J. Biomedical Science and Engineering, 2011, 4, 94-99
doi:10.4236/jbise.2011.42013 Published Online February 2011 (http://www.SciRP.org/journal/jbise/
JBiSE
).
Published Online February 2011 in SciRes. http://www.scirp.org/journal/JBiSE
Thermal stability of proteins in the presence of aprotic ionic
liquids
Hidetaka Noritomi, Ken Minamisawa, Reo Kamiya, Satoru Kato
Department of Applied Chemistry, Tokyo Metropolitan University, Minami-Ohsawa, Tokyo, Japan.
Received 6 December 2010; revised 24 December 2010; accepted 28 December 2010.
ABSTRACT
Thermal stability of lysozyme dissolved in aqueous
solutions was examined in the presence of wa-
ter-miscible aprotic ionic liquids consisting of 1-ethyl-
3-methylimidazolium cation and several kinds of
anions. Addition of ionic liquids to an aqueous solu-
tion containing lysozyme prevented unfolded proteins
from aggregating irreversibly at high temperatures.
The thermal denaturation curve of lysozyme with
ionic liquids was entirely shifted to higher tempera-
ture, compared with that without ionic liquids. The
remaining activity of lysozyme after the heat treat-
ment was markedly dependent upon the kind and
concentration of ionic liquids. The remaining activi-
ties of lysozyme with 1.5 M 1-ethyl-3-methylimida-
zolium tetrafluoroborate ([emim][BF4]) and 0.1 M 1-
ethyl-3-methylimidazolium trifluoromethanesulfonate
([emim][Tf]) exhibited 88 and 68% after the heat
treatment at 90oC for 30 min, respectively, although
that without ionic liquids was perfectly lost.
Keywords: Thermal Stability; Lysozyme; Ionic Liquid;
Remaining Activity
1. INTRODUCTION
In resent years, the production and applications of pro-
teins have rapidly increased, not only in biochemical re-
search, but also in the chemical, food, and pharmaceuti-
cal industries, since proteins can exhibit exquisite bio-
logical activities. Three-dimensional structure of proteins
is kept by several weak interactions such as ionic effects,
hydrogen bonds, and hydrophobic interactions. When
these weak interactions are disrupted by changes of sev-
eral different kinds in the environment of proteins, pro-
teins are denatured, and inactivated via their unfolding
[1-3]. In particular, modest heating can easily disrupt
several of these stabilizing interactions. Thermal denatu-
ration is a serious problem not only in the separation and
storage of proteins but also in the processes of biotrans-
formation, drug production, and food manufacturing.
Several strategies have so far been proposed in order to
prevent thermal denaturation [4-11,21]. They include
chemical modification, immobilization, genetic modifi-
cation, and addition of stabilizing agents. The addition of
stabilizing agents to an aqueous solution containing pro-
teins is one of the most convenient methods for minimiz-
ing thermal denaturation. It has been reported that poly-
ols, sugars, amino acids, amino acid derivatives, me-
thylamines, and inorganic salts are available for improv-
ing protein stability. However, these additives do not suf-
ficiently prevent irreversible protein aggregation or some
of them are no longer stable at high temperatures.
Ionic solvent that is liquid at room temperature has at-
tracted increasing attention as a green solvent for the
chemical processes because of the lack of vapor pressure,
the thermal stability, and the high polarity [12,13].
Chemical and physical properties of ionic liquids can be
changed by the appropriate modification of organic
cations and anions, which are constituents of ionic liquids.
Biotransformation in ionic liquids has increasingly been
studied [14,15,34]. We have found that the activity of
protease is highly maintained not only in wa-
ter-immiscible aprotic ionic liquids but also in wa-
ter-miscible aprotic ionic liquids as well [22,23]. On the
other hand, it has been reported that protic ionic liquids
keep the stability of proteins in an aqueous solution at
high temperatures [24,25], and amyloid fibrils of proteins
are dissolved in protic ionic liquids and are refolded by
dilution with an aqueous solution [32]. Moreover, aprotic
ionic liquids can refold the denatured protein [33].
