Tài liệu Báo cáo khoa học: Prevention of thermal inactivation and aggregation of lysozyme by polyamines docx

Prevention of thermal inactivation and aggregation of lysozyme
by polyamines
Motonori Kudou
1
, Kentaro Shiraki
1
, Shinsuke Fujiwara
2
, Tadayuki Imanaka
3
and Masahiro Takagi
1
1
School of Materials Science, Japan Advanced Institute of Science and Technology, Ishikawa, Japan;
2
Department of Bioscience,
School of Science and Technology, Kwansei Gakuin University, Hyogo, Japan;
3
Department of Synthetic Chemistry
and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan
Proteins tend to form inactive aggregates at high tempera-
tures. We show that polyamines, which have a relatively
simple structure as oligoamids, effectively prevent thermal
inactivation and aggregation of hen egg lysozyme. In the
presence of additives, including arginine and guanidine
(100 m
M
), more than 30% of 0.2 mgÆmL
)1
lysozyme in
sodium phosphate buffer (pH 6.5) formed insoluble aggre-

gates by heat treatment (98 °C for 30 min).
1
However, in the
presence of 50 m
M
spermine or spermidine, no aggregates
were observed after the same heat treatment. The residual
activity of lysozyme after this heat treatment was very low
(< 5%), even in the presence of 100 m
M
arginine and
guanidine, while it was maintained at  50% in the presence
of 100 m
M
spermine and spermidine. These results imply
that polyamines are new candidates as molecular additives
for preventing the thermal aggregation and inactivation of
heat-labile proteins.
Keywords: protein misfolding; protein aggregation; poly-
amine; thermal inactivation.
Proteins fold into their unique native structure, even in vitro.
However, they tend to form undesirable and uncontrollable
aggregates during the unfolding and refolding processes,
both in the laboratory and even in their natural environ-
ment in living cells. Protein aggregation is a major problem
in the large-scale production of recombinant proteins [1–3],
as well as in living cells, where it may lead to the occurrence
of fatal diseases
2
[4,5]. Various techniques have been

developed to prevent the formation of protein aggregates.
One of the major approaches used to prevent protein
aggregation is the addition of small molecules to the
solution. This is a relatively simple method compared with
using chaperon systems [6–8].
The small molecular additives used to prevent the
formation of protein aggregates are classified as protein-
denaturing reagents or others. Denaturants, typically
guanidine and urea, weaken the hydrophobic intermole-
cular interaction of proteins [9,10]. Detergents, such as
Triton-X100 and SDS, are stronger protein-denaturing
reagents than denaturants [10,11]. Not only do these
reagents dissolve aggregates and inclusion bodies but they
also unfold the native structure of proteins. Accordingly, the
concentration at which this type of reagent is effective at
preventing the aggregation and inactivation of proteins is
hard to determine.
3
Arginine (Arg) is a nondenaturing reagent that has been
used widely as an additive to prevent protein aggregation
[9–12]. Arg does not destabilize the native structure, having
only a minor effect on protein stability [11,13], and enhances
the solubility of aggregate-prone molecules. Because of its
beneficial properties, Arg has been used for various proteins
and situations. However, the effect of Arg and other
nondenaturing additives does not completely solve the
aggregation problem. The development of better additives
for preventing protein aggregation has been long awaited.
In this article, we focus on naturally occurring poly-
amines [putrescine, NH

2
(CH
2
)
4
NH
2
; spermidine, NH
2
(CH
2
)
3
NH(CH
2
)
4
NH
2
;spermine,NH
2
(CH
2
)
3
NH(CH
2
)
4
NH(CH

2
)
3
NH
2
] as small molecular additive candidates
for preventing heat-induced aggregation and inactivation of
proteins. There are a large number of different polyamines
4
in hyperthermophiles [14–16], which suggests that poly-
amines have a biophysical role in the adaptation of
hyperthermophilic proteins to high temperature environ-
ments. Although it has been reported that polyamines bind
to biomolecules (DNA, RNA, and platelets) by electrostatic
interactions [17–19], at present no research has been
published regarding the role of polyamines on thermal
aggregation and inactivation of proteins.
Materials and methods
Materials
Hen egg white lysozyme and betaine/HCl were purchased
from Sigma Chemical Co. All amino acids, guanidine/HCl,
urea, putrescine/2HCl, spermidine/3HCl, and spermine/
4HCl were purchased from Wako Pure Chemical Industries
(Osaka, Japan). Micrococcus lysodeikticus for the kinetics
Correspondence to K. Shiraki, School of Materials Science,
Japan Advanced Institute of Science and Technology,
1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292, Japan.
Fax: + 81 761 51 1655, Tel.: + 81 761 51 1657,
E-mail:
Abbreviations: DCp, heat capacity change; DH, enthalpy change;

