International Journal of Biological Macromolecules 87 (2016) 563569

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Charge state of arginine as an additive on heat-induced protein

aggregation.
Takumi Miyatake a , Shunsuke Yoshizawa a , Tsutomu Arakawa b , Kentaro Shiraki a,
a
b

Institute of Applied Physics, University of Tsukuba, Tsukuba, Ibaraki 305-8573, Japan

Alliance Protein Laboratories, San Diego, CA 92121, United States

a r t i c l e

i n f o

Article history:
Received 29 January 2016
Received in revised form 8 March 2016
Accepted 9 March 2016
Available online 14 March 2016
Keywords:
Arginine
Charge state
Protein aggregation
Solvent additive
Lysozyme

a b s t r a c t
Arginine (Arg) is one of the most versatile solvent additives, such as suppressing protein aggregation,
increasing solubility of small aromatic compounds and peptides, and preventing protein binding on
solid surfaces. In this study, we investigated the role of the charged state of -amino group of Arg for
the prevention of protein aggregation. As expected, Arg effectively suppressed thermal aggregation of
hen egg-white lysozyme at neutral pH, whereas the suppression effect diminished at and above pH
9.0, which corresponds to the pK of Args -amino group. The pH dependence of Arg as an aggregation
suppressor was conrmed by additional experiments with neutral proteins, bovine hemoglobin and
bovine -globulin. Interestingly, N-acetylated arginine, which lacks the -amino group, showed a weaker
suppressive effect on protein aggregation than Arg, even at neutral pH. These results indicate that both
positively charged -amino group and guanidinium group play important roles in suppressing heatinduced protein aggregation by Arg. The elucidated limitation of Arg at alkaline pH provides new insight
in the application as well as the mechanism of Arg as a solvent additive.
2016 Elsevier B.V. All rights reserved.

1. Introduction
Arginine (Arg) is one of the most commonly used additives for
preventing protein aggregation without altering or destabilizing
the tertiary structure of the protein [13]. Since Arg was found to
be a chemical chaperone, as reported by Buchner and Rudolph [4],
various applications of Arg as a solvent additive have been reported,
including the aggregation suppression of chemically reduced [5,6]
and thermally denatured [68] proteins, reduction of the viscosity of concentrated protein solution [9,10], solubilization of poorly
soluble compounds [1113], and improvement of column chromatography performance [14,15]. To date, Arg is one of the most
versatile additives used to improve the stability and solubility of
proteins, peptides, and chemicals [16].
What is the mechanism behind these effects of Arg? The main
driving force has been considered to be the interactions of the
guanidinium group of Arg with aromatic or charged residues
through cation- interactions [1719]. In addition, it was reported
that the aliphatic moiety of the Arg side chain interacts with the
aromatic ring of 4-mercaptoethylpyridine [20]. Thus, multimodal
interactions seem to occur between Arg and the target solutes.

Corresponding author.
E-mail address: shiraki@bk.tsukuba.ac.jp (K. Shiraki).
http://dx.doi.org/10.1016/j.ijbiomac.2016.03.015
0141-8130/ 2016 Elsevier B.V. All rights reserved.

Moreover, we have previously reported that both the guanidinium

and -amino groups of Arg play an indispensable role in protein stabilization [21]. The depletion of the negative charge by the chemical
modication of the carboxyl group improved the effect of Arg in stabilizing against the thermal aggregation of proteins [2224]. In this
study, we focused on the role that the -amino group of Arg plays
in suppressing protein aggregation. The results presented here are
the rst reported about the effects of the -amino group of Arg on
protein aggregation.

2. Materials and method

2.1. Materials
Ultra-pure water was obtained from a Milli-Q SP TOC
system water purier (Millipore Corp., Bedford, Mass., USA). larginine (Arg base), l-arginine hydrochloride (ArgHCl), l-lysine
hydrochloride (Lys), glycine (Gly), guanidine hydrochloride (Gdn),
hydrochloric acid (HCl), and sodium hydroxide (NaOH) were
obtained from Wako Pure Chemical Industries, Ltd. (Osaka,
Japan). N-acetyl-l-arginine (AcArg), hen egg-white lysozyme,
hemoglobin from bovine blood, and -globulin from bovine blood
were obtained from SigmaAldrich Corp. (St Louis, Mo., USA).
Sodium chloride (NaCl) and sodium phosphate were obtained from