In our present work, we have focused on the remaining
activity of proteins after heat treatment in order to ad-
dress a question of whether or not water-miscible aprotic
ionic liquids consisting of 1-ethyl-3-methylimidazolium
cations and several kinds of anions affect the thermosta-
bility of proteins in aqueous solutions. As a model protein,
chicken egg-white lysozyme has been employed, since it
is well investigated regarding its structure, properties,

H. Noritomi et al. / J. Biomedical Science and Engineering 4 (2011) 94-99 95
functions, and thermostability [16-18,24,25].
2. EXPERIMENTAL
2.1. Materials
Lysozyme from chicken egg while (EC 3.2.1.17, 46400
units/mg solid, MW=14300, pI=11.1) and Micrococcus
lysodeikticus (ATCC No. 4698) were purchased from
Sigma-Aldrich Co. (St. Louis, USA). 1-Ethyl-3-me-
thylimidazolium trifluoromethanesulfonate ([emim][Tf])
(98% purity) was supplied from Shikoku Kasei Co.
(Kagawa, Japan). 1-Ethyl-3-methylimidazolium tetra-
fluoroborate ([emim][BF4]) (99% purity) and 1-ethyl-3-
methylimidazolium chloride ([emim][Cl]) (99% purity)
were obtained from Kanto Chemical Co. (Tokyo, Japan).
The structures of ionic liquids used in the present work
are shown in Figure 1. The other reagents were pur-
chased from Sigma-Aldrich Co. (St. Louis, USA). All
solvents used were of guaranteed grade and commer-
cially available, and were used without further purifica-
tion.
2.2. Heat Treatment of Lysozyme
In a typical experiment, the aqueous solution containing
100 μM lysozyme was prepared by dissolving lysozyme
to 0.01 M phosphate buffer solution at pH 7.0. One mL
of lysozyme aqueous solution with or without a requisite
quantity of ionic liquids in a 4-mL screw-cap vial was
placed in thermostated silicone oil bath at 90oC for 30
min.
2.3. Measurement of Remaining Activity of
Lysozyme
Lysozyme catalyzes hydrolysis of the β-1,4 glycosidic
linkage between the N-acetylmuramic acid and N-ace-
tylglucosamine components of peptidoglycan. This causes
breakdown and removal of peptidoglycan from the bac-
terium which results in cell bursting or lysis in natural
hypotonic solutions [16]. After the heat treatment, an
aqueous solution of lysozyme was cooled in thermo-
stated water bath at 25oC for 10 min. After 10 μL of the
cooled aqueous solution of lysozyme was added to 3mL
of 0.01 M phosphate buffer solution at pH 7 containing
200 mg/L Micrococcus lysodeikticus at 25oC, the ab-
sorbance was continuously measured at 450 nm by
Figure 1. Structures of ionic liquids used in the present work.
UV/vis spectrophotometer (Ubest-55, Japan Spectro-
scopic Co. Ltd.). Bacterial lysis obeys a first order reac-
tion. The lysis rate constant (k) is calculated by


0
450 450
ln
A
Akt
(1)
where t, Ao
450, and A450 are the reaction time, the ab-
sorbance of the substrate solution at 450 nm at T = 0,
and the absorbance of the substrate solution at 450 nm at
T = t, respectively. The remaining activity (R. A.) is de-
fined as
0
.. 100RAxk k

(2)
where ko and k are the lysis rate constants of native and
heat-treated enzymes at 25oC, respectively. Data for re-
maining activity is the average of triple measurements.