DSC, differential scanning calorimetry; T
m
, midpoint temperature
of thermal unfolding.
Enzymes: lysozyme (EC 3.2.1.17).
(Received 29 July 2003, revised 27 August 2003,
accepted 23 September 2003)
Eur. J. Biochem. 270, 4547–4554 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03850.x
assay of lysozyme was purchased from Nacalai Tesque, Inc.
(Kyoto, Japan). Trimethylamine-N-oxide was purchased
from Aldrich Chemical Company, Inc. All other chemicals
used were of high-quality analytical grade.
Heat-induced aggregation of lysozyme
Heat-induced aggregates of lysozyme were quantified as
follows.
5
Solutions, containing 0.2 mgÆmL
)1
lysozyme in
50 m
M
sodium-phosphate buffer (pH 6.5) and different
concentrations of additives, were prepared. All stock
solutions for additives and protein were dissolved in
50 m
M
sodium-phosphate buffer (pH 6.5)
6
and adjusted to
pH 6.5 by NaOH or HCl before sample preparation. After

heat treatment at 98 °C, the samples were centrifuged at
15 000 g for 20 min. The absorbance of the supernatants
was monitored by using a Jasco spectrophotometer model
V-550 (Japan Spectroscopic Company, Tokyo, Japan) to
determine the concentration of soluble lysozyme, using an
extinction coefficient of 2.63 cm
)1
per mgÆmL
)1
7
[20].
Residual activity of lysozyme after heat treatment
The bacteriolytic activity of lysozyme was estimated as
follows [21]. A 1.5 mL volume of 0.5 mgÆmL
)1
M. lys-
odeikticus solution prepared in 50 m
M
sodium-phosphate
buffer (pH 6.5) was mixed with 20 lL of the heat-treated
samples containing 0.2 mgÆmL
)1
lysozyme and 100 m
M
additive. The decrease in light scattering intensity of the
solution was monitored by measuring the absorbance (A)at
600 nm. The rate constant of inactivation was determined
by fitting the data to a linear extrapolation.
CD spectra
Far-ultraviolet CD spectra were measured using a Jasco

spectropolarimeter, model J-720 W, equipped with a
thermal incubation system. The far-ultraviolet CD spectrum
of lysozyme was measured at a protein concentration of
0.1 mgÆmL
)1
, in a 2-mm cuvette.
Differential scanning calorimetry
Differential scanning calorimetry (DSC) for a mixture of
lysozyme and an additive was performed using a nano-
DSCII Differential Scanning Calorimeter 6100 (Calori-
metry Sciences Corporation, UT, USA) with a cell
volume of 0.299 mL and at a scanning rate of
2 °CÆmin
)1
. Degassing during the calorimetric experiment
was prevented by maintaining an additional constant
pressure of 2.5 bars over the liquid in the cell. The
samples were 4.0 mgÆmL
)1
lysozyme in 50 m
M
sodium-
phosphate buffer (pH 6.5) or sodium-acetate buffer
(pH 4.4) together with 100 m
M
additive. Solutions con-
taining additives were dialysed to adjust their pH to 6.5
or 4.4. The enthalpy change (DH), heat capacity change
(DC
p

), and midpoint temperature of thermal unfolding
(T
m
) were determined by a conventional method, as
described previously [22].
Results
Heat-induced aggregation of lysozyme
Hen egg white lysozyme (pI ¼ 11) was used as a model
protein because its mechanism of refolding and misfolding
has been extensively studied [12,13,23–27]. Lysozyme can
preferentially refold into its native structure from thermally
unfolded states, while under neutral pH it tends to form
irreversible aggregates during heating [23–25].
The amount of heat-induced aggregates produced from
0.2 mgÆmL
)1
lysozyme,whenheatedto98°C for 30 min,
was measured in the presence of various additives at
pH 6.5 (Fig. 1). The amount of aggregates gradually
Fig. 1. The amount of heat-induced aggregates produced in the presence of various additives. Solutions containing 0.2 mgÆmL
)1
lysozyme (pH 6.5)
and various concentrations of additives were heated at 98 °C for 30 min. After heat treatment, the amount of aggregates was calculated by
determining the soluble concentration of lysozyme by centrifugation. (A) Arginine (Arg), (d); glycine (Gly), (s); guanidine, (h). (B) Betaine, (d);
trimethylamine-N-oxide, (s); putrescine, (m); spermidine, (h); spermine, (j). (C) NaCl, (d); KCl, (s); urea, (j).
4548 M. Kudou et al. (Eur. J. Biochem. 270) Ó FEBS 2003
decreased with increasing concentrations of Arg or
guanidine from 0 to 0.5
M
(Fig. 1A). In contrast,

