Tài liệu Báo cáo khoa học: Arginine ethylester prevents thermal inactivation and aggregation of lysozyme pptx

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-

cules. The data suggest that ArgEE could be used as an
additive to prevent inactivation and aggregation of heat-
labile proteins.
Keywords: arginine; arginine ethylester; lysozyme; protein
aggregation; thermal inactivation.
Protein a ggregation is a serious problem for both biotech-
nology and cell biology. Diseases such a s prion misfolding,
Alzheimer’s, and other amyloidoses are phenomena f or
which protein aggregation in our living cells is of consid-
erable relevance [1–4]. In the field of biotechnology,
aggregation, resulting in the formation of inclusion bodies,
is a major problem in bacterial recombinant systems [5–7].
Under unfolding conditions, irreversible a ggregation
competes with correct folding. The classical model by
Lumry–Eyring has b een used to describe protein aggrega-
tion [8–10]:
N $ A ! Agg ð1Þ
where N, A, and Agg represent a native state, a non-native
state, and aggregates. Equation (1) involves a first-order
reversible folding/unfolding reaction and subsequent inter-
molecular association w ith a higher-order irreversible pro-
cess. The kinetics and equiliblium of Eqn (1) are dependent
on solution conditions, such as temperature, pH, and the
presence of additives. The additives may influence both the
solubility and the stability o f proteins in the N a nd A states.
They also may change the folding rate to prevent or
accelerate the nonspecific aggregation from A to Agg.
Guanidine and urea are well established as aggregation
suppressors that weaken the hydrophobic intermolecular
interaction of p roteins [11,12]. In particular, these denatu-

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

2
PO
4
were from Wako Pure
Chemical Industries Ltd. All chemicals used were of high
quality analytical grade.
Time course of thermal inactivation and aggregation
Heat treatment o f lysozyme w as performed a s f ollows:
500 lL of the sample solutions containing 1.0 mgÆmL
)1
or
0.2 m gÆmL
)1
lysozyme and 100 m
M
sodium phosphate
buffer pH 7.1 in the presence or absence of 100 m
M
Correspondence to K. Shiraki, School of Materials Science, Japan
Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai,
Tatsunokuchi, Ishikawa 923-1292, Japan. Tel.: +81 761 51 1657,
E-mail:
Abbreviations: ArgEE, arginine ethylester; Gdn, guanidine; T
m
,
midpoint temperature of thermal unfolding.
Enzymes: lysozyme (EC 3.2.1.17); ribonuclease A (EC 3.1.27.5).
(Received 4 April 2004, revised 7 June 2004, accepted 17 June 2004)
Eur. J. Biochem. 271, 3242–3247 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04257.x
additives were prepared in 1.5 mL microtubes. The samples

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

scattering intensity of the solution was monitored by
absorbance at 600 nm. The re sidual activity was e stimated
by fitting the data to a linear extrapolation.
pH dependence of thermal inactivation and aggregation
Heat treatment of lysozyme was performed according to t he
following procedure: 500 lL of the sample solutions
containing 1.0 mgÆmL
)1
lysozyme and at variou s pH values
(adjusted by t he addition of 100 m
M
phosphate borate
buffer) in the presence or absence of Arg or ArgEE were
prepared in 1.5 mL microtubes. The samples were heated at
98 °C for 10 min. After the heat treatment, the samples
were cooled on ice for 4 h. The samples w ere centrifuged at
15 000 g for 20 min at 4 °C, and then the concentration of
soluble protein and residual activity were determined. After
these measurements, the precise pH was d etermined using
the residual sample.
Thermal unfolding by DSC
Thermal unfolding curves of lysozyme were measured by
DSC, using a nano-DSCII differential scanning calorimeter
6100 (Calorimetry Sciences Corporation) with a cell v olume
of 0.299 m L at a scanning rate of 2 °CÆmin
)1
. Degassing
during the calorimetric experiment was prevented by
maintaining an additional constant pressure of 2.5 bar over
the liquid in the cell. Solution contained 4.0 mg ÆmL