564

T. Miyatake et al. / International Journal of Biological Macromolecules 87 (2016) 563569

Nacalai Tesque Inc. (Tokyo, Japan). All chemicals used were of

reagent grade and used as received.
2.2. Turbidity measurements
The heat-induced aggregation of proteins was measured as
follows. Stock protein solutions containing 1.0 mg/ml protein in
50 mM sodium phosphate, 50 mM glycine, and specied amounts
of additives were prepared and adjusted to pH 7.010.0 by the
addition of NaOH or HCl. A 2 mL aliquot of the stock solution was
introduced into a 1 cm path-length glass cell and incubated at indicated temperatures using a Jasco temperature controller ETC-550T
(Japan Spectroscopic Co., Ltd., Tokyo, Japan). For lysozyme experiments, the sample solutions were incubated at 90 C and the light
scattering intensity at 400 nm was monitored using a Jasco spectrophotometer model V-550 (Japan Spectroscopic Co., Ltd., Tokyo,
Japan). For hemoglobin, the experimental conditions were modied to 70 C and 500 nm; note that hemoglobin has an absorbance
peak at 400 nm.
2.3. Analysis of protein aggregation kinetics curves
To analyze the protein aggregation kinetics curves measured by
the increase in light scattering intensity (I) as a function of time (t)
and characterize the effect of Arg on protein aggregation, the lag
period was calculated by tting the section of the sharply increase
of light scattering intensity. We use the Eq. (1) as below,
I = I0 + v(t t0 )2 ,

(1)

where I0 is the initial value of the light scattering intensity, t0 is

the length of the lag period, and v is a parameter characterizing
the initial rate of aggregation. The empirical Eq. (1) was proposed
after the inspection of the initial parts of the kinetic curves [25].
When the additive suppresses protein aggregation, the lag period
t0 will become longer than that in the absence of an additive. In this
study, we investigated the length of the lag period to evaluate the
suppression effect of the additives.
2.4. Circular dichroism spectroscopy
Circular dichroism (CD) spectroscopy was performed using a
Jasco CD spectropolarimeter model J-720W with a Peltier cell
holder model PTC-348W. Heat-induced unfolding was monitored
at 288.5 nm with a heating rate of 1 C/min using a 1 cm path-length
cell. The native lysozyme was solubilized in 50 mM glycine and
50 mM sodium phosphate buffer at pH 8.0 or 9.5 containing 200 mM
additive. The nal protein concentration was adjusted to 0.1 mg/ml.
3. Results
We investigated the effects of ArgHCl on heat-induced protein
aggregation as a function of pH in the range 7.010.0, which causes
the charge state of Arg to vary. To the best of our knowledge,
lysozyme is the best model protein for this experiment, because
lysozyme aggregates under a broad range of pH, from neutral to
alkaline, owing to its high isoelectric point of 11.0 [26]. Fig. 1
shows representative light scattering intensity kinetic data for a
1.0 mg/ml lysozyme solution incubated at 90 C in the absence
or presence of ArgHCl at various pH. Fig. 1A shows the results
at pH 7.0. In the absence of ArgHCl (solid line), turbidity sharply
increased with incubation time, with nearly no lag period. When
100 mM ArgHCl was added, the turbidity increase was still sharp
(dotted line) but with a slightly increased lag period. Upon further
increasing ArgHCl concentration to 500 mM (broken line), the lag
period greatly increased and turbidity increase became less sharp.