 50 m
M
spermidine and spermine completely prevented
the thermal aggregation of lysozyme (Fig. 1B). The
aggregation curve for putrescine, the smallest polyamine
used in this study,
8
was essentially identical to those of Arg
and guanidine. On the other hand, small ammonium ions
(trimethylamine-N-oxide and betaine) did not prevent
heat-induced aggregation (Fig. 1B). Other additives, such
as glycine (Gly), urea, NaCl, or KCl did not prevent heat-
induced aggregation (Fig. 1A,C). These data indicate that
spermidine and spermine prevent heat-induced aggrega-
tion of lysozyme better than the other additives tested in
this study.
Aggregation of lysozyme as a function of heating
time and protein concentration
Figure 2A shows the time course of heat-induced aggrega-
tion with 100 m
M
of each additive. After 4 min, the amount
of deposited aggregates showed an increase in the absence of
any additives. However, in the presence of Arg, the amount
of aggregates gradually increased after 10 min. In contrast
9
,
with spermine, no aggregates were observed, even after heat
treatmentat98°C for 40 min.
Figure 2B shows the dependence of aggregation upon the

initial concentration of lysozyme. In the absence of any
additives, the concentration of soluble proteins reached a
plateau at  0.07 mgÆmL
)1
. Further increase of the protein
concentration resulted in a gradual increase in the soluble
concentration of lysozyme, from 0.07 mgÆmL
)1
to
0.14 mgÆmL
)1
. In the presence of 100 m
M
spermidine or
spermine, no aggregates were observed at a protein
concentration of < 0.4 mgÆmL
)1
. With an increasing con-
centration of lysozyme, the curve reached a plateau at
 0.7 mgÆmL
)1
. Interestingly, putrescine was clearly less
effective than spermidine, whereas spermine was as effective
as spermidine. This implies that an important factor
required for polyamines to prevent protein aggregation is
the presence of a secondary amine, rather than the number
of cations or molecular mass.
Aggregation by cooling
We examined the heat-induced aggregation of lysozyme
during cooling (Fig. 3A). Protein solutions of 0.2 mgÆmL

)1
lysozyme, prepared in 50 m
M
sodium-phosphate buffer
(pH 6.5) containing 100 m
M
additive, were heated at 98 °C
for 30 min and then cooled from 98 °Cto50°Cbyusinga
thermal controller. The concentration of soluble protein
slightly decreased with cooling time. The slightly negative
correlation between cooling time and concentration of
soluble protein may be explained by the prolonged therm-
ally unfolded state of the protein with longer cooling times.
At temperatures above 84 °C, lysozyme was fully unfolded
by heating (Fig. 3B). These data indicate that lysozyme
aggregated during the heat treatment, rather than during the
cooling.
Fig. 2. Heat-induced aggregation of lysozyme was dependent on the incubation time and protein concentration. (A) The solutions containing
0.2 mgÆmL
)1
lysozyme (pH 6.5) and 100 m
M
arginine (Arg) (s), spermine (j), or no additive (d), were heated at 98 °C. After heat treatment, the
amount of aggregates was calculated by determining the soluble concentration of lysozyme by centrifugation. (B) The horizontal axis shows the
concentration of lysozyme in samples containing different concentrations of lysozyme and 100 m
M
additive at pH 6.5. After heat treatment at 98 °C
for 30 min, the soluble concentrations of lysozyme were determined and plotted on the vertical axis. No additive, (d); Arg, (s); putrescine, (m);
spermidine, (h); or spermine (j).
Ó FEBS 2003 Prevention of thermal aggregation by polyamines (Eur. J. Biochem. 270) 4549

