Arginine ethylester prevents thermal inactivation and aggregation
of lysozyme
Kentaro Shiraki
1
, Motonori Kudou
1
, Shingo Nishikori
1,2
, Harue Kitagawa
1,2
, Tadayuki Imanaka
3
and Masahiro Takagi
1,2
1
School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), Tatsunokuchi, Japan;
2
Innovation plaza
Ishikawa, Japan Science and Technology Agency (JST), Tatsunokuchi, Japan;
3
Department of Synthetic Chemistry and Biological
Chemistry, Graduate School of Engineering, Kyoto University, Japan
Arginine is a versatile additiv e to prevent p rotein aggrega-
tion. This paper shows that arginine ethylester (ArgEE)
prevents heat-induced inactivation and agg regation o f h en
egg lysozyme more effectively than arginine or guanidine.
The addition of ArgEE decreased the melting temperature o f
lysozyme. This data could be interpreted in terms of A rgEE
binding to unfolded lysozyme, possibly through the ethyl-
ated carboxyl group, which leads to effective prevention of
intermolecular interaction a mong aggregation-prone mole-

rants increase the solubility of aggregation-prone unfolded
molecules, but decrease the s tability of the native state.
Among nondenaturing reagents, a rginine is t he most widely
used additive for increasing refolding yields by decreasing
aggregation, for example when it is used in experiments wit h
a single chain antibody [11,13]. Arginine does not facilitate
refolding, but suppresses aggregation, with only a minor
effect on protein stability [14], while it enhances the
solubility of aggregates-prone molecules, leading to an
increase in refolding yields [15–17]. Although other addi-
tives, such as proline, glycerol, glycine, and ethylene glycol,
have been used [12], these are not enough t o solve the
problems of protein aggregation and misfolding. Recently
we reported that polyamines, typically spermine and
spermidine, prevent heat-induced inactivation and aggre-
gation of lysozyme [18,19]. As p art of a series of studies to
develop additives, this paper shows a new candidate,
arginine ethylester (ArgEE), as a superior additive to
prevent heat-induced inact ivation and aggregation of lyso-
zyme as a model protein.
Materials and methods
Materials
Bovine pancreas RNaseA, hen egg white lysozyme, horse
myoglobin, Arginine/HCl (Arg), and A rgEE were from
Sigma Chemical Co. Guan idine h ydrochloride (GdnHCl),
NaCl, Na
2
HPO
4
,andNaH

were heat treated at 98 °C for various periods. After the
heat treatment, th e samples were immediately cooled on ice
for 4 h. The samples were centrifuged at 15 000 g for
20 min at 4 °C, and then the concentration o f soluble
protein and residual activity were determined. The protein
concentration o f the supernatants was determined by
measuring absorbance at 280 nm with the appropriate
blank, using e xtinction co efficients of 2.63 cm
)1
per mgÆml
)1
.
Measurements of protein concentration and residual
activity
The concentration of soluble protein was monitored with a
Jasco spectrophotometer model V-550 (Japan Spectro-
scopic Company), using an extinction coefficient of
2.63 cm
)1
per mgÆml
)1
[20]. The residual activity of the
soluble fraction was determined as follows [9,18]: 1.5 mL of
0.5 mgÆmL
)1
Micrococcus lysod eikticus solution contain ing
50 m
M
sodium phosphate buffer pH 6.5 was mixed with
20 lL o f the protein solution. The decrease in light

