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Stabilization against Thermal Inactivation Promoted by Sugars on Enzyme Structure and Function: Why Is Trehalose More Effective Than Other Sugars? Original Research Article
Archives of Biochemistry and Biophysics, Volume 360, Issue 1, 1 December 1998, Pages 10-14
Mauro Sola-Penna, José Roberto Meyer-Fernandes

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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
1 To whom correspondence should be addressed. E-mail:
maurosp@pharma.ufrj.br.
2 Abbreviations used: glucose 6-phosphate dehydrogenase (EC
1.1.1.49); Pi, inorganic pyrophosphatase (EC 3.6.1.1); Tris, tris[hydroxymethyl]
aminomethane.
10 0003-9861/98 $25.00
Copyright © 1998 by Academic Press
All rights of reproduction in any form reserved.
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 360, No. 1, December 1, pp. 10–14, 1998
Article No. BB980906
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,
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.
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, (n) maltose,
(‚

glucose, or (Œ

fructose. Values are the mean of 3 independent
measurements and standard errors were always less than 5%.
STABILIZATION OF ENZYMES BY TREHALOSE 11
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
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 (n), 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.
12 SOLA-PENNA AND MEYER-FERNANDES
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.

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ljj200511(金币+3): 2010-09-17 18:18:13

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.
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 (n), 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.
STABILIZATION OF ENZYMES BY TREHALOSE 13
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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