In the presence of additives, including arginine and guanidine 100 mM, more than 30% of 0.2 mgÆmL1 lysozyme in sodium phosphate buffer pH 6.5 formed insoluble aggre-gates by heat treatmen
Trang 1Prevention of thermal inactivation and aggregation of lysozyme
by polyamines
Motonori Kudou1, Kentaro Shiraki1, Shinsuke Fujiwara2, Tadayuki Imanaka3and Masahiro Takagi1 1
School of Materials Science, Japan Advanced Institute of Science and Technology, Ishikawa, Japan;2Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, Hyogo, Japan;3Department 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 mM), 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)
presence of 50 mMspermine 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 mM arginine and guanidine, while it was maintained at 50% in the presence
of 100 mM 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
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, NH2(CH2)4NH2; spermidine, NH2 (CH2)3NH(CH2)4NH2; spermine, NH2(CH2)3NH(CH2)4 NH(CH2)3NH2] 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: kshiraki@jaist.ac.jp
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)
Trang 2assay 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 mM sodium-phosphate buffer (pH 6.5) and different
concentrations of additives, were prepared All stock
solutions for additives and protein were dissolved in
50 mMsodium-phosphate buffer (pH 6.5)
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)1per mgÆmL)1
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 mM sodium-phosphate
buffer (pH 6.5) was mixed with 20 lL of the heat-treated
samples containing 0.2 mgÆmL)1 lysozyme and 100 mM
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 mM sodium-phosphate buffer (pH 6.5) or sodium-acetate buffer (pH 4.4) together with 100 mM additive Solutions con-taining additives were dialysed to adjust their pH to 6.5
or 4.4 The enthalpy change (DH), heat capacity change (DCp), and midpoint temperature of thermal unfolding (Tm) were determined by a conventional method, as described previously [22]
Results
Heat-induced aggregation of lysozyme Hen egg white lysozyme (pI¼ 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)1lysozyme, when heated to 98°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)1lysozyme (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).
Trang 3decreased with increasing concentrations of Arg or
guanidine from 0 to 0.5M (Fig 1A) In contrast,
50 mM spermidine and spermine completely prevented
the thermal aggregation of lysozyme (Fig 1B) The
aggregation curve for putrescine, the smallest polyamine
used in this study,
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 mMof 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
with spermine, no aggregates were observed, even after heat
treatment at 98°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 mM 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 mM sodium-phosphate buffer (pH 6.5) containing 100 mMadditive, were heated at 98°C for 30 min and then cooled from 98°C to 50 °C by using a 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)1lysozyme (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).
Trang 4Heat 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)1lysozyme (black circles in Fig 4A), were resolved by the addition of guanidine/HCl (to a final concentration of 4.0M)
These samples were diluted 10-fold by 50 mM 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 °C to 50 °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.
Trang 5unresolved 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 mM putrescine, the inactivation
curves of 0.2 and 1.0 mgÆmL)1lysozyme 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 mM 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 mM spermidine and
spermine, the inactivation curve of 1.0 mgÆmL)1lysozyme
was identical to that of 0.2 mgÆmL)1lysozyme 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)1lysozyme in the absence
of additives In the presence of 100 mMArg, the inactivation
rate constants for 0.2 and 1.0 mgÆmL)1lysozyme 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)1lysozyme, were
recovered by the addition of 100 mM spermidine and
spermine, respectively On the other hand, the residual
activity was < 5%, for most of the other additives (even
100 mMArg),
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 mM 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 (DCp) are listed only
at pH 4.4
At pH 6.5, the Tmof lysozyme was 77.3°C in the absence
of additives The addition of 100 mMpolyamines and Arg slightly increased the Tmof lysozyme by 1 °C and 0.5 °C, respectively, whereas addition of other additives did not alter the values compared with no additive
Tm values at neutral pH are responsible for preventing irreversible aggregation during DSC measurement At
pH 4.4, the presence of polyamines slightly decreased the
Tmby
15 1.9–2.7°C Gly exhibited the best results, judging by the increased Tm The decreased Tmvalue, as conferred by polyamines, implies that they bind to the unfolded mole-cules DH and DCpvalues 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 mMadditive 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
a
Residual activities of 0.2 mgÆmL)1lysozyme containing 100 m M
additive (pH 6.5) after heat treatment at 98 °C for 30 min.
b
Residual activities of 1.0 mgÆmL)1lysozyme 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, ,.
Trang 6The 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 mMspermidine
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:
where, U represents the unfolded molecule that can
refold after heat treatment, A represents the irreversibly
denatured molecule and An 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
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
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 pKa 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
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)1lysozyme 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
a 50-m M sodium-phosphate buffer (pH 6.5); b 50 m M sodium-acetate buffer (pH 4.4).
Trang 7a 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
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
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.
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