Heat inactivation of lysozyme
The recovery of enzymatic activity after heat treatment is
another criterion used to estimate the effect of additives
because it is the most reliable measure of whether additives
prevent irreversible misfolding as well as aggregation.
Figure 4 shows the residual activity of lysozyme after heat
treatment at 98 °C. In the absence of additives, the
inactivation curves of 0.2 and 1.0 mgÆmL
)1
lysozyme
fitted well to single-exponential equations (Fig. 4A). The
inactivation rate constants for 0.2 and 1.0 mgÆmL
)1
lyso-
zyme were 0.067 and 0.21 min
)1
, respectively. The heated
samples, containing 1.0 mgÆmL
)1
lysozyme (black circles in
Fig. 4A), were resolved by the addition of guanidine/HCl
(to a final concentration of 4.0
M
)
10
and vortexing for 15 min.
These samples were diluted 10-fold by 50 m
M
sodium-
phosphate buffer (pH 6.5), after which the residual activities

of the samples were measured (Fig. 4A). However, the
inactivation rate constant of the resolved sample was
0.23 min
)1
, which was almost identical to that of the
Fig. 3. Heat-induced aggregation is influenced by cooling time. (A) After heat treatment at 98 °C for 30 min, the protein solutions were cooled from
98 °Cto50°C for respective periods of time, and then the concentrations of soluble lysozyme were determined. The solutions were 0.2 mgÆmL
)1
lysozyme and 50 m
M
sodium phosphate buffer (pH 6.5). The line shows the least-square fit for the raw data. (B) Far-ultraviolet CD spectra of
lysozyme in 50 m
M
sodium-phosphate buffer (pH 6.5) at different temperatures.
Fig. 4. Effect of additives on the time course of heat inactivation. Samples containing 0.2 mgÆmL
)1
(open symbols) or 1.0 mgÆmL
)1
(closed symbols)
lysozyme, in 50 m
M
sodium phosphate buffer (pH 6.5) containing 100 m
M
additive, were heated at 98 °C. (A) Circles, no additives; crosses, samples
of the closed circles resolved by 4.0
M
guanidine/HCl and refolded by dilution. (B) Circles, putrescine; squares, spermidine. (C) Circles, spermine;
squares, arginine (Arg). The continuous curves show least-square fitting of the respective data with single-exponential equations.
4550 M. Kudou et al. (Eur. J. Biochem. 270) Ó FEBS 2003
unresolved sample. These facts indicate that the inactivated

molecules, under the experimental conditions used in this
study
11
, were mainly stabilized by covalent bonds (probably
disulfide exchanges) rather than by noncovalent inter-
actions.
In the presence of 100 m
M
putrescine, the inactivation
curves of 0.2 and 1.0 mgÆmL
)1
lysozyme depended on the
protein concentration, as shown by single-exponential
equations
12
(Fig. 4B). The inactivation rate constants for
0.2 and 1.0 mgÆmL
)1
lysozyme with 100 m
M
putrescine
were 0.023 and 0.11 min
)1
, respectively, which were two- to
threefold slower than those in the absence of additives.
Interestingly, in the presence of 100 m
M
spermidine and
spermine, the inactivation curve of 1.0 mgÆmL
)1

lysozyme
was identical to that of 0.2 mgÆmL
)1
lysozyme. The rate
constants of inactivation in the presence of spermidine and
spermine were 0.020 and 0.034 min
)1
, respectively
(Fig. 4B,C); these constants were one order of magnitude
slower than those of 1.0 mgÆmL
)1
lysozyme in the absence
of additives. In the presence of 100 m
M
Arg, the inactivation
rate constants for 0.2 and 1.0 mgÆmL
)1
lysozyme were 0.045
and 0.19 min
)1
, respectively (Fig. 4C). Arg also prevented
the heat inactivation of lysozyme; however, the preventive
effect was clearly lower than that induced by spermidine or
spermine.
In order to elucidate the versatility of polyamines, various
additives were tested by measuring the residual activities
(Table 1). After heat treatment for 30 min, total residual
activities of 52% and 57%, for 0.2 mgÆmL
)1
lysozyme, were

recovered by the addition of 100 m
M
spermidine and
spermine, respectively. On the other hand, the residual
activity was < 5%, for most of the other additives (even
100 m
M
Arg),
13
which is an order of magnitude lower than
for spermidine and spermine. Also, at a protein concentra-
tion of 1.0 mgÆmL
)1
and heat treatment for 10 min,
polyamines prevented heat inactivation of lysozyme more
effectively than the other additives (Table 1).
DSC analysis
To reveal the effect of additives on protein stability,
thermodynamic parameters were determined using DSC.
Representative DSC curves of lysozyme in the presence of
100 m
M
additive are shown in Fig. 5, and the thermo-
dynamic parameters derived from nonlinear least-squares fit
of the DSC data are listed in Table 2. DSC curves showed
full reversibility at pH 4.4, but not at pH 6.5, so enthalpy
change (DH) and heat capacity changes (DC
p
) are listed only
at pH 4.4.