At 1000 mM ArgHCl (broken and dotted line), the lag period drastically increased and turbidity increase was much shallower. Fig. 1B
shows the results at pH 8.0. The lag period was short and the turbidity increase was sharp in the absence of ArgHCl (solid line), similar
to the results obtained at pH 7.0. A similar trend to the results at
pH 7.0 was observed at pH 8.0 in the presence of ArgHCl. Increasing ArgHCl concentration resulted in a longer lag period and a
shallower turbidity increase. These results clearly demonstrate the
suppressive effects of Arg on the thermal aggregation of lysozyme
at these pH. The results at pH 9.0 (Fig. 1C) and pH 10.0 (Fig. 1D) are in
sharp contrast to those at pH 7.0 and 8.0. The turbidity increase was
sharp regardless of the ArgHCl concentration. Only a small increase
at pH 9.0 and a negligible increase at pH 10.0 were observed in
the lag period. Thus, Arg exhibited little suppression of the heatinduced aggregation of lysozyme at pH 9.0 and 10.0. The lag periods
were determined by tting the data in Fig. 1 to Eq. (1) and are plotted versus pH in Fig. 2A for different ArgHCl concentrations. The
lag periods thus determined were 214, 112, 79, and 30 s at pH 7.0,
8.0, 9.0, and 10.0, respectively, slowly decreasing with increasing
pH (closed circles). Fig. 2A also shows the pH dependence at different ArgHCl concentrations. At any ArgHCl concentration, the lag
periods decreased with increasing pH, demonstrating that higher
pH accelerates aggregation regardless of ArgHCl concentration. It is
evident in Fig. 2A that a strong ArgHCl concentration dependence in
the lag period at pH 7.0 and 8.0 exists, but such dependence appears
to diminish at pH 9.0 and 10.0. All data points at different ArgHCl
concentrations converge into each other. Such an ArgHCl concentration dependence in the lag period is more clearly seen in Fig. 2B,
wherein the relative lag period is plotted against ArgHCl concentration. At pH 7.0 (closed circles), the lag period sharply increased
with ArgHCl concentration, as is the case at pH 8.0 (closed squares).
Such ArgHCl concentration dependence is weak at pH 9.0 (open
circles) and 10.0 (open squares). The small increase in relative lag
period at pH 9.0 and 10.0 may be due to the suppressive effects of
the guanidinium group on aggregation. Arg suppressed the protein
aggregation at pH 7.0 and 8.0, but it did not at pH 9.0 and 10.0.
Arg has three functional groups, an -carboxyl group (pK 1.8), a
guanidinium group (pK 12.5), and an -amino group (pK 9.0) [27].
Thus, the pH-dependence data show that the charge state of the
-amino group of Arg plays an important role in the inhibition of
protein aggregation.
We examined the lag period of the thermal aggregation of
lysozyme in the presence of 200 mM NaCl, Gly, Lys, Gdn, and ArgHCl
at different pH; note that 200 mM ArgHCl exhibited strong suppressive effects at pH 7.0 and 8.0 (see Fig. 2B). Fig. 3A shows the
results at pH 8.0. It is evident that NaCl is ineffective and Gly, Lys,
and Gdn are effective but to a much lesser extent than ArgHCl.
NaCl increases the ionic strength of the solution, thus indicating
that higher ionic strength neither suppresses nor enhances aggregation. Gly is dipolar at pH 8.0 and thereby may weakly suppress
aggregation by dipoledipole interaction with lysozyme. Lys is positively charged at pH 8.0, indicating a contribution of net positive
charge to aggregation suppression, which is weaker than ArgHCl.
The data for Gdn are similar to those of Gly, perhaps owing to the
200 mM concentration being too low to be effective; the effects
of Gdn as a protein denaturant normally occur above 2 M. Interestingly, the effects of additives at 200 mM concentration are far
weaker at pH 9.5 (Fig. 3B), as seen with ArgHCl at pH 9.0 and 10.0
(Fig. 2). NaCl showed no effects at pH 9.5 as well as at pH 8.0,
and Gly and Lys were marginally effective. Both Gdn and ArgHCl
showed small suppressive effects with identical magnitude, suggesting that their guanidinium groups play a major role at pH 9.5,
where the contribution of the positively charged -amino group of
Arg diminishes, as indicated earlier in Fig. 2B. These results suggest
that both the guanidinium and positively charged -amino groups
of Arg play an important role in suppressing protein aggregation.

T. Miyatake et al. / International Journal of Biological Macromolecules 87 (2016) 563569

565

Fig. 1. Representative data for the thermal aggregation of lysozyme with the ArgHCl concentrations of 0 mM (solid line), 100 mM (dotted line), 500 mM (broken line), and
1000 mM (broken and dotted line). Samples containing 1.0 mg/ml lysozyme, 50 mM Gly and 50 mM sodium phosphate buffer were prepared at pH 7.0 (A), pH 8.0 (B), pH 9.0
(C), and pH 10.0 (D) and heated at 90 C.

Fig. 2. Lag periods calculated from Fig. 1 using Eq. (1). Samples containing 1.0 mg/ml lysozyme and 50 mM Gly and 50 mM sodium phosphate buffer were prepared at pH
7.010.0 and heated at 90 C. (A) Lag periods of samples in the absence (closed circles) or presence of ArgHCl: 25 mM (closed squares), 50 mM (closed triangles), 100 mM
(closed downward-pointing triangles), 200 mM (open circles), 500 mM (open squares), and 1000 mM (open triangles). (B) Relative lag periods calculated from Fig. 2A at pH
7.0 (closed circles), pH 8.0 (closed squares), pH 9.0 (open circles), and pH 10.0 (open squares). (C) Relative lag periods of samples containing Arg base (closed circles) or ArgHCl
(open circles) at pH 10.0.

