This paper shows that arginine ethylester ArgEE prevents heat-induced inactivation and aggregation of hen egg lysozyme more effectively than arginine or guanidine.. As part of a series of
Trang 1Arginine ethylester prevents thermal inactivation and aggregation
of lysozyme
Kentaro Shiraki1, Motonori Kudou1, Shingo Nishikori1,2, Harue Kitagawa1,2, Tadayuki Imanaka3
and Masahiro Takagi1,2
1
School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), Tatsunokuchi, Japan;2Innovation plaza Ishikawa, Japan Science and Technology Agency (JST), Tatsunokuchi, Japan;3Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Japan
Arginine is a versatile additive to prevent protein
aggrega-tion This paper shows that arginine ethylester (ArgEE)
prevents heat-induced inactivation and aggregation of hen
egg lysozyme more effectively than arginine or guanidine
The addition of ArgEE decreased the melting temperature of
lysozyme This data could be interpreted in terms of ArgEE
binding to unfolded lysozyme, possibly through the
ethyl-ated carboxyl group, which leads to effective prevention of
intermolecular interaction among 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 aggregation is a serious problem for both
biotech-nology and cell biology Diseases such as prion misfolding,
Alzheimer’s, and other amyloidoses are phenomena for
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 aggregation
competes with correct folding The classical model by
Lumry–Eyring has been used to describe protein
aggrega-tion [8–10]:
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 with 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 of proteins in the N and 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 proteins [11,12] In particular, these
denatu-rants increase the solubility of aggregation-prone unfolded
molecules, but decrease the stability of the native state Among nondenaturing reagents, arginine is the most widely used additive for increasing refolding yields by decreasing aggregation, for example when it is used in experiments with
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 to 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 part 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 inactivation 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 ArgEE were from Sigma Chemical Co Guanidine hydrochloride (GdnHCl), NaCl, Na2HPO4, and NaH2PO4 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 of lysozyme was performed as follows:
500 lL of the sample solutions containing 1.0 mgÆmL)1or 0.2 mgÆmL)1 lysozyme and 100 mM sodium phosphate buffer pH 7.1 in the presence or absence of 100 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: kshiraki@jaist.ac.jp
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)
Trang 2additives were prepared in 1.5 mL microtubes The samples
were heat treated at 98C for various periods After the
heat treatment, the samples were immediately cooled on ice
for 4 h The samples were centrifuged at 15 000 g for
20 min at 4C, and then the concentration of soluble
protein and residual activity were determined The protein
concentration of the supernatants was determined by
measuring absorbance at 280 nm with the appropriate
blank, using extinction coefficients of 2.63 cm)1per 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)1Micrococcus lysodeikticussolution containing
50 mM sodium phosphate buffer pH 6.5 was mixed with
20 lL of the protein solution The decrease in light
scattering intensity of the solution was monitored by
absorbance at 600 nm The residual activity was estimated
by fitting the data to a linear extrapolation
pH dependence of thermal inactivation and aggregation
Heat treatment of lysozyme was performed according to the
following procedure: 500 lL of the sample solutions
containing 1.0 mgÆmL)1lysozyme and at various pH values
(adjusted by the addition of 100 mM phosphate borate
buffer) in the presence or absence of Arg or ArgEE were
prepared in 1.5 mL microtubes The samples were heated at
98C for 10 min After the heat treatment, the samples
were cooled on ice for 4 h The samples were centrifuged at
15 000 g for 20 min at 4C, and then the concentration of
soluble protein and residual activity were determined After
these measurements, the precise pH was determined 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 volume
of 0.299 mL at a scanning rate of 2CÆmin)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 mgÆmL)1 lysozyme, various concentrations of Arg or ArgEE, and
100 mM sodium phosphate buffer pH 6.5 The apparent melting temperature (Tm) 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 thermal incubation system The samples containing 1.0 mgÆmL)1 protein, 500 mM additive, and 100 mM sodium phosphate buffer pH 6.5 were prepared The thermal unfolding was measured by
CD at 280 nm intensity with increasing temperature of
1CÆmin)1 The data obtained were fitted to a conventional two-state equation and determined the apparent Tm
Results
Thermal inactivation and aggregation of lysozyme
in the presence of additives Figure 1 shows the thermal inactivation and aggregation of lysozyme at pH 7.1 In the absence of any additives, lysozyme was inactivated and aggregated 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, showing that the higher-order processes of aggregates can be described by single-exponential equations [26,27] In the presence of
100 mMArg, 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 aggregation under several different conditions are shown in Table 1 The rates of inactivation and aggregation depend on the protein
Fig 1 Thermal inactivation and aggregation of lysozyme in the presence of additives The samples containing 1.0 mgÆmL)1lysozyme with no additive (A), 100 m M Arg (B), and 100 m M ArgEE (C) at pH 7.1 were heated at 98 C for the times shown After the heat treatment, the residual activity (s) and the amount of aggregate calculated by the concentration of soluble protein (d) were determined and plotted The continuous and broken lines show the theoretical curves fitted to the closed and open circles with single exponential equations.