At pH 6.5, the T
m
of lysozyme was 77.3 °C in the absence
of additives. The addition of 100 m
M
polyamines and Arg
slightly increased the T
m
of lysozyme by  1 °Cand0.5°C,
respectively, whereas addition of other additives did not
alter the values compared with no additive
14
.Theincreased
T
m
values at neutral pH are responsible for preventing
irreversible aggregation during DSC measurement. At
pH 4.4, the presence of polyamines slightly decreased the
T
m
by
15
1.9–2.7 °C. Gly exhibited the best results, judging by
the increased T
m
. The decreased T
m
value, as conferred by
polyamines, implies that they bind to the unfolded mole-
cules. DH and DC

p
values were approximately the same
(within 5%) in the presence or absence of additives. These
results suggest that the thermodynamic equilibrium of
lysozyme is not influenced by 100 m
M
additive and that the
molecular mechanism of spermidine and spermine as
aggregation suppressors cannot be explained by the slight
change in the thermodynamic parameters.
Table 1. Residual activity of lysozyme after heat treatment.
Additive
Residual activity
(%)
a
Residual activity
(%)
b
Putrescine 15.1 ± 2.4 67.6 ± 4.8
Spermidine 52.4 ± 5.0 82.2 ± 6.1
Spermine 56.6 ± 4.3 90.5 ± 4.7
Lysine 2.3 ± 0.4 32.1 ± 6.6
Arginine 4.3 ± 1.3 30.9 ± 7.1
Glycine 1.5 ± 1.2 15.4 ± 2.8
Guanidine 0.8 ± 0.8 23.5 ± 2.9
Urea 0.8 ± 0.3 11.8 ± 3.1
NaCl 2.3 ± 0.4 13.2 ± 1.9
KCl 1.0 ± 0.7 11.8 ± 2.8
Glucose 0.9 ± 0.6 11.8 ± 1.3
Maltose 1.5 ± 0.7 13.1 ± 0.9

No additive 0.8 ± 0.8 21.3 ± 4.1
Before heating 100.0 ± 4.8 100.0 ± 6.1
a
Residual activities of 0.2 mgÆmL
)1
lysozyme containing 100 m
M
additive (pH 6.5) after heat treatment at 98 °C for 30 min.
b
Residual activities of 1.0 mgÆmL
)1
lysozyme containing 100 m
M
additives (pH 6.5) after heat treatment at 98 °C for 10 min.
Fig. 5. Differential scanning calorimetry (DSC) measurement. The
samples contained 4.0 mgÆmL
)1
lysozyme and 100 m
M
additive in
50 m
M
sodium-acetate buffer (pH 4.4). No additive, s;arginine(Arg),
h;putrescine,n; spermidine, ,.
Ó FEBS 2003 Prevention of thermal aggregation by polyamines (Eur. J. Biochem. 270) 4551
Discussion
The present study indicates two points regarding the heat-
induced aggregation and inactivation of lysozyme, namely
(a) in the absence of additives, loss of activity is dependent
on the protein concentration (Fig. 4A), indicating that the

rate-limiting step of the heat inactivation of lysozyme is
involved in an intermolecular interaction and (b) the
resolved and refolded samples did not increase the activity
(Fig. 4A). These two facts imply that the heat-induced
inactivation of lysozyme is caused by covalent interactions
among molecules, probably disulfide reshuffling. Interest-
ingly, in the presence of spermidine and spermine, the
inactivation rates were not dependent on the protein
concentration (Fig. 4B,C). This implies that spermidine
and spermine prevent intermolecular interactions. More-
over, after heat treatment at 98 °C for 30 min, no aggre-
gates were observed in the presence of 100 m
M
spermidine
or spermine (Fig. 2A), while 50% of the molecules were
inactivated (Fig. 4B,C).
These facts propose the following mechanism, whereby
the heat-induced aggregation and inactivation of lysozyme
is considered to follow two steps of an irreversible reaction
at high temperatures:
U ! A Eqn ð1Þ
A þ A
n
! A
nþ1
Eqn ð2Þ
where, U represents the unfolded molecule that can
refold after heat treatment, A represents the irreversibly
denatured molecule and A
n

