifa´cio (FUJB/UFRJ); Fundac¸a˜o de Amparo a` Pesquisa do Estado do Rio de Janeiro (FAPERJ); Programa de Nu´cleos de Exceleˆncia (PRONEX), and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq).
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