566

T. Miyatake et al. / International Journal of Biological Macromolecules 87 (2016) 563569

Fig. 3. Lag periods for the thermal aggregation of lysozyme in the absence and presence of additives. Samples containing 1.0 mg/ml lysozyme, 50 mM Gly and 50 mM
sodium phosphate buffer, and 200 mM additive (NaCl, Gly, Lys, Gdn, or ArgHCl) at
pH 8.0 (A) or pH 9.5 (B) were heated at 90 C. Data are the mean of three independent
experiments and error bars represent one standard deviation.

Although 200 mM NaCl showed no apparent effects at both pH

8.0 and 9.5, there will be 725 mM NaCl in 1000 mM ArgHCl at pH
10.0, which is required to increase the pH of 1000 mM Arg solution
made with ArgHCl. We have prepared Arg solution in the same
buffer using an Arg base so that no extra NaCl is generated. Fig. 2C
compares the relative lag periods of Arg made with ArgHCl (open
circles) and Arg base (closed circles). A clear difference exists in
the relative lag period above 500 mM Arg, with the value being
signicantly higher in the absence of NaCl. It thus appears that NaCl
inhibits the effectiveness of Arg in suppressing protein aggregation.
As described above, ArgHCl strongly suppressed the heatinduced aggregation of lysozyme at pH 8.0 but not above pH 9.0.
The observed suppression may be in part due to the stabilization
of the protein structure by ArgHCl. Fig. 4 shows the change in the
CD signal at 288.5 nm, which follows the unfolding of the tertiary
structure. The thermal denaturation pattern is highly similar in the
absence and presence of 200 mM ArgHCl at pH 8.0 (Fig. 4A). No signicant difference in the mid-point temperature (Tm ) of thermal
unfolding was observed with or without ArgHCl at 72 C. Similarly, no effect of 200 mM ArgHCl was observed at pH 9.5 (Fig. 4B). In
addition, little pH-dependence of Tm exists at pH 8.0 and 9.5. These
results indicate that ArgHCl does not affect the thermal stability of
lysozyme.
The loss of the aggregation suppression of Arg above pH 9.0 was
ascribed to titration of its -amino group. Thus, we further investigated the suppressive effect of acetylarginine (AcArg), which lacks
a positive charge on its -amino group even at pH 8.0 (Fig. 5A).

Fig. 5B shows a comparison of 200 mM ArgHCl and AcArg. At pH

7.0, AcArg signicantly increased the lag period, suggesting that
the guanidinium group plays an important role. However, its effect
is weaker than ArgHCl, indicating that the elimination of the Nterminal charge reduces the suppressive effects of Arg. A similar
trend was observed at pH 8.0. At pH 9.0, no difference between
ArgHCl and AcArg was seen, consistent with there being no terminal charge for both additives. Interestingly, at pH 10.0, AcArg
appears to be more effective. In the absence of a charged -amino
group, the more hydrophobic AcArg may bind more strongly to
heat-denatured lysozyme and suppress its aggregation, suggesting
that hydrophobic interactions play a role in additive binding and
lysozyme aggregation.
The loss of ArgHCl effects at a higher pH may be somewhat
biased, as higher pH also enhances the aggregation of lysozyme
(Fig. 2A) owing to its high pI. Specically, aggregation is too strong
to observe any of the suppression effects of the additives. Thus,
we tested hemoglobin, which has a pI of 6.8 [28] and thereby
may be less aggregating at higher pH. Fig. 6A shows the lag
period of hemoglobin incubated at 70 C at different pH. The lag
period increased slightly, indicating that aggregation is slower, as
expected from the increase in net charge at higher pH. The small
increase of lag period from pH 79 might be due to destabilization of the tetrameric structure by higher pH, leading to enhance
aggregation. ArgHCl greatly increased the lag period, depending on
concentration, increasing from 50 to 200 s for 1000 mM ArgHCl
at pH 7.0, similarly to the heat-induced aggregation of lysozyme at
the same pH. An identical trend was observed at pH 8.0. At pH 9.0
and 10.0, the suppressive effects largely diminished, as seen with
lysozyme. A similar trend was observed for -globulin (Fig. 6B),
which contains a broad range of pI, although the suppressive effects
of ArgHCl are not as dramatic as with lysozyme and hemoglobin.
Nevertheless, it should be emphasized that ArgHCl lost its effectiveness at pH 9.0. These results support the above data, thereby
indicating that the reduction of the aggregation suppressing effect
of ArgHCl at alkaline pH is independent of the type of protein.
4. Discussion
Arg is currently extensively used in pharmaceutical industries.
Its effects on proteins are primarily mediated through the suppression of proteinprotein [3,29] or proteinsurface [30] interactions.
Arg has been shown to strongly suppress thermal aggregation of
proteins. This is not due to the stabilization of the protein structure
against thermal denaturation. Arg neither stabilizes nor destabilizes the protein structure, as has been experimentally shown
before [13], and has also been shown here for lysozyme (Fig. 4).
The weak interactions of Arg with folding or unfolding intermedi-

Fig. 4. Thermal transitions curve of native lysozyme monitored by CD at 288.5 nm. The samples containing 0.1 mg/mL lysozyme and 50 mM Gly and 50 mM sodium phosphate
buffer at pH 8.0 (A) or pH 9.5 (B) were heated with a heating rate of 1 C/min in the absence (closed circles) or presence of ArgHCl (open squares). The continuous curves
were calculated using the two-state thermal unfolding equation.