3. RESULTS AND DISCUSSION
3.1. Thermal Inactivation of Lysozyme
When proteins dissolved in an aqueous solution are
placed at high temperatures, most of proteins are imme-
diately unfolded due to the disruption of weak interac-
tions, including ionic effects, hydrogen bonds, and hy-
drophobic interactions, which are prime determinants of
protein tertiary structures. In addition, the intermolecular
aggregation among unfolded proteins, the incorrect
structure formation, and the chemical deterioration reac-
tions in unfolded proteins proceed as shown in Figure 2
[1-3,19,20]. In particular, protein aggregation easily oc-
curs upon the exposure of the hydrophobic surfaces of a
protein, and this phenomenon becomes the major prob-
lem because of the irreversible inactivation. On the other
hand, when a heated solution of denatured proteins with-
out protein aggregation is slowly cooled back to its nor-
mal biological temperature, the reverse process, which is
renaturation with restoration of protein function, often
occurs. Accordingly, if stabilizing agents can sufficiently
prevent irreversible aggregation of unfolded proteins, it is
expected that unfolded proteins are refolded by cooling
Figure 2. Schematic illustration of thermal denaturation of
proteins.
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H. Noritomi et al. / J. Biomedical Science and Engineering 4 (2011) 94-99 96
treatment, and the high remaining activity is obtained.
Table 1 represents the remaining activities of ly-
sozyme in the presence of various kinds of additives
after heat treatment at 90oC for 30 min. Lysozyme with-
out additives lost its activity perfectly after heat treat-
ment. Native lysozyme solution immedieately became
turbid due to the formation of protein aggregation, as
soon as heat treatment was carried out, as shown in Fig-
ure 3(b). It has been reported that the precipitation due
to protein aggregation is observed above 10 μM ly-
sozyme [18]. As lysozyme concentration in the present
work was 100 μM (1.4 mg/mL) which was ten times
higher than that, the formation of protein aggregation
was dramatically accelerated. Inorganic salts and glyc-
erol used as a conventional stabilizing agent inhibited
the formation of protein aggregation, and exhibited
thermal stabilization to some extent. On the other hand,
[emim][BF4] and [emim][Tf] showed high remaining
activities. The lysozyme solution in the presence of ionic
liquids was transparent after heat treatment, as seen in
Table 1. Remaining activities of lysozyme in the presence of
various kinds of additives after heat treatment at 90oC for 30 min.
Additive Remaining activity (%)
None 0
1.0 M Sodium chloride 29
0.3 M Ammonium sulfate 41
2.8 M Glucose 8
5.4 M Glycerol 15
7 mM β-Cyclodextrin 0
0.01 M Triton-X 0
2 % Pectin 0
0.7 M Urea 6
0.1 M [emim][Tf] 68
1.5 M [emim][BF4] 88
1.5 M [emim][Cl] 3.4
(a) (b)
Figure 3. Photographs of lysozyme solutions after heat treat-
ment at 90oC for 30 min: (a) lysozyme solution with 1.5 M
[emim][BF4], (b) lysozyme solution without [emim][BF4].
Figure 3(a). When lysozyme solution in the presence of
protic ionic liquids (alkylammonium formates) is heated
at 90oC, protein aggregation is prevented, and any
cloudy appearance is absent [25]. The hydrophobic core
of lysozyme unfolded by heat interacts with the cation of
ionic liquids, and cation adsorption results in acquisition
of a net positive charge preventing aggregation via elec-
trostatic repulsion [24].
Figure 4 shows the relationship between temperature
and the remaining activity of lysozyme in aqueous solu-
tions containing water-miscible ionic liquids after the
heat treatment for 30 min. As seen in the figure, the de-
pendence of the remaining activity on the temperature
exhibited the sigmoid curve. The remaining activity of
lysozyme without ionic liquids gradually decreased with
an increase in temperature below 70oC, accompanied
with the formation of precipitation due to protein aggre-
gation, drastically dropped in the range from 70 to 80oC,
and was then lost at temperatures of 80oC or higher. The
transition temperature was exhibited around 75oC, simi-
lar to the case measured by differential scanning calo-
rimetry [24]. On the other hand, the remaining activity
of lysozyme with 1.5 M [emim][Cl] gradually decreased
with an increase in temperature below 75oC, and drasti-
cally dropped in the range from 80 to 90oC. The remain-
ing activity of lysozyme with 1.5 M [emim][BF4] was
highly maintained below 80oC, gradually decreased with
temperature, and the remaining activity depicted 60% at
98oC. Similarly, the remaining activity of lysozyme with
0.1 M [emim][Tf] was highly retained below 80oC,
gradually decreased with temperature below 92oC, dras-
tically dropped in the range from 92 to 98oC, and was
then lost at 98oC. These results indicated that the addi-
tion of aprotic ionic liquids to an aqueous solution of
lysozyme effectively improved the thermal stability of
lysozyme at high temperatures.
