From the Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India
Received for publication, January 24, 2003 , and in revised form, April 15, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Trehalose has also been found to be very effective in the stabilization of labile proteins during lyophilization (15, 16) and exposure to high temperatures in solution (2, 8, 9). Sugars in general protect proteins against dehydration by hydrogen bonding to the dried protein by serving as water substitute (15, 17). Several studies carried out by Timasheff and coworkers (9, 18) show that sugars and polyols stabilize the folded structure of proteins in solution as a result of greater preferential hydration of the unfolded state compared with the native state. The mechanism is fundamentally different from stabilization in the dried state and points toward the different origins of protein denaturation under different stress conditions (17). In solution, trehalose has been observed to stabilize RNase A by increasing the surface tension of the medium, which leads to the preferential hydration of the protein (8, 9). These studies have been carried out using a representative protein at a few selected conditions only. Different proteins are expected to interact with cosolvent molecules in varied ways depending on their physico-chemical properties. In general, trehalose has been observed to provide protection to different proteins to various extents and the efficacy of protection depends on the nature of the protein (2, 4). Despite the availability of such data, the exact role of proteins and their physico-chemical properties in trehalose-mediated stability is still not clear. Studies done earlier by Gekko (19, 20), using polyol osmolytes and free energy of transfer studies, suggested that unfavorable interactions of the amino acid side chains with polyols dominate the stability effect, and peptide-polyol interactions contribute negligibly to the stability mediated by polyols. However, recently, Bolen and coworkers (2123), based on carefully conducted transfer studies of amino acids and model compounds, have shown that cumulative interactions between amino acid side chains and osmolytes (including sucrose) favor protein unfolding, whereas their overall stabilization is achieved due to unfavorable peptide-osmolyte interactions. The exact nature of interactions that govern the osmolyte-mediated stability of proteins is, therefore, not yet very clear. Overall, protein stability should depend upon a fine balance between favorable and unfavorable interactions of the native and the denatured protein states with the cosolvent molecules (24). The stabilizing effect would, thus, depend on the nature of both the proteins as well as the cosolvent molecules and generalization of the effect may not be possible.
To understand the mechanism of trehalose-mediated thermal stability of
proteins in detail, we have studied its effect on the thermal stability of a
set of five well characterized globular proteins, viz., ribonuclease
A (RNase A),1
lysozyme, cytochrome c (cyt c), -chymotrypsinogen
(
-CTgen), and trypsin-inhibitor (Trp-Inh) at varying cosolvent
concentrations and pH values. These proteins vary in their molecular size,
ranging from 12.3 kDa for cyt c to 25.7 kDa for
-CTgen, their
hydrophobicities, and the net charges, with pI values in the range of 4.1 for
Trp-Inh to 10.7 for lysozyme. The data have been analyzed in the light of the
role of bulk properties of the solvent environment and the physico-chemical
properties of proteins. Because trehalose in addition to imparting
thermodynamic stability to proteins also helps in the retention of activity of
enzymes during storage at high temperatures, we have carried out activity
studies for RNase A at elevated temperatures and denaturing conditions to
understand the stability-activity relationship of the enzyme in the presence
of trehalose.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Thermal Denaturation ExperimentsFor monitoring the
unfolding of the tertiary structure, thermal denaturation experiments were
carried out using a Cecil 599 UV-visible spectrophotometer to which a linear
temperature programmer (CE-247, Cecil) was attached. The concentration of the
protein solutions was 0.5 mg/ml, except for cyt c where 0.1
mg/ml protein was used. The protein solutions were loaded in a 0.5-ml masked
and Teflon-stoppered quartz cuvette (Hellma, Germany). A temperature scan rate
of 1 °C/min was used in all the experiments. The wavelengths for
monitoring conformational changes related to the tertiary structure were 287
nm for RNase A, 293 nm for
-CTgen, 301 nm for lysozyme, 285 nm for
Trp-Inh, and 394 nm for cyt c based on their difference spectra. The
reversibility of the thermal transitions recorded for the proteins was
ascertained by reheating the protein solutions.
To investigate the unfolding of secondary structure elements, thermal denaturation was monitored by far-UV CD measurements using a Jasco J715 spectropolarimeter at selected pH conditions, one for each protein, at a scan rate of 1 °C/min. A wavelength of 222 nm was used to specifically probe the opening up of helical regions in the proteins.