)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

in the presence of additives
Figure 1 s hows the thermal inactivation and aggregation of
lysozyme at pH 7.1. In the absence of any additives,
lysozyme was inactivated and a ggregated with a single-
exponential manner after a lag period of  200 s (Fig. 1A).
The presence of lag phase on the inactivation curve implies
that the inactivation is affected by the formation of
aggregates during heating. This is consistent with previous
data showing that the thermal inactivation of lysozyme has
a single rate-limiting step [21–25]. Actually, more than 10
types of protein have been analysed, s howin g that the
higher-order processes of aggregates can be described by
single-exponential equations [26,27]. In the presence of
100 m
M
Arg, the inactivation and aggregation rates were
slightly decelerated (Fig. 1B). In the presence of ArgEE, the
heat-induced inactivation rate of lysozyme was one-sixth
that in the absence of additives (Fig. 1C).
The rates of inactivation and aggr egation under several
different conditions are shown in Table 1. The rates o f
inactivation and aggregation d epend on the protein
Fig. 1. Thermal inactivation and aggregation of lysozyme in the presence of additives. T he samples containin g 1.0 mgÆmL
)1
lysozyme with no
additive (A), 100 m
M
Arg (B), and 100 m
M
ArgEE (C) at pH 7.1 were h eated a t 9 8 °C for the times shown. After t he heat treatment, the residual

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-

gation of lysozyme. After heat treatment at 98 °Cfor
10 min, 1.0 m gÆmL
)1
lysozyme without additives was
completely inactivated above pH 7.2 (Fig. 2A, s). Several
reports of the heat-induced inactivation of lysozyme have
shown that t he inactivation at alkaline pH is highly related
to the intermolecular noncovalent interactions, followed by
covalent modification, mainly caused by disulfide exchange
[21,22,24]. This is because the pI of lysozyme is around
pH 11.
After the sa me heat trea tment, the sigmoidal a ctivity
curve was slightly improved by the addition of 100 m
M
Arg
(Fig. 2 A, h). On the other hand, the sigmoidal activity
curve was clearly shifted to alkaline p H by the addition of
100 m
M
ArgEE ( Fig. 2A, n). For example, 80% or more of
the enzymes retained the active form after heat treatment
at 98 °C for 10 min in the presence of 100 m
M
ArgEE
at pH 6.5; under the same conditions, only 30% of the
enzymes retained the active form even in the presence of the
same concentration of Arg.
ArgEE p revents heat-induced aggregation (Fig. 2B) as
well as inactivation (Fig. 2A). After heat t reatment at 9 8 °C
for 10 min, the amount of aggregates increased with

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

additive were measured as shown i n Fig. 1 . The
inactivation and aggregation profiles were fitted to single exponential
equations and th e a pparent r ate c onstants w ere c alculated. n d, N o data
due to the slow rate of aggregation under the conditions used.
Protein concentration Additive
Inactivation
(· 10
)3
Æs
)1
)
Aggregation
(· 10
)3
Æs
)1
)
1.0 mgÆmL
)1
(pH 7.1) None 7.04 ± 0.56 4.42 ± 0.43
NaCl 5.85 ± 0.24 4.54 ± 0.23
GdnHCl 4.81 ± 0.44 2.61 ± 0.38
Spermine 1.22 ± 0.13 0.57 ± 0.14
Arg 4.24 ± 0.39 2.17 ± 0.25
ArgEE 1.15 ± 0.15 0.42 ± 0.08
0.2 mgÆmL
)1
(pH 7.1) None 4.03 ± 0.24 1.11 ± 0.06
Arg 1.62 ± 0.25 1.09 ± 0.06
ArgEE 0.84 ± 0.11 0.35 ± 0.08

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

the thermal unfolding curve of lysozyme in the presence of
ArgEE as m onitored by DSC. In the absence of a dditives,
the T
m
value of lysozyme w as 78.1 °CatpH6.5.TheT
m
increased a s the concentration of ArgEE increased from 0 to
60 m
M
. The maximum T
m
of lysozyme is 79.8 °Cat60m
M
ArgEE. The increase in T
m
may correspond to the increase
in solubility of t he aggregation-prone molecules caused by
the addition of ArgEE. With further increases in the
concentration of ArgEE, the T
m
decreased from 100 to
600 m
M
. The decreasing T
m
corresponds to the unfolding
effect of ArgEE on lysozyme. Figure 3B summarizes the T
m
of lysozyme in the presence of Arg and A rgEE. Unlike with
ArgEE, the T