Figure 4. Thermal denaturation curves of lysozyme with or
without ionic liquid. The aqueous solution of 100 μM ly-
sozyme with or without ionic liquids was incubated in a sili-
cone oil bath thermostated at requisite temperature for 30 min.
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H. Noritomi et al. / J. Biomedical Science and Engineering 4 (2011) 94-99 97
3.2. Refolding of Lysozyme with Ionic Liquids
Figure 5 shows the time course of remaining activity in
the presence of ionic liquids at 25oC after the heat treat-
ment at 90oC for 30 min. The remaining activity of ly-
sozyme with 1.5 M [emim][BF4] or 0.1 M [emim][Tf]
increased with incubation time, and reached the equilib-
rium at 2 and 7 min, respectively. In thermal denaturation
of lysozyme without protein aggregation, when the hy-
drophobic core of proteins is exposed, but the disulfide
bonds keep intact, denatured proteins spontaneously re-
fold to their native structures on cooling after thermal
denaturation [26-30]. The refolding of thermally-dena-
tured proteins is enhanced in the presence of protic ionic
liquids such as alkylammonium nitrate and alkylammo-
nium formates [24,25]. Moreover, N’-alkyl and N’-(ω-
hydroxyalkyl) N-methylimidazolium chlorides refold
denatured proteins such as hen egg white lysozyme and
the single-chain antibody fragment ScFvOx [33].
3.3. Dependence of the Remaining Activity of
Lysozyme on the Concentration of Ionic
Liquids via Heat Treatment
Figure 6 shows the plot of the remaining activity of ly-
sozyme against the concentration of ionic liquids after
the heat treatment at 90oC for 30 min. The remaining
activity was strongly dependent on the concentration of
[emim] [BF4] or [emim][Tf], while the effect of concen-
tration of [emim][Cl] was not observed. The remaining
activity in the presence of [emim][BF4] increased with
an increase in the concentration of [emim][BF4] and
reached a plateau around 0.8 M. The remaining activity
in the presence of [emim][Tf] dramatically increased with
increasing the concentration of [emim][Tf], the maximal
remaining activity was obtained at 0.1 M [emim][Tf], and
Figure 5. Time dependence of remaining activity of lysozyme
with ionic liquids on cooling at 25oC after heat treatment at
90oC for 30 min. After heat treatment, the aqueous solution of
100 μM lysozyme with 0.1 M [emim][Tf] or 1.5 M [emim][BF4]
then decreased steeply. After heat treatment, the
was incubated in a water bath thermostated at 25oC.
remaining
emaining Activity of
Figu ty of ly-
activity of lysozyme increases with an increase in the
concentration of ethylammonium formate and 2-meth-
oxyethylammonium formate, while the remaining activity
increases at low concentration of propylammonium for-
mate, but at higher concentrations of propylammonium
formate the protein spontaneously denatures [25]. Thus,
the dependence of concentration of ionic liquids on the
remaining activity of proteins changes by switching from
one ionic liquid to another.
3.4. Dependence of the R
Lysozyme on the Concentration of Ionic
Liquids after the Incubation at 25oC
re 7 shows the plot of the remaining activi
sozyme against the concentration of ionic liquids after the
incubation at 25oC for 30 min without the heat treatment.
The remaining activity in the presence of [emim][Cl] or
[emim][BF4] was undependent on the concentration of
Figure 6. Effect of concentration of ionic liquids on remaining
activity of lysozyme after heat treatment at 90oC for 30 min. The
aqueous solution of 100 μM lysozyme with requisite concentra-
tion of ionic liquids was incubated in a silicone oil bath thermo-
stated at 90oC for 30 min.
