J. Biol. Macromol., 16 (1), 3-7 (2016)

ArticleThe effects of N-acetyltryptophan and caprylic acid on protein aggregation
Shogo Oki1, Tsutomu Arakawa2, Kentaro Shiraki1,*1

Institute of Applied Physics, University of Tsukuba, Tsukuba, Ibaraki 305-8573, Japan2
Alliance Protein Laboratories, San Diego, CA 92121, United State

Received May 18, 2016; Revised June 29, 2016; Accepted July 4, 2016
N-acetyltryptophan (AT) and caprylic acid (Cap) are known to suppress the heat-induced aggregation ofserum albumins. In this study, we examined the commonality of AT and Cap on the protein aggregation using hen
egg-white lysozyme, ovalbumin, and bovine γ-globulin (BGG), as well as bovine and human serum albumin. ATand Cap as additives increased the unfolding temperatures of human and bovine albumins analyzed by far-UV
circular dichroism. Both AT and Cap highly suppressed the heat-induced aggregation of BGG as well as albumins,
while they enhanced the aggregation of lysozyme. In all cases, the addition of 100 mM ArgHCl showed nosynergistic effects with AT or Cap in suppressing the aggregation of BGG. The small chemicals of AT and Cap
will be useful additives toward the thermal stabilization of albumins and γ-globulin.

Key words: N-acetyltryptophan, caprylic acid, aggregation, ArgHCl, protein.
1. Introduction
Both N-acetyltryptophan (AT) and caprylic acid (Cap)have been used to suppress the heat-induced formation ofsoluble oligomers of serum albumins [1–3]. Fig.1 shows
the chemical structures of AT and Cap. Cap is a type offatty acid with a carboxyl end, found in coconut, babassu,
and milk fat [4]. AT is a tryptophan derivative modified
in the amino group by acetyl group. The effects of these
additives are derived from their bindings to the albumins[