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

Arg
(74.2 ± 0.4 °C). These data, being consistent with the
DSCanalysesoflysozyme,suggestthatArgEEhasa
destabilizing effect on proteins.
The thermal unfolding curves of myoglobin in the
absence of a dditive and in the presence of GdnHCl could
not measured by near-UV CD due to the aggregation
that occurred under these conditions. An identical
experiment was performed with lysozyme but the
near-UV CD data generated were too noisy to allow
evaluation of the T
m
.
Charged states of Arg and ArgEE
In order to understand the importance of amphiphilicity,
titration curves of Arg and ArgEE were compared
(Fig. 5 A). The pK
a
of amino groups on Arg and ArgEE
were determined as pH 9.2 and 7.4, respectively. The
decreased pK
a
of amino group of ArgEE compare to Arg
is related to the ethylation of the main chain of the carboxyl
group. Figure 5B shows the amount of aggregation of
lysozyme at pH 6.5 and 10.0 in the presence of Arg and
ArgEE after heat treatment a t 98 °C for 30 min. At pH 6.5
Arg and ArgEE possess positive charges on their amino
group, while at pH 10.0 they lose the positive charges. W ith
increasing concentration of ArgEE, the amount of aggre-

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

values of amino groups on Arg and ArgEE. A
small q uantity of 1.0
M
NaOH wa s added to 10 mL of 1.0
M
Arg/HCl
(s)orArgEE-2HCl(h) solution. (B) Heat-induced aggregation o f
lysozyme in the presence of A rg (circles) or A rgEE (squares) at pH 6.5
(open symbols) or pH 10.0 (closed symbols). T he samples containing
0.2 mgÆmL
)1
lysozyme with additives a t pH 6.5 or 10.0 were he ated at
98 °C for 30 min. The samples were centrifuged at 15 000 g for
30 min, and then the amount of aggregates was determined.
Ó FEBS 2004 Aggregation of lysozyme with additive (Eur. J. Biochem. 271) 3245
prominent effect of ArgEE w as observed when m onitoring
at pH 10.0 (Fig. 5B). These data suggest that ArgEE
prevents heat-induced aggregation only in the charged state
of the amino group.
Discussion
From early studies on protein aggregation, it is known t hat
denaturing reagents, such as GdnHCl and u rea, increase the
solubility o f a ggregate-prone molecule s, leading to i mprove-
ment in refolding yield [12]. On the other hand, Arg is a
nondenaturing reagent that prevents protein aggregation
[14–16]. Although Arg is one of the m ost w idely u sed
additives f or the prevention of p rotein aggregation a nd
improvement of refoldin g yield, only a few papers have
reported the molecular mechanism of Arg as an additive.
The following properties are shown: (a) Arg is the best

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.
Hen egg white lysozyme is inactivated irreversibly b y
heat treatment a t n eutral pH [21–24]. The rate- limiting
step of inactivation at around pH 7 is the intermolecular
interaction between exposed hydrophobic surfaces, fol-
lowed by irreversible disulfide exchange [24,25]. The
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

citrate synthase [29], bovine alpha A-crystallin [30], a nd
ovalbumin [31], although the process of aggregation is
expected to be second- or higher-order kinetics [8,32–34].
This is because t he rate-limiting s tep of thermal aggre-
gation is th e nucleation with g rowth of aggregates,
leading to p seudo-first-order kinetics after a lag period
of  200 s. The p resence of t he lag phase observed i n
both inactivation and aggregation results from the
structural change to aggregation-prone molecules. The
aggregation-prone molecules m ust possess low solubility
and a large hydrophobic region on the surface in
comparison with the soluble unfolded molecules
[22,35,36]. Our data described by the pseudo-first-order
kinetics also support the same conclusion even in the
presence of Arg and ArgEE.
In summary, this paper shows that ArgEE prevents
heat-induced inactivation and aggregation of lysozyme.
Although Arg has been used as a n additive to prevent
protein aggregation f or several d ecade s, ArgEE, as well as
spermine [18], is considered as a new candidate chemical
chaperone for heat-induced inactivation and aggregation of
proteins.
Acknowledgements
This work was partly supported by a Grant-in-Aid for Scientific
Research from the Ministry of E ducation, Science, Sports and Culture
of Japan (14350433, 14045229) and The Japan S ecurities Scholarship
Foundation.
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