Trang 3concentration, indicating that intermolecular interaction is
the rate-limiting step in both inactivation and aggregation
of lysozyme by heat treatment As the data points were
well fitted to the single-exponential equation, 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, two (or several) protein molecules transform
the aggregation-prone unfolded molecules to the
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 thermal inactivation and aggregation of lysozyme
[18] These data show that ArgEE 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 98C for
10 min, 1.0 mgÆmL)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 the 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 same heat treatment, the sigmoidal activity curve was slightly improved by the addition of 100 mMArg (Fig 2A, h) On the other hand, the sigmoidal activity curve was clearly shifted to alkaline pH by the addition of
100 mMArgEE (Fig 2A, n) For example, 80% or more of the enzymes retained the active form after heat treatment
at 98C for 10 min in the presence of 100 mM 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 prevents heat-induced aggregation (Fig 2B) as well as inactivation (Fig 2A) After heat treatment at 98C for 10 min, the amount of aggregates increased with increasing pH (Fig 2B, s) In the presence of 100 mM Arg, the profile was slightly improved However, in the presence of 100 mMArgEE, the profile was clearly shifted to alkaline pH (Fig 2B, n)
Interestingly, in the presence of 1.0MNaCl, the inacti-vation and aggregation curves in the presence of Arg are the same as those in the absence of additives (Fig 2C,D) This indicates that the 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 interaction between exposed hydrophobic regions, Arg may play a role in the prevention of intermolecular interactions due to electrostatic interactions
On the other hand, the inactivation and aggregation curves obtained with 100 mM ArgEE are clearly different in the presence of 1.0M NaCl; ArgEE prevents both thermal inactivation and aggregation at high pH (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 in Fig 1 The
inactivation and aggregation profiles were fitted to single exponential
equations and the apparent rate constants were calculated nd, No data
due to the slow rate of aggregation under the conditions used.
Protein concentration Additive
Inactivation (· 10)3Æs)1)
Aggregation (· 10)3Æs)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)1lysozyme and 0 M (A,B) or 1.0 M (C,D) NaCl with 100 m M additives at various pHs were prepared The additives are none (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.