)1
lysozyme, various concentrations of Arg or ArgEE, and
100 m
M
sodium phosphate buffer pH 6.5. The apparent
melting temperature ( T
m
) was determined at the peak of the
DSC curve.
Thermal unfolding by near-UV CD
Thermal unfolding curves of myoglobin and RNaseA were
measured by near-UV CD, with a Jasco spectropolarimeter
model J-720 W equipped with t hermal incubation system.
The samples containing 1.0 m gÆmL
)1
protein, 500 m
M
additive, and 100 m
M
sodium phosphate buffer pH 6.5
were prepared. The thermal unfolding was measured by
CD at 280 nm intensity with increasing temperature of
1 °CÆmin
)1
. The data obtained w ere fitted to a conventional
two-state equation and determined the apparent T
m
.
Results
Thermal inactivation and aggregation of lysozyme

activity (s) and the amount o f aggregate c alculated by the concentration of solub le protein ( d) were determined and p lotted. The c ontinu ous and
broken lines show the theoretical curves fitted to the closed and open circles with single exponen tial equations.
Ó FEBS 2004 Aggregation of lysozyme with additive (Eur. J. Biochem. 271) 3243
concentration, indicating that intermolecular interaction is
the rate-limiting step in both inactivation and aggregation
of lysozyme by heat treatment. As the d ata points were
well fitted to the single-exponential eq uation, the heat-
induced aggregation and inactivation of lysozyme appar-
ently follow first-order kinetics. However, the fact that the
rates of aggregation and inactivation depend on protein
concentration allows us to consider the processes as
pseudo-first order, as reported previously [18,26,27]. These
data imply that the rate-limiting step of aggregation and
inactivation is the stage of irreversible unfolding, which is
affected by the additives. After the obligatory process of
unfolding, t wo (or several) p rotein molecule s transform
the a ggregation-prone un folded molecules to t he aggre-
gates.
ArgEE lowered the dependence of the rate of aggregation
on protein concentration, implying that ArgEE prevents
intermolecular interactions. The rates of inactivation and
aggregation of lysozyme in the presence of ArgEE were
similar to those in spermine, which is a favourable additive
to prevent t hermal inactivation and aggregation of l ysozyme
[18]. These data s how that ArgE E is a new candidate
additive for the prevention of thermal inactivation of
lysozyme.
pH dependence of the inactivation and aggregation
in the presence of additives
Figure 2 shows the pH-dependent inactivation and aggre-

increasing pH (Fig. 2B, s). In the p resence of 100 m
M
Arg, the profile was slightly improved. However, in the
presence of 100 m
M
ArgEE, the p rofile was clearly shifted to
alkaline pH (Fig. 2B, n).
Interestingly, in the presence of 1.0
M
NaCl, the inacti-
vation and agg regation c urves i n the presence of Arg are the
same as those in the absence of additives (Fig. 2C,D). Th is
indicates that t he prevention of inactivation and aggregation
by Arg can be explained solely by electrostatic interactions.
On the basis that heat-induced aggregation is due to the
intermolecular interactio n between exposed hydrophobic
regions, Arg may play a role in the prevention of
intermolecular interactions due to electrostatic interaction s.
On the other hand, the inactivation and aggregation curves
obtained with 100 m
M
ArgEE are clearly different in the
presence of 1.0
M
NaCl; ArgEE prevents both t hermal
inactivation and aggregation at high p H (Fig. 2B,D). The
Table 1. Rates of thermal inactivation and aggregation in the presence
of additives. Thermal inactivation and aggregation in the presence or
absence of 100 m
M

0.2 mgÆmL
)1
(pH 6.5) None 1.01 ± 0.09 0.41 ± 0.21
Arg 0.76 ± 0.16 0.28 ± 0.17
ArgEE 0.11 ± 0.03 nd
Fig. 2. pH-dependent thermal inactivation and aggregation of lysozyme
in the presence of additives. Samples containing 1.0 mgÆmL
)1
lysozyme
and 0
M
(A,B) or 1.0
M
(C,D) N aCl w ith 100 m
M
additives at vario us
pHswereprepared.Theadditivesarenone(s), Arg (h), or ArgEE
(n). These samples were heated at 98 °C for 10 min and residual
activity (A,C) and amount of aggregates (B,D) were determined.
Continuous, dotted, and broken lines show the fitted curves to no
additives, Arg, and ArgEE with sigmoidal equations.
3244 K. Shiraki et al. (Eur. J. Biochem. 271) Ó FEBS 2004
data obtained in the presence of NaCl suggest that the
molecular mechanism of ArgEE in preventing thermal
inactivation and aggregation is different from that of Arg.
Thermal unfolding profile of proteins in the presence
of ArgEE
In order to investigate whether or not ArgEE destabilizes
protein structure, we analysed the thermal unfolding profile
of lysozyme in the presence of additives. Figure 3A shows