T. Miyatake et al. / International Journal of Biological Macromolecules 87 (2016) 563569

567

Fig. 5. (A) Chemical structures of ArgHCl and AcArg. (B) Lag period for thermal aggregation of lysozyme in the presence of ArgHCl or AcArg. Samples containing 1.0 mg/ml
lysozyme, 50 mM Gly and 50 mM sodium phosphate buffer, and 200 mM additive at pH 7.010.0 were heated at 90 C.

Fig. 6. Lag periods for aggregation of hemoglobin (A) and -globulin (B) from bovine
blood in the absence and presence of ArgHCl. Samples containing 1.0 mg/ml protein,
50 mM Gly and 50 mM sodium phosphate buffer, and 01000 mM ArgHCl at pH
7.010.0 were heated at 70 C (A) or 80 C (B).

ates have been implicated as one of the mechanisms by which Arg

suppresses protein aggregation. However, the detailed mechanism
of aggregation suppression by Arg still remains to be elucidated.
The guanidinium group of Arg plays a key role in weak interactions between Arg and proteins [1719]. Here, we have attempted
to investigate the role that the -amino group of Arg plays in suppressing protein aggregation. It is evident in this study that the
suppressive effects of Arg diminish at higher pH, where the amino group is deprotonated and hence loses its positive charge.
Such pH dependence has been observed by Hamada et al. [5] and
Shiraki et al. [22]. What is the mechanism of the loss of the aggregation suppression by Arg at higher pH? Two possibilities may be
considered: the loss of weak binding or loss of effectiveness of
bound Arg molecules. The effects of Arg on thermal stability were
identical at pH 8.0 and 9.5, i.e., no effects (Fig. 4), consistent with
previous observations [5,22], whereas Arg strongly suppressed the
heat-induced aggregation of lysozyme and two other proteins at pH
8.0. This means that no change in Arg binding upon thermal unfolding exists. Here, we hypothesized identical Arg binding both to the
native and thermally unfolded proteins through cation- interaction between guanidinium group of Arg and aromatic rings of
protein surface, as schematically depicted in Fig. 7. Proteins unfold
upon heating and subsequently aggregate in the absence of Arg.
Arg is shown to bind to the native protein, which is most likely
to be associated with the suppression of the colloidal aggregation
of native proteins at high protein concentrations. Arg also binds
to the unfolded protein (or unfolding intermediates) equally well
[31]. One question is that can Arg also bind to the unfolded protein
at higher pH? Although no direct evidence exists, we assume that
binding occurs at higher pH as well, based on the similar effects

of Arg on the thermal stability of lysozyme at pH 9.5 (Fig. 4). Furthermore, Tomita et al. reported that Arg transformed the limiting
regime of the aggregation of lysozyme at pH 10 [32], which supports our hypothesis. Based on the above assumption, the observed
difference in the effects of Arg between pH 8.0 and above pH 9.0 is
ascribed to the effectiveness of bound Arg molecules.
At pH 7.0 and 8.0, the binding of Arg leads to an increase in the
apparent net charge of the unfolded molecules owing to the contribution of the positive charge on Arg. The increased net charge
would effectively increase the electrostatic repulsion between the
aggregation-prone unfolded molecules and the reduced aggregation (Fig. 7). While this may be true for a positively charged
lysozyme, this mechanism is not, however, fully applicable to
hemoglobin, which should have a net negative charge. The binding of positively charged Arg reduces the net negative charge.
This may be explained by more Arg binding to oppositely charged
hemoglobin, leading to electrostatic repulsion. In fact, more binding of Arg is observed for BSA, which has a net negative charge
[16,33]. Thus, similar phenomenon is likely to occur in the case
of hemoglobin. This mechanism is consistent with the effect of
NaCl, which suppressed the effects of Arg on protein aggregation,
as reported before [22,34] and also shown in Fig. 2C. NaCl screens
the electrostatic repulsion. More importantly, this mechanism can
explain the suppression of the effects of Arg at higher pH. Arg loses
its net positive charge above pH 9.0 and hence charge repulsion
owing to the bound Arg molecules. Alternative contributions, such
as hydration, are also possible with pH dependent change [35].
Non-charged Arg may also be more hydrophobic and hence its
binding may make the protein more aggregation-prone state. This
study proposes the general structure of additives as aggregation
suppressors, which is composed of both binding and functional (e.g.
charged) regions.
Residual suppression of Arg (Fig. 2B) at higher pH may be due
to its guanidinum group. This molecular mechanism implies the
modulation of the effectiveness of Arg by the chemical modication of charged groups. As shown in Fig. 5, the AcArg showed a
weaker suppressive effect on the thermal aggregation of lysozyme
than Arg. On the contrary, Arg chemically modied at its -carboxyl
group was found to be superior aggregation suppressors, such as
arginine ethylester, arginine methylester [21,22,36], and arginine
amide [23]. Hamada et al. reported a positive relation between the
refolding yield owing to aggregation suppression and the positive
charge of Arg analogs [23]. These data are consistent with the idea
that the total net charge of additives plays an important role in
suppressing thermal aggregation. This mechanism can also explain