Analysis of DataThe evaluation of thermodynamic parameters
from the thermal denaturation curves was based on the equilibrium constant
K, for N D conversion for a two-state
reversible transition, where N represents the native state and
D is the denatured state. The equilibrium constant was deduced from
the equation,
![]() | (Eq. 1) |
![]() | (Eq. 2) |
RNase A Activity AssayRNase A catalyzed hydrolysis of 2',3'-cCMP was measured by the change in the absorbance at 286 nm (27). Two sets of experiments were conducted in the presence of 1.5 M trehalose and 20 mM Tris, pH 7.0. In set 1, RNase A was incubated at high temperatures (66 °C and 60 °C, with 1 M GdmCl) for 13 h followed by cooling to room temperature and monitoring the activity by addition of 2',3'-cCMP from the stock. In set 2, RNase was added to the reaction buffer and allowed to equilibrate at high temperatures (63 °C, 56 °C, and 52 °C, with 1 M GdmCl) at which the activity was monitored by the addition of 2',3'-cyclic CMP. All the reactions were carried out in a 1.0-ml Teflon-stoppered quartz cuvette. The temperature of the cuvette was maintained by using a programmable thermoelectric cuvette holder.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Effect of Trehalose on the Thermodynamics of Protein
DenaturationThermodynamic parameters for protein denaturation in
trehalose solutions at different conditions, obtained from the data in
Fig. 1, have been presented in
Table I.
Tm and
G0 are the
increments in the midpoint of transition, Tm, and Gibbs
free energy of stabilization,
G0, respectively.
G0 in the presence of trehalose has been
calculated at the Tm of the control, where
G0 for control is zero. For all the proteins, both
Tm and
G0
increase linearly with an increase in the trehalose concentration.
Hm and
Sm were also
observed to be increasing with trehalose concentration in the case of RNase A,
-CTgen, Trp-Inh, and cyt c at pH 4.0. However, these
parameters show a decrease in the case of lysozyme at pH 4.0 and 7.0, and cyt
c at pH 7.0, which could be due to partial irreversible aggregation
of the proteins in the post denaturation region. Overall, barring these
exceptions involving protein aggregation, trehalose was observed to gradually
increase the
Hm and
Sm
of protein denaturation. The experimental errors in
Hm were in the range of ±14% for
RNase and cyt c and ±57% for
-CTgen, lysozyme,
and Trp-Inh. The error in Tm measurements was within
±0.5 °C for all the proteins. The fitting errors were always within
the experimental error limits. The CD data presented as insets in
Fig. 1 have not been used in
Table I. However, the
transition temperatures and the various thermodynamic parameters obtained by
CD measurements matched well with those evaluated by absorption
spectroscopy.
|
The slopes of the curves (Hm versus
Tm) plotted in Fig.
2 represent the heat capacity of protein denaturation,
Cp for RNase A obtained in the presence of
trehalose and are 0.87 kcal mol1
K1 at pH 2.5 and 4.0, and 1.1 kcal
mol1 K1 at pH 7.0,
respectively. The values are considerably lower than the spectroscopically
obtained
Cp values of 2.07 kcal
mol1 K1 and 2.2 kcal
mol1 K1 evaluated by
varying the Tm of RNase A using GdmCl
(26) and urea
(31), respectively, and
calorimetric values of 1.74 kcal mol1
K1 in buffer and 2.16 kcal
mol1 K1 in 1
M GdmCl reported by Liu and Sturtevant
(32). Previously, several
other osmolytes like sarcosine
(29) and polyols
(25) have also been observed
to decrease the apparent heat capacity of denaturation of globular proteins.
Neutral salts, including carboxylic acid salts
(26), which increase the
thermal stability of proteins, also lead to a decrease in the denaturation
heat capacity of several proteins just like osmolytes. For proteins other than
RNase A, e.g. for
-CTgen and lysozyme at pH 2.5, cyt
c at pH 4.0, and Trp-Inh at pH 7.0,
Hm
also increases as a function of Tm, though marginally
(Table I), and results in much
lower
Cp values than the corresponding values
reported for these proteins without trehalose.
|
Solution Surface Tension and Protein StabilityFig. 3 presents data showing the effect of the surface tension of trehalose solutions on the thermal stability, as monitored by Tm of various proteins studied. Surface tension of aqueous trehalose solutions has been observed to increase linearly with the concentration resulting in a slope of 1.34 dyne cm1 mol1 at 20 °C (33). The data presented in Fig. 3 suggest a good correlation of the effect of the increased surface tension of trehalose solutions with the increase in the Tm for all the proteins studied. Studies done by us earlier using a series of polyols (25) and carboxylic salts (26) also indicate a strong correlation of the surface tension effect with the thermal stability of proteins, suggesting an important role of water and the solvent environment in the stability of proteins.