represents the insoluble
aggregates. Under Eqn (1), spermidine and spermine
prevent the intermolecular interactions shown in Eqn
(2). As shown by Klibanov and co-workers [23,28], all
proteins inactivate by heat; however, spermidine and
spermine prevent heat inactivation of lysozyme as a
result of inhibiting intermolecular interactions – the
rate-limiting step.
Some research has reported that the heat inactivation of
proteins is caused by both noncovalent and covalent
modifications, including disulfide exchanges [23,24,28],
b-elimination of disulfide bonds [24,28], and deaminations
of Gln and/or Asn [25,28]. At neutral pH values, the rate-
limiting step of covalent modification is disulfide exchange
[23,24,28]. Volkin & Klibanov showed that the half-lives of
destruction of the disulfide bonds in 14 proteins at 100 °C
were 0.6–1.4 h at pH 8 and 9–16 h at pH 6; lysozyme was
no exception for the inherent thermal instability of disulfide
bonds [
16
23]. In view of these facts, we conclude that
spermidine and spermine prevent intermolecular inter-
actions, including disulfide exchanges and aggregation
17
.
During early studies on protein aggregation, it was
found that denaturing reagents of tertiary structures, such
as guanidine and urea, increased the solubility of protein
and improved the yield of refolding [10,11]. Other
denaturing substances, such as lauryl maltoside micells

[29] and surfactants [30], were found to promote the
correct folding of proteins. When using these additives it
is important to use the appropriate nondenaturing con-
centration, but this may be difficult because the native
state is easily destabilized in the presence of these
additives.
18
On the other hand, Arg has a favorable
property – it is not a denaturant, yet it enhances the
solubility of the aggregate-prone form of unfolded protein
[10,12,31]. For this reason, Arg has been commonly used
as an aggregation suppressor. However, we report, in this
study, the new finding that spermine and spermidine are
more effective for preventing heat-induced aggregation
than other, well-known additives (Table 1).
Many researchers have reported the biological role of
polyamines in enhancing growth or cell proliferation [32,33].
Polyamines are relatively simple structures that are com-
posed of multivalent amines. The pK
a
values of the
secondary amines in putrescine, spermidine, and spermine
were 8.0–8.5, whereas those of the primary amines were
10.0–11.1 [34]. In biophysical aspects, polyamines can bind
with nucleic acids and phospholipids, and stabilize and
regulate their tertiary structures [17–19]. In this article, we
report that polyamines prevent aggregation of lysozyme, a
positively charged protein
19
(pI ¼ 11). Under acidic condi-

tions, lysozyme can recover its active form, even after heat
treatment for several hours [23,25], suggesting that some
degree of additional charge neutralization may be at work.
Although the present report did not investigate the precise
mechanism of formation and inhibition of aggregates, our
data imply that the formation of ion pairs with local
negative charges would effectively increase the net charge of
the protein, leading to increased electrostatic repulsion and
Table 2. Thermodynamic parameters of lysozyme with additives. DCp, heat capacity change; DH, enthalpy change; T
m
, midpoint temperature of
thermal unfolding. Thermodynamic parameters were determined by differential scanning calorimetry measurement of 4.0 mgÆmL
)1
lysozyme with
100 m
M
additives in buffer.
Additive
T
m
at pH 6.5
(°C)
a
T
m
at pH 4.4
(°C)
b
DH at pH 4.4
(kJÆmol

)1
)
b
DC
p
at pH 4.4
(kJÆmolÆK
)1
)
b
No additive 77.3 81.0 452 20.7
Putrescine 78.4 79.1 436 21.7
Spermidine 78.4 78.7 437 22.2
Spermine 78.6 78.3 448 22.8
Arginine 77.8 79.7 439 21.6
Glycine 77.7 81.3 442 20.6
Guanidine 77.1 79.1 437 22.0
NaCl 77.7 79.9 441 21.6
a
50-m
M
sodium-phosphate buffer (pH 6.5);
b
50 m
M
sodium-acetate buffer (pH 4.4).
4552 M. Kudou et al. (Eur. J. Biochem. 270) Ó FEBS 2003
a reduction of intermolecular interaction. It is worth
mentioning that hyperthermophiles, which can grow at
temperatures of > 90 °C, synthesize several kinds of

multivalent polyamines as their culture temperature increa-
ses [14–16]. Our results imply that polyamines play a
significant role in preventing heat-induced aggregation and
inactivation of proteins in vivo.
In conclusion, our results indicate that polyamines are a
new class of additives which can prevent the aggregation
and inactivation of heat-labile proteins. We propose that the
following two questions should be addressed during future
investigations of the efficacy of spermidine and spermine on
protein aggregation.
20
First, can polyamines prevent the
aggregation and inactivation of other proteins? Although it
is still unclear, preliminary data has been obtained that heat
inactivation of trypsin is effectively inhibited by polyamines.
Second, can polyamines prevent the formation of
21
other
types of aggregates, such as fibril formation or refolding-
induced aggregation? Recently, Hoyer et al. reported that
polyamines induce fibril aggregation of a-synuclein [35].
Our preliminary data using a model peptide reached the
same conclusion.
Acknowledgements
We thank Dr D. Hamada, Y. Mitsukami, and S. Uchida for helpful
comments and critical reading of the manuscript, and H. Kitagawa for
assistance with experiments. This work was supported by a Grant-
in-Aid for Scientific Research from the Ministry of Education, Science,
Sports and Culture of Japan (14350433, 14045229), a grant from the
Science and Technology Incubation Program in Advanced Region by