568

T. Miyatake et al. / International Journal of Biological Macromolecules 87 (2016) 563569

Fig. 7. Proposed molecular mechanism for the effect of Arg on protein aggregation at neutral pH or alkaline pH.

why the naturally occurring polyamines (putrescine, spermidine,

and spermine) are good aggregation suppressors [5,37].
Although various effective applications of Arg have been
reported, the limitations of Arg as a solvent additive are as follows.
(i) pH range: As mentioned above, Arg is not effective at alkaline
pH or in the presence of salts. This is related to the charged state of
the additive. (ii) Freezing: At alkaline pH, Arg does not prevent the
inactivation of -galactosidase during freezing and freezedrying
[38]. (iii) Concentration: Arg up to 1000 mM can show undesirable effects on protein structure: e.g., Arg-induced aggregation
[39], inactivation [40,41], and destabilization of oligomeric protein
[42,43]. Because of its effects on proteinprotein interactions, Arg
not only suppresses undesirable protein aggregation and association but also induces the dissociation of oligomers to monomeric
proteins.
Arg is effective at suppressing protein aggregation; however,
limitations exist as described above. To overcome such limitations, combination with other additives should be considered. For
example, poly(ethylene glycol) has a synergistic effect with Arg
on protein aggregation because of the inhibition of hydrophobic
interactions between the proteins [44]. These data indicate that different additives may compensate for the lost effectiveness of Arg
on protein aggregation. A similar idea has been reported where
the combination of Arg and Hofmeister salts more effectively prevented protein aggregation than Arg alone [45]. Kosmotropes with
Arg not only stabilized protein structure [42] but also promoted the
clusterization of Arg, leading together to enhanced the inhibition
of protein aggregation.
In conclusion, we have shown here that the charge state of the
-amino group of Arg plays an important role in suppressing the
thermal aggregation of lysozyme. Specically, the positive charge of
Arg is essential in preventing heat-induced protein aggregation and
should provide information useful for the development of additives

that suppress undesirable protein aggregation under a broad range

of conditions.

Acknowledgements
The authors are grateful for nancial support from University of Tsukuba and JSPS KAKENHI Grant Numbers 15H03583 and
15K13812.

References
[1] K. Shiraki, M. Kudou, S. Fujiwara, T. Imanaka, M. Takagi, Biophysical effect of
amino acids on the prevention of protein aggregation, J. Biochem. 132 (2002)
591595.
[2] T. Arakawa, K. Tsumoto, The effects of arginine on refolding of aggregated
proteins: not facilitate refolding but suppress aggregation, Biochem. Biophys.
Res. Commun. 304 (2003) 148152.
[3] T. Arakawa, K. Tsumoto, Y. Kita, B. Chang, D. Ejima, Biotechnology applications
of amino acids in protein purication and formulations, Amino Acids 33
(2007) 587605.
[4] J. Buchner, R. Rudolph, Renaturation, purication and characterization of
recombinant Fab-fragments produced in Escherichia coli, Nat. Biotechnol. 9
(1991) 157162.
[5] H. Hamada, R. Takahashi, T. Noguchi, K. Shiraki, Differences in the effects of
solution additives on heat- and refolding-induced aggregation, Biotechnol.
Prog. 24 (2008) 436443.
[6] E.M. Lyutova, A.S. Kasakov, B.Y. Gurvits, Effects of arginine on kinetics of
protein aggregation studied by dynamic laser light scattering and tubidimetry
techniques, Biotechnol. Prog. 23 (2007) 14111416.
[7] U. Das, G. Hariprasad, A.S. Ethayathulla, P. Manral, T.K. Das, S. Pasha, A. Mann,
M. Ganguli, A.K. Verma, R. Bhat, S.K. Chandrayan, S. Ahmed, S. Sharma, P. Kaur,
T.P. Singh, A. Srinivasan, Inhibition of protein aggregation: supramolecular
assemblies of arginine hold the key, PLoS One 2 (2007) e1176.
[8] D. Shukla, B.L. Trout, Interaction of arginine with proteins and the mechanism
by which it inhibits aggregation, J. Phys. Chem. B 114 (2010) 1342613438.
[9] N. Inoue, E. Takai, T. Arakawa, K. Shiraki, Arginine and lysine reduce the high
viscosity of serum albumin solutions for pharmaceutical injection, J. Biosci.
Bioeng. 117 (2014) 539543.