|
Wyman Linkage and Interaction of Trehalose with Proteins
Trehalose stabilizes proteins by shifting the equilibrium constant in favor of
the native state. To analyze the effect of trehalose on the denaturation
reaction, the Wyman linkage equation
(24) given below
(Equation 3) was used to
determine the relative preferential interaction of trehalose with the two end
states of the proteins,
![]() | (Eq. 3) |
|
Activity of RNase A in the Presence of TrehaloseRNase A was taken as a model enzyme to analyze the effect of trehalose on its bioactivity at high temperatures. The relative activity of RNase A in the presence of trehalose presented in Table II has been calculated by dividing the slopes of the linear zone of the corresponding activity plots by the slope of the data for control (buffer). The stabilization factor, fNt/fNc presented in Table II, wherein fNt is the fraction of the protein in the native state in the trehalose solution and fNc is the fraction of the native protein in the control buffer, was calculated from the thermal denaturation curves for RNase A in the respective solvent systems at the indicated temperatures. The data indicate a remarkable retention of activity of the enzyme in the presence of trehalose under various conditions of the experiment compared with control. Both the storage (set 1) as well as the operational (set 2) stabilities of the enzyme increased in the presence of trehalose as suggested by activity measurements under different conditions. Interestingly, greater relative retention of activity was observed in the presence of a mixture of 1.5 M trehalose and 1.5 M GdmCl compared with trehalose alone. We have obtained similar results earlier using lysozyme as a model enzyme (34).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Thermodynamic Basis for Protein Stabilization by
TrehaloseIt has been suggested that preferential interactions of
cosolvents with the native and the denatured state of a protein govern their
stabilization effect (24). The
values of n, the difference in the cosolvent molecules bound
to the denatured and the native proteins, that we obtained for RNase A,
lysozyme, and
-CTgen clearly indicate preferential exclusion of
trehalose from the vicinity of the denatured proteins
(Fig. 4). The variations in the
slopes for different proteins could be indicative of the subtle variations in
the physico-chemical properties of proteins and hence varied protein-solvent
interactions. The variations in the slopes for a given protein have also been
observed to arise from changes in the pH of the trehalose solutions
(Fig. 4 legend), indicating the
dependence of the degree of trehalose exclusion on the charge status of the
protein.
A decrease in the pH is also known to cause an increase in the
hydrophobicity of proteins
(35). This suggests that a
decrease in pH should be accompanied by an increase in the degree of exclusion
of trehalose due to the more hydrophobic nature of the protein. This is quite
evident from the increased value of n in the case of
RNase A as the pH decreases from 7.0 to 2.5
(Fig. 4). Interestingly, a
decrease in the pH of the protein solutions also results in an increase in
Tm in the presence of trehalose. A relatively
larger stability effect at low pH in the case of RNase A can be ascribed to an
increased exclusion of trehalose from the vicinity of the denatured protein as
compared with the native protein.
Trehalose has been observed to decrease the heat capacity of denaturation
considerably for all the five proteins studied. Heat capacity is a sensitive
thermodynamic parameter that can reflect upon the subtle changes in
protein-solvent interactions
(32,
36). Positive
Cp of protein unfolding is known to originate from
the ordering of polar solvent around the exposed nonpolar groups in proteins,
whereas the buried polar groups contribute in the opposite way
(36).
Cp can, thus, be related to the total change in the
accessible surface area (
ASA) upon protein unfolding
(37). Trehalose is expected to
cause more ordering of the solvent upon protein denaturation by inducing
preferential hydration and lead to a positive value of
Cp. However, our results are not consistent with
this assumption. Because
Cp depends upon
ASA
or the extent of unfolding
(38,
39), any residual structure
present in the unfolded state could lead to a decrease in the
Cp value. The presence of trehalose could result in
incomplete exposure of hydrophobic groups, and consequently a decrease in the
value of
Cp. Osmolytes and other salting-out agents
responsible for protein stabilization are known to induce the formation of
secondary structures in proteins under denaturing conditions
(4042).