Trang 4data 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 monitored by DSC In the absence of additives,
the Tmvalue of lysozyme was 78.1C at pH 6.5 The Tm
increased as the concentration of ArgEE increased from 0 to
60 mM The maximum Tmof lysozyme is 79.8C at 60 mM
ArgEE The increase in Tmmay correspond to the increase
in solubility of the aggregation-prone molecules caused by
the addition of ArgEE With further increases in the
concentration of ArgEE, the Tm decreased from 100 to
600 mM The decreasing Tmcorresponds to the unfolding
effect of ArgEE on lysozyme Figure 3B summarizes the Tm
of lysozyme in the presence of Arg and ArgEE Unlike with
ArgEE, the Tmdid not change with increasing
concentra-tions of Arg
Figure 4 shows thermal unfolding curves of RNaseA
(Fig 4A) and myoglobin (Fig 4B) in the presence or
absence of additives as monitored by near-UV CD The
Tm value of RNaseA in the absence of additives was
64.7 ± 0.1C In the presence of 500 mM GdnHCl and
Arg, Tm values of RNaseA were 59.6 ± 0.1C and
60.8 ± 0.2C, respectively, which were 5.1 C and 3.9 C
lower than those obtained without the additives The Tmof
RNaseA with 500 mMArgEE (56.1 ± 0.2C) was 8.6 C
lower than without additives Similarly, the Tmof
myoglo-bin in the presence of 500 mMArgEE (58.3 ± 0.4C) was
clearly lower than in the presence of 500 mM Arg
(74.2 ± 0.4C) These data, being consistent with the
DSC analyses of lysozyme, suggest that ArgEE has a
destabilizing effect on proteins
The thermal unfolding curves of myoglobin in the
absence of additive 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 Tm
Charged states of Arg and ArgEE
In order to understand the importance of amphiphilicity, titration curves of Arg and ArgEE were compared (Fig 5A) The pKa of amino groups on Arg and ArgEE were determined as pH 9.2 and 7.4, respectively The decreased pKaof 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 at 98C 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 With increasing concentration of ArgEE, the amount of aggre-gates steeply decreased from 0 to 30 mM The addition of
30 mMArgEE 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 curves of lysozyme in the presence of additives
monitored by DSC The samples containing 4.0 mgÆmL)1lysozyme
with various concentrations 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) or ArgEE (s).
Fig 4 Thermal unfolding curves of proteins in the presence of additives monitored by near-UV CD The samples containing 4.0 mgÆmL)1 RNaseA (A) or myoglobin (B) in the presence or absence of 500 m M
additive at pH 6.5 were measured by CD at 280 nm The additives are
no additive (s), GdnHCl (h), Arg (n), and ArgEE (·) The data were fitted to conventional two-state equations.
Fig 5 Differences in the chemical properties of Arg and ArgEE (A) Determination of pK a values of amino groups on Arg and ArgEE A small quantity of 1.0 M NaOH was added to 10 mL of 1.0 M Arg/HCl (s) or ArgEE-2HCl (h) solution (B) Heat-induced aggregation of lysozyme in the presence of Arg (circles) or ArgEE (squares) at pH 6.5 (open symbols) or pH 10.0 (closed symbols) The samples containing 0.2 mgÆmL)1lysozyme with additives at pH 6.5 or 10.0 were heated 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.
Trang 5prominent effect of ArgEE was observed when monitoring
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 that
denaturing reagents, such as GdnHCl and urea, increase the
solubility of aggregate-prone molecules, leading to
improve-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 most widely used
additives for the prevention of protein aggregation and
improvement of refolding 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 acids [15] and (b) Arg does not
stabilize proteins against heat treatment [16] In addition,
this paper shows that (c) Arg prevents heat-induced
aggregation by an electrostatic 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 as additive because it is an Arg derivative that
possesses guanidium group on its side chain Although we
have examined several Arg derivatives, only ArgEE shows a
strong effect in preventing 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 lysozyme by the
introduced hydrophobic end on the carboxyl group, 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 by
heat treatment at neutral 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], and
ovalbumin [31], although the process of aggregation is
expected to be second- or higher-order kinetics [8,32–34]
This is because the rate-limiting step of thermal
aggre-gation is the nucleation with growth of aggregates,
leading to pseudo-first-order kinetics after a lag period
of 200 s The presence of the lag phase observed in
both inactivation and aggregation results from the
structural change to aggregation-prone molecules The
aggregation-prone molecules must 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 an additive to prevent protein aggregation for several decades, 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 Education, Science, Sports and Culture
of Japan (14350433, 14045229) and The Japan Securities Scholarship Foundation.
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