T. Miyatake et al. / International Journal of Biological Macromolecules 87 (2016) 563569

[10] N. Inoue, E. Takai, T. Arakawa, K. Shiraki, Specic decrease in solution
viscosity of antibodies by arginine for therapeutic formulations, Mol. Pharm.
11 (2014) 18891896.
[11] K. Tsumoto, M. Umetsu, I. Kumagai, D. Ejima, T. Arakawa, Solubilization of
active green uorescent protein from insoluble particles by guanidine and
arginine, Biochem. Biophys. Res. Commun. 312 (2003) 13831386.
[12] J. Li, M. Garg, D. Shah, R. Rajagopalan, Solubilization of aromatic and
hydrophobic moieties by arginine in aqueous solutions, J. Chem. Phys. 133
(2010) 054902.
[13] D. Shah, J. Li, A.R. Shaikh, R. Rajagopalan, Argininearomatic interactions and
their effects on arginine-induced solubilization of aromatic solutes and
suppression of protein aggregation, Biotechnol. Prog. 28 (2012) 223231.
[14] T. Arakawa, D. Ejima, K. Tsumoto, M. Ishibashi, T. Masao, Improved
performance of column chromatography by arginine: dye-afnity
chromatography, Protein Expr. Purif. 52 (2007) 410414.
[15] T. Arakawa, K. Tsumoto, K. Nagase, D. Ejima, The effects of arginine on protein
binding and elution in hydrophobic interaction and ion-exchange
chromatography, Protein Expr. Purif. 54 (2007) 110116.
[16] T. Arakawa, Y. Kita, Multi-faceted arginine: mechanism of the effects of
arginine on protein, Curr. Protein Pept. Sci. 15 (2014) 608620.
[17] M.M. Flocco, S.L. Mowbray, Planar stacking interactions of arginine and
aromatic side-chains in proteins, J. Mol. Biol. 235 (1994) 709717.
[18] J.L. Pellequer, B. Zhao, H.I. Kao, C.W. Bell, K. Li, Q.X. Li, A.E. Karu, V.A. Roberts,
Stabilization of bound polycyclic aromatic hydrocarbons by a pi-cation
interaction, J. Mol. Biol 302 (2000) 691699.
[19] L. Ito, K. Shiraki, T. Matsuura, M. Okumura, K. Hasegawa, S. Baba, H.
Yamaguchi, T. Kumasaka, High-resolution X-ray analysis reveals binding of
arginine to aromatic residues of lysozyme surface: implication of suppression
of protein aggregation by arginine, Protein Eng. Des. Sel. 24 (2011) 269274.
[20] A. Hirano, T. Maruyama, K. Shiraki, T. Arakawa, T. Kameda, Mechanism of
protein desorption from 4-mercaptoethylpyridine resins by arginine
solutions, J. Chromatogr. A 1373 (2014) 141148.
[21] T. Matsuoka, H. Hamada, K. Matsumoto, K. Shiraki, Indispensable structure of
solution additives to prevent inactivation of lysozyme for heating and
refolding, Biotechnol. Prog. 25 (2009) 15151524.
[22] K. Shiraki, M. Kudou, S. Nishikori, H. Kitagawa, T. Imanaka, M. Takagi, Arginine
ethylester prevents thermal inactivation and aggregation of lysozyme, Eur. J.
Biochem. 271 (2004) 32423247.
[23] H. Hamada, K. Shiraki, l-Argininamide improves the refolding more
effectively than l-arginine, J. Biotechnol. 130 (2007) 153160.
[24] K. Shiraki, S. Tomita, N. Inoue, Small amine molecules: solvent design toward
facile improvement of protein stability against aggregation and inactivation,
Curr. Pharm. Biotechnol. 17 (2016) 116125.
[25] B.I. Kurganov, Antiaggregation activity of chaperones and its quantication,
Biochemistry (Mosc). 78 (2013) 15541566.
[26] Y. Wang, C. Li, G.J. Pielak, Effects of proteins on protein diffusion, J. Am. Chem.
Soc. 132 (2010) 93929397.
[27] R.M.C. Dawson, D.C. Elliott, W.H. Elliott, K.M. Jones, Data for Biochemical
Research, 3rd ed., Oxford University Press, New York, 1986.
[28] W. Lei, Z. Meng, W. Zhang, L. Zhang, M. Xue, W. Wang, Induced t recognition
of proteins by surface imprinted silica with soft recognition sites, Talanta 99
(2012) 966971.