JST (Japan Science and Technology Corporation), and the Sasakawa
Scientific Research Grant from The Japan Science Society.
References
1. Fink, A.L. (1998) Protein aggregation: folding aggregates, inclu-
sion bodies and amyloid. Fold. Des. 3, 9–23.
2. Lilie, H., Schwarz. E. & Rudolph, R. (1998) Advances in refolding
of proteins produced in E. coli. Curr. Opin. Biotechnol. 9, 497–501.
3. Carrio, M.M. & Villaverde, A. (2002) Construction and decon-
struction of bacterial inclusion bodies. J. Biotechnol. 96, 3–12.
4. Bucciantini, M., Giannoni, E., Chiti, F., Baroni, F., Formigli, L.,
Zurdo, J., Taddei, N., Ramponi, G., Dobson, C.M. & Stefani, M.
(2002) Inherent toxicity of aggregates implies a common
mechanism for protein misfolding diseases. Nature 416, 507–511.
5. Chiti, F., Calamai, M., Taddei, N., Stefani, M., Ramponi, G. &
Dobson, C.M. (2002) Studies of the aggregation of mutant pro-
teins in vitro provide insights into the genetics of amyloid diseases.
Proc.NatlAcad.Sci.USA99, 16419–16426.
6. Rozema, D. & Gellman, S.H. (1995) Artificial chaperones: protein
refolding via sequential use of detergent and cyclodextrin. J. Am.
Chem. Soc. 117, 2373–2374.
7. Altamirano, M.M., Golbik, R., Zahn, R., Buckle, A.M. & Fersht,
A.R. (1997) Refolding chromatography with immobilized mini-
chaperones. Proc.NatlAcad.Sci.USA94, 3576–3578.
8. Izumi, M., Fujiwara, S., Shiraki, K., Takagi, M., Fukui, K. &
Imanaka, T. (2001) Utilization of immobilized archaeal chaper-
onin for enzyme stabilization. J. Biosci. Bioeng. 91, 316–318.
9. Buchner, J. & Rudolph, R. (1991) Renaturation, purification and
characterization of recombinant Fab-fragments produced in
Escherichia coli. Biotechnology 9, 157–162.
10. Rudolph, R. & Lilie, H. (1996) In vitro folding of inclusion body

proteins. FASEB J. 10, 49–56.
11. Taneja, S. & Ahmad, F. (1994) Increased thermal stability of
proteins in the presence of amino acids. Biochem. J. 303, 147–153.
12. Shiraki,K.,Kudou,M.,Fujiwara,S.,Imanaka,T.&Takagi,M.
(2002) Biophysical effect of amino acids on the prevention of
protein aggregation. J. Biochem. (Tokyo) 132, 591–595.
13. Arakawa, T. & Tsumoto, K. (2003) The effects of arginine on
refolding of aggregated proteins: not facilitate refolding, but
suppress aggregation. Biochem. Biophys. Res. Commun. 304, 148–
152.
14. Oshima, T. (1979) A new polyamine, thermospermine, 1,12-di-
amino-4,8-diazadodecane, from an extreme thermophile. J. Biol.
Chem. 254, 8720–8722.
15. Oshima, T. (1982) A pentaamine is present in an extreme therm-
ophile. J. Biol. Chem. 257, 9913–9914.
16. Oshima, T., Hamasaki, N., Senshu, M., Kakinuma, K. &
Kuwajima, I. (1987) A new naturally occurring polyamine con-
tainingaquaternaryammoniumnitrogen.J. Biol. Chem. 262,
11979–11981.
17. Pelta, J., Livolant, F. & Sikorav, J.L. (1996) DNA aggregation
induced by polyamines and cobalthexamine. J. Biol. Chem. 271,
5656–5662.
18. Raspaud, E., Chaperon, I., Leforestier, A. & Livolant, F. (1999)
Spermine-induced aggregation of DNA, nucleosome, and chro-
matin. Biophys. J. 77, 1547–1555.
19. De la Pena, N.C., Sosa-Melgarejo, J.A., Ramos, R.R. & Mendez,
J.D. (2000) Inhibition of platelet aggregation by putrescine, sper-
midine, and spermine in hypercholesterolemic rabbits. Arch. Med.
Res. 31, 546–550.
20. Wetlaufer, D., Kwok, E., Anderson, W.L. & Johnson, E.R. (1974)