m
did not change with increasing concentra-
tions of Arg.
Figure 4 shows thermal unfolding curves of RNaseA
(Fig. 4 A) and myoglobin (Fig. 4B) in the presence or
absence of additives as monitored by near-UV CD. The
T
m
value of RNaseA in the absence of additives was
64.7 ± 0.1 °C. In the presence of 500 m
M
GdnHCl and
Arg, T
m
values of RNaseA were 59.6 ± 0.1 °Cand
60.8 ± 0.2 °C, respectively, which were 5.1 °Cand3.9°C
lower than those obtained without the a dditives. The T
m
of
RNaseA with 50 0 m
M
ArgEE (56.1 ± 0.2 °C) was 8 .6 °C
lower than without additives. Similarly, the T
m
of myoglo-
bin in the presence of 500 m
M
ArgEE (58.3 ± 0 .4 °C) w as
clearly lower than in the presence of 500 m
M

gates steeply decreased from 0 to 30 m
M
. The addition of
30 m
M
ArgEE completely prevents heat-induced aggrega-
tion of lysozyme at pH 6.5. The preventive effect of ArgEE
was clearly higher than that of Arg (Fig. 5B). However, no
Fig. 3. Thermal unfolding cu rves of ly so zyme in th e p rese nce o f additiv es
monitoredbyDSC.The samples containing 4.0 mgÆmL
)1
lysozyme
with various con centrations of Arg or ArgEE at pH 6.5 were measured
by DSC. (A) Representative curves in the presence of ArgEE. The
concentrations of ArgEE were shown in the figure. (B) T
m
in the
presence of Arg (d)orArgEE(
s).
Fig. 4. Thermal unfolding curves of proteins in the presence of add itives
monitoredbynear-UVCD.The samples cont aining 4.0 mgÆmL
)1
RNaseA (A) or myoglobin (B) in the presence or absence of 500 m
M
additive at pH 6.5 w ere measured by CD at 280 nm. The ad ditives are
no additive ( s), Gd nHCl (h), Arg (n), and ArgEE (·). The data were
fitted to conventional two-state equations.
Fig. 5. Differences in the c hemi cal properties of Arg and ArgEE. (A)
Determ ina tio n of p K
a

additive for the prevention of heat-induced aggregation of
lysozyme out of 15 amino a cids [15] and ( b) Arg does not
stabilize proteins against heat treatment [16]. In addition,
this paper shows t hat (c) Arg p revents heat-induced
aggregation by an e lectrostatic interaction between protein
molecules (Fig. 2).
This paper focused on the ArgEE as a new additive to
prevent heat inactivation and aggregation. We selected
ArgEE a s additive b ecause it i s an Arg derivative that
possesses g uanidium group on its side c hain. Although we
have examined several Arg derivatives, only ArgEE shows a
strong effect in preventin g protein inactivation. The
molecular mechanism of ArgEE in preventing heat-induced
aggregation is different from that of Arg. ArgEE may bind
preferentially to unfolded molecules of l ysozyme by the
introduced hydrophobic e nd on the carboxyl grou p, leading
to an increase in the apparent net charge of the unfolded
molecules. The increased net charge caused by binding of
the additives would effectively increase the electrostatic
repulsion between unfolded or partially unfolded molecules
that are prone to form irreversible aggregates and reduce
aggregation and misfolding.
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irreversible aggregation of protein caused by heat treat-
ment usually follows pseudo first-order kinetics at the
terminal phase, such as seen with beef catalase [28], beef

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