Figure 7. Effect of concentration of ionic liquids on remaining
activity of lysozyme during incubation at 25oC. The aqueous
solution of 100 μM lysozyme with requisite concentration of
ionic liquids was incubated in a water bath thermostated at 25oC
for 30 min.
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H. Noritomi et al. / J. Biomedical Science and Engineering 4 (2011) 94-99 98
ionic liquids till 1.2 M [emim][Cl] or 2.0 M [emim][BF4]
of
Figur aining activity of ly-
and gradually droped, while it in the presence of [emim]
[Tf] decreased with an increase in the concentration of
[emim][Tf]. These results indicate that [emim][Tf] tends
to strongly function as a denaturant, compared with
[emim][Cl] and [emim][BF4]. Electrolytes promote or
inhibit the stability of proteins according to the kind of
electrolytes [35]. Moreover, chemical denaturants, such
as urea and guanidine hydrochloride, can promote disso-
lution of inclusion bodies, which are protein aggregation
formed by prokaryotic expression systems [31]. Similarly,
[emim][Tf] inhibits the formation of protein aggregation
at low [emim][Tf] concentrations, but it mainly denatures
proteins at higher [emim][Tf] concentrations.
3.5. Time Course of Remaining Activity
Lysozyme via Heat Treatment with or
without Ionic Liquids
e 8 shows time course of rem
sozyme with or without ionic liquids through the heat
treatment at 90oC. The remaining activity of lysozyme
without ionic liquids dramatically decreased with an in-
crease in time, accompanied with the formation of pro-
tein aggregation, and was almost lost at 10 min. It has
been reported that the remaining activity in the thermal
denaturation process accompanied with the formation of
protein aggregation follows first-order kinetics [18]. As
seen in the figure, the relationship of the remaining activ-
ity of proteins in the absence of ionic liquids with heat
treat time could be correlated by first-order kinetics. On
the other hand, 1.5 M [emim][BF4] or 0.1 M [emim][Tf]
prevented the thermal inactivation of lysozyme. In the
presence of ionic liquids the turbidity of solutions due to
protein aggregation was not observed through heat treat-
ment. This indicates that the thermal inactivation mainly
Figure 8. Time dependence of remaining activity with or
on of ly-
Rate constant (min-1) Half life (min)
without ionic liquids after heat treatment at 90oC. The aqueous
solution of 100 μM lysozyme with or without ionic liquids was
incubated in a silicone oil bath thermostated at 90oC.
Table 2. Rate constants and half lives of inactivati
sozyme at 90oC.
Ionic liquid
none 0.43 1.6
1.5 M [emim][Cl] 0.065 11
0.1 M [emim][Tf] 0.0081 86
1.5 M [emim][BF4]0.0049 141
sults from the covalent change as shown in Figure 2.
t the remaining activity of ly-
Klibanov, A.M. (1989) Minimizing
re
The plots of remaining activity versus heat treatment time
on thermal inactivation of lysozyme in the presence of
ionic liquids followed first-order kinetics on linearity. It
has been reported that the thermal inactivation of ly-
sozyme obeyed first-order kinetics when it irreversibly
proceeded by the covalent change without the formation
of protein aggregation [17]. Ta b l e 2 represents rate con-
stants and half lives of inactivation of lysozyme with or
without ionic liquids calculated from the fitting curves.
The half lives with 1.5 M [emim][BF4], 0.1 M [emim]
[Tf], and 1.5 M [emim][Cl] were 88, 54, or 6.9 times
longer than that without ionic liquids, respectively.
4. CONCLUSIONS
We have demonstrated tha
sozyme is sufficiently maintained after heat treatment at
high temperatures, since aprotic ionic liquids prevented
unfolded proteins from aggregating. The remaining activ-
ity of lysozyme markedly depended upon the kind and
concentration of ionic liquids. Specifically, [emim][Tf]
exhibited thermostabilization effect of proteins at low
concentrations, but denatured proteins at high concentra-
tions. When the heat treatment was carried out at 90oC,
the half lives with 1.5 M [emim][BF4] and 0.1 M [emim]
[Tf] were much superior to that with 1.5 M [emim][Cl].
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