Kinetic determinism of lysozyme folding at high temperatures.
Biochem. Biophys. Res. Commun. 56, 380–405.
21. Wetlaufer, D.B. & Saxena, V.P. (1970) Formation of three-
dimensional structure in proteins. I. Rapid nonenzymic reactiva-
tion of reduced lysozyme. Biochemistry 9, 5015–5023.
22. Hagihara, Y., Shiraki, K., Nakamura, T., Uegaki, K., Takagi, M.,
Imanaka, T. & Yumoto, N. (2002) Screening for stable mutants
with amino acid pairs substituted for the disulfide bond between
residues 14 and 38 of bovine pancreatic trypsin inhibitor (BPTI).
J. Biol. Chem. 277, 51043–51048.
23. Volkin, D.B. & Klibanov, A.M. (1987) Thermal destruction
processes in proteins involving cystine residues. J. Biol. Chem. 262,
2945–2950.
24. Tomizawa, H., Yamada, H., Tanigawa, K. & Imoto, T. (1995)
Effects of additives on irreversible inactivation of lysozyme at
neutral pH and 100 degrees C. J. Biochem. (Tokyo) 117, 369–373.
25. Tomizawa, H., Yamada, H., Wada, K. & Imoto, T. (1995) Sta-
bilization of lysozyme against irreversible inactivation by
suppression of chemical reactions. J. Biochem. (Tokyo) 117,635–
640.
26. Maachupalli-Reddy, J., Kelly, B.D. & Clark, E.D.B. (1997) Effect
of inclusion body contaminants on the oxidative renaturation of
hen egg white lysozyme. Biotechnol. Prog. 13, 144–150.
27. Ueda, T., Nagata, M. & Imoto, T. (2001) Aggregation and che-
mical reaction in hen lysozyme caused by heating at pH 6 are
depressed by osmolytes, sucrose and trehalose. J. Biochem.
(Tokyo) 130, 491–496.
28. Zale, S.E. & Klibanov, A.M. (1986) Why does ribonuclease irre-
versibly inactivate at high temperatures? Biochemistry 25, 5432–
5444.

29. Zardeneta, G. & Horowitz. P.M. (1992) Micelle-assisted protein
folding. Denatured rhodanese binding to cardiolipin-containing
lauryl maltoside micelles results in slower refolding kinetics but
greater enzyme reactivation. J. Biol. Chem. 267, 5811–5816.
30. Wetlaufer, D.B. & Xie, Y. (1995) Control of aggregation in pro-
tein refolding: a variety of surfactants promote renaturation of
carbonic anhydrase II. Protein Sci. 4, 1535–1543.
Ó FEBS 2003 Prevention of thermal aggregation by polyamines (Eur. J. Biochem. 270) 4553
31. Umetsu, M., Tsumoto, K., Hara, M., Ashish, K., Goda, S.,
Adschiri, T. & Kumagai, I. (2003) How additives influence the
refolding of immunoglobulin-folded proteins in a stepwise dialysis
system. Spectroscopic evidence for highly efficient refolding of a
single-chain Fv fragment. J. Biol. Chem. 278, 8979–8987.
32. Rozov, A. & Burnashev, N. (1999) Polyamine-dependent facil-
itation of postsynaptic AMPA receptors counteracts paired-pulse
depression. Nature 401, 594–598.
33. Freire de Lima, C.G., Nascimento, D.O., Soares, M.B., Bozza,
P.T., Castro-Faria-Neto, H.C., de Mello, F.G., DosReis, G.A. &
Lopes, M.F. (2000) Uptake of apoptotic cells drives the growth of
a pathogenic trypanosome in macrophages. Nature 403, 199–203.
34. Takeda, Y., Samejima, K., Nagano, K., Watanabe, M., Sugeta,
H. & Kyogoku, Y. (1983) Determination of protonation sites in
thermospermine and in some other polyamines by 15N and 13C
nuclear magnetic resonance spectroscopy. Eur. J. Biochem. 130,
383–389.
35. Antony, T., Hoyer, W., Cherny, D., Heim, G., Jovin, T.M. &
Subramaniam, V. (2003) Cellular polyamines promote the
aggregation of a-synuclein. J. Biol. Chem. 278, 3235–3240.
4554 M. Kudou et al. (Eur. J. Biochem. 270) Ó FEBS 2003