569

[29] B.M. Baynes, D.I.C. Wang, B.L. Trout, Role of arginine in the stabilization of
proteins against aggregation, Biochemistry 44 (2005) 49194925.
[30] Y. Shikiya, S. Tomita, T. Arakawa, K. Shiraki, Arginine inhibits adsorption of
proteins on polystyrene surface, PLoS One 8 (2013) 39.
[31] K.R.C. Reddy, H. Lilie, R. Rudolph, C. Lange, l-Arginine increases the solubility
of unfolded species of hen egg white lysozyme, Protein Sci. 14 (2005)
929935.
[32] S. Tomita, H. Yoshikawa, K. Shiraki, Arginine controls heat-induced
clustercluster aggregation lysozyme at around the isoelectric point,
Biopolymer 95 (2011) 695701.
[33] C.P. Schneider, B.L. Trout, Investigation of cosolute-protein preferential
interaction coefcients: new insight into the mechanism by which arginine
inhibits aggregation, J. Phys. Chem. B 113 (2009) 20502058.
[34] M.M. Nuhu, R. Curtis, Arginine dipeptides affect insulin aggregation in a pHand ionic strength-dependent manner, Biotechnol. J. 10 (2015) 404416.
[35] A.C. Dumetz, A.M. Chockla, E.W. Kaler, A.M. Lenhoff, Effects of pH on
proteinprotein interactions and implications for protein phase behavior,
Biochemica et Biophysica Acta 1784 (2008) 600610.
[36] M.T. Gao, X.Y. Dong, Y. Sun, Interactions between l-arginine/l-arginine
derivatives and lysozyme and implications to their inhibition effects on
protein aggregation, Biotechnol. Prog. 29 (2013) 13161324.
[37] M. Kudou, K. Shiraki, S. Fujiwara, T. Imanaka, M. Takagi, Prevention of thermal
inactivation and aggregation of lysozyme by polyamines, Eur. J. Biochem. 270
(2003) 45474554.
[38] K. Izutsu, S. Yoshioka, Y. Takeda, The effects of additives on the stability of
freeze-dried -galactosidase stored at elevated temperature, Int. J. Pharm. 71
(1991) 137146.
[39] T.B. Eronina, N.A. Chebotareva, N.N. Sluchanko, V.V. Mikhaylova, V.F.
Makeeva, S.G. Roman, S.Y. Kleymenov, B.I. Kurganov, Dual effect of arginine
on aggregation of phosphorylase kinase, Int. J. Biol. Macromol 68 (2014)
225232.
[40] D. Lu, Z. Liu, M. Zhang, Z. Liu, H. Zhou, The mechanism of PNIPAAm-assisted
refolding of lysozyme denatured by urea, Biochem. Eng. J. 24 (2005) 5564.
[41] M. Ishibashi, K. Tsumoto, M. Tokunaga, D. Ejima, Y. Kita, T. Arakawa, Is
arginine a protein-denaturant? Protein Expr. Purif. 42 (2005) 16.
[42] A. Fujimoto, A. Hirano, K. Shiraki, Ternary system of solution additives with
arginine and salt for refolding of beta-galactosidase, Protein J. 29 (2010)
161166.
[43] V. Vagenende, A.X. Han, M. Mueller, B.L. Trout, Protein-associated cation
clusters in aqueous arginine solutions and their effects on protein stability
and size, ACS Chem. Biol. 8 (2013) 416422.
[44] S. Tomita, H. Hamada, Y. Nagasaki, K. Shiraki, Synergistic effect of
polyethylene glycol with arginine on the prevention of heat-induced
aggregation of lysozyme, J. Phys. Conf. Ser. 106 (2008) 012022.
[45] C.P. Schneider, D. Shukla, B.L. Trout, Arginine and the Hofmeister series: the
role of ionion interactions in protein aggregation suppression, J. Phys. Chem.
B 115 (2011) 74477458.