Naturally occurring osmolytes like sarcosine, proline, sugars, and
trimethylamine-N-oxide have been shown to result in the contraction
of the denatured state proportional to their stabilizing power
(22,
43). A relatively compact
denatured state would have lesser hydrophobic surface in contact with the
solvent leading to a decrease in the apparent heat capacity of protein
denaturation. This is supported by our observation of a comparatively higher
value of
Cp for RNase A at pH 7.0 in trehalose
solution where 1.5 M GdmCl was also added. GdmCl is a strong
chaotropic agent and unfolds the protein to a more extended conformation
leading to the greater exposure of the buried hydrophobic groups.
The CD data presented as insets in
Fig. 1 for all the five
proteins studied also indicate the retention of a considerable amount of
secondary structure in the presence of 1 M trehalose relative to
control at the Tm for the proteins in buffer. Even at very
high temperatures (7080 °C), where the proteins are extensively
denatured, there is slight retention of the secondary structure in trehalose
solutions relative to that in the buffer alone. The increase in the negative
ellipticity for -CTgen at pH 2.5 with an increase in temperature is
surprising. A similar observation has been made earlier by Chalikian et
al. (44). Nonetheless,
there is an increase in the Tm of the protein in the
presence of trehalose and the data match well with those obtained from UV
melting curves for the protein.
A decrease in the Cp value can have a marked
affect on the protein stability, because it results in the flattening of the
stability curve (
G versus T) leading to an increase in the
free energy of stabilization over a broad range of temperatures.
Interestingly, this seems to be one of the common strategies adopted by
several hyperthermophilic proteins to enhance their overall stability
(4547).
Also, several studies (22,
29,
43,
48) have shown clearly that
natural osmolytes like sugars, sarcosine, and trimethylamine-N-oxide
essentially reduce the entropy of the denatured state, thereby increasing the
relative stability of the native state. In a similar manner, trehalose may
lead to protein stabilization as the result of a decrease in the entropy of
the denatured state, because the lower heat capacity of denaturation observed
suggests that the denatured state of the proteins has regions that are still
not fully exposed to the solvent. Therefore, it appears that decreasing the
entropy of the denatured state of proteins is a common thermodynamic approach
adopted to achieve higher stability and is closely related to a decrease in
the heat capacity of protein denaturation.
Effect of Surface Tension and Other Physico-chemical Properties of
WaterSurface tension of solvent is known to exert its affect on
protein stability by increasing the energy requirement for cavity formation in
the solvent to accommodate the increased surface area of proteins upon
denaturation (6,
8,
24). A linear correlation of
the increase in the Tm of proteins with increase in
the surface tension of trehalose suggests this property of water to be an
important factor contributing toward protein stability.
It has been observed that, at identical concentrations, trehalose increases the surface tension of water by much larger amounts compared with other sugars and polyols. It is interesting to note that thermodynamic properties of water like partial molal heat capacity and volume, related to the structure of aqueous solutions, also show a considerable increase in the presence of trehalose (49). These values are higher in magnitude compared with those for several other mono- and disaccharides as well as polyols (49, 50). It has also been reported that trehalose has a larger hydrated volume compared with other sugars (51). The increase in the values of these parameters has been attributed to stronger and more extensive hydrogen bonding between hydroxyl groups of trehalose and water molecules. Protein denaturation in such a solution would need additional energy to accommodate its increased surface area. In addition, cosolvents increasing the surface tension of water also get depleted at the protein-solvent interface leading to the preferential hydration of proteins (24). The preferential hydration effect should lead to a loss in the entropy of solvation upon protein denaturation, rendering the unfolded state even more unstable, and resulting in a shift of the equilibrium in favor of the native state. Although the surface tension of water increases to a much larger extent for certain simple electrolytes and carboxylic acid salts (26) compared with trehalose at identical concentrations, it seems that nature has preferred trehalose over these salts as their charged nature could have inhibitory effect on the activity of enzymes.2 These data suggest why trehalose has been selected by nature as an exceptional stabilizing agent under various stress conditions.
Role of Physico-chemical Properties of
ProteinsTable I shows clearly that trehalose does not affect the stability of various proteins
to the same extent. The increase in Tm of various proteins
as a function of surface tension of the medium
(Fig. 3) to different extents
suggests that the nature of the protein also plays an important role in the
trehalose-mediated thermal stability of proteins. We attempted to investigate
the role of the hydrophobicity of proteins by taking into account the changes
in hydrophobic interactions due to changes in nonpolar accessible surface area
(ASAnp) and polar accessible surface area (
ASAp) upon protein
denaturation, i.e.
ASAnp plus
ASAp, where
(42.5 cal/mol.Å2) and
(3.1
cal/mol.Å2) are weights of each contribution
(52). For simplicity, the term
ASAnp plus
ASAp can be considered to reflect the
effective hydrophobic energy driving the polypeptide chain to fold due to
unfavorable interactions with the polar solvent (water). The values of
ASAnp and
ASAp for various proteins have been calculated from
Myers et al. (37). A
plot (not shown) between the increments in Tm
(
Tm) due to the addition of trehalose and
ASAnp plus
ASAp of various proteins did not show
any correlation suggesting that the side chains, which determine the polarity
or nonpolarity of a protein, do not predominantly affect the net stability
provided by trehalose. However, these results cannot be considered conclusive
due to the limited number of proteins used in this study. Also, in the
presence of trehalose all the proteins may not unfold to a complete random
coil, hence limiting the calculation of the true changes in the accessible
surface area upon denaturation. Another reason for the lack of correlation
could be that these proteins also differ from each other in other properties
like net charge at a given pH (with pI values ranging from 4.1 for Trp-Inh to
10.7 for lysozyme), which should also contribute to protein-solvent
interactions as suggested by the dependence of
Tm
values in the presence of trehalose as a function of pH for a given
protein.
Bolen and coworkers
(2123)
have proposed that solvophobic (osmophobic) interaction between the peptide
backbone and osmolytes, including sucrose, is the main force driving the
protein to a more compact state. To test the validity of the above
proposition, the Tm values were plotted against the
number of peptide bonds in proteins, and a good correlation was observed at pH
7.0 (Fig. 5). This suggests
that protein backbone-cosolvent interactions are unfavorable and do contribute
toward protein stability. However, the lack of such a correlation at pH 2.5
and 4.0 (plots not shown) suggests that these unfavorable interactions cannot
be considered the sole factor leading to protein stabilization as has been
suggested in the case of other osmolytes wherein only one pH condition was
used
(2123).
Because trehalose is a neutral molecule, the pH-dependent changes in protein
stability mediated by trehalose should, therefore, have their origin in the
nature of proteins.
|
Recent studies show that hydrophobic side chains favor unfolding, whereas peptide bonds favor folding of proteins in the presence of sucrose (21) and other osmolytes (22, 23). On the contrary, extensive studies on transfer of amino acids and diglycine to aqueous polyol solutions carried out by Gekko (19, 20) show the dominance of unfavorable interactions between polyols and hydrophobic side chains. The transfer free energy of peptide bond has been observed positive for small chain polyols, which becomes negligible for longer chain polyols like sorbitol (19). Based on these results it has been suggested that peptide-water interactions dominate the stabilization of chymotrypsinogen by polyols (53), whereas intensification of hydrophobic interactions dominates the polyol-induced stabilization of lysozyme (54). Recently, studies carried out by Weatherly and Pielak (55) suggest that osmolytes can interact differently with proteins and that simple models are not sufficient to understand protein-osmolyte interactions. The present study involving several proteins varying in their molecular size does indicate the contribution of unfavorable peptide-trehalose interactions in protein stability. However, in addition, the contribution of charge status of proteins based on the pH-dependent stability effect mediated by trehalose is also evident.
Activity of RNase A in the Presence of TrehaloseRNase A was taken as a model system to analyze, in general, the thermostabilization effect of trehalose especially on the bioactivity of enzymes at high temperatures. The relative activity of RNase A in the presence of trehalose (Table II) has been calculated by dividing the slope of the linear zone of the corresponding activity plots by the slopes of the data for control.
In set 1, the activity retention in the presence of trehalose is seen to depend upon its effectiveness in locking the protein molecule in its native state even under denaturing conditions as has been suggested for the action of chaperonins (56). In buffer at 66 °C, only 7% molecules of RNase A are present in the folded state. The addition of 1.5 M trehalose raises the Tm of RNase A to 67.7 °C and the population of the folded molecules to 70%. At high temperatures, trehalose is known to preferentially bind weakly to the native state of RNase A (9) and could, thus, protect against any deleterious temperature-induced kinetic reactions like aggregation and preserve the overall activity on cooling. Trehalose has also been known to suppress the aggregation of unfolded proteins in vivo (57) as well as heat-denatured proteins in vitro (41).
In the second set, the greater effectiveness of trehalose at 63 °C as compared with that at 56 °C may be explained on the basis of the extent of unfolding at the two temperatures. In the absence of any additive, RNase A has a Tm of 61.6 °C at pH 7.0 (26). There is only a marginal difference in the population of the native RNase A in the presence (fNt) and the absence (fNc) of trehalose at 56 °C, i.e. the stabilization factor fNt/fNc is 1.04. However, this ratio increases to 2.82 at 63 °C resulting in an increase in the relative activity retention from 1.47 at 56 °C to 2.41 at 63 °C upon addition of 1.5 M trehalose. This is essentially due to a marked thermostabilization effect of trehalose on protein conformation at the higher temperature. At 52 °C, the relative activity term, in the presence of a mixture of 1.5 M trehalose and 1 M GdmCl, was much higher than the ratio of the native states at the same temperature. This could be due to a decrease in the deleterious effect of heat on enzymes at lower temperature. The greater protective action of trehalose in the presence of GdmCl could be ascribed to the fact that GdmCl being a protein solubilizer can prevent inactivation due to aggregation of the enzyme molecules at higher temperatures (34). Solubilizing agents like GdmCl and urea have been known to increase the refolding yields of proteins (58, 59). The higher activity obtained at 25 °C after incubation of RNase A at 60 °C in the presence of 1 M GdmCl in comparison to that incubated at 66 °C without GdmCl clearly demonstrates the role of GdmCl in inhibiting the aggregation of the protein molecules during incubation. A much smaller value of relative activity compared with the corresponding stabilization factor indicates that RNase A refolds to a large extent to its native state even in the absence of trehalose.
Unlike trehalose, other mono- and disaccharides and several of the polyols
have not been observed to provide thermostabilization and thermoprotection of
proteins to such an extent as trehalose
(1,
2,
10,
11). This is essentially due
to the differences in their cosolvent molecular structure and their solution
physico-chemical properties as described earlier. Most of the polyols and
sugars studied so far lead to preferential hydration of proteins at low
temperatures (2025 °C). However, studies carried out at higher
temperatures (50 °C) show considerable differences in the mode of
protein-cosolvent interactions
(9,
18). At low temperatures
sorbitol and trehalose lead to preferential hydration of native RNase A,
whereas at higher temperatures, sorbitol as well as trehalose have been
observed to bind weakly to RNase A
(9,
18). Trehalose in comparison
to sorbitol binds to a greater extent to the native state at higher but
nondenaturing temperatures. Fundamentally, both of these cosolvents stabilize
the native state of RNase A. However, as far as bioactivity is concerned, the
varying extent of binding of the cosolvent at elevated temperatures may have
perceptible effect on the retention of activity. Strong binding of trehalose
to the native state at high temperatures may provide a more compatible
environment and protection from heat inactivation.
This study clearly demonstrates that trehalose-induced thermostabilization of the protein structure is also helpful in the retention of biological activity of proteins at high temperatures. It is concluded that surface tension effect dominates the stability effect of trehalose, and, although unfavorable peptide-trehalose interactions contribute to protein stability as proposed, the interactions of trehalose with various side chains of proteins also contribute to the stability effect. Even though the nature of protein molecules contributes to protein-trehalose interaction in aqueous solutions to some extent, trehalose can be expected to work as a universal protein stabilizer and could be effectively used to increase the stability of many of the industrial and therapeutic enzymes without fail.
![]() |
FOOTNOTES |
---|
Present address: Molecular Biology Unit, National Dairy Research Institute,
Karnal 132001, India.
To whom correspondence should be addressed. Tel.: 91-11-2670-4086; Fax:
91-11-2616-7261; E-mail:
rajivbhat{at}hotmail.com.
1 The abbreviations used are: RNase A, ribonuclease A; ASA, change in
accessible surface area;
Cp, apparent heat capacity
of denaturation;
Hm, enthalpy of denaturation;
G0, Gibbs energy;
G0, free energy of stabilization;
Tm, midpoint of thermal denaturation;
-CTgen,
-chymotrypsinogen; cyt c, cytochrome c; Trp-Inh,
Trypsin Inhibitor; GdmCl, guanidinium chloride; CD, circular dichroism; MOPS,
4-morpholinepropanesulfonic acid.
2 D. P. Kumar and R. Bhat, unpublished data.
3 A. Tiwari and R. Bhat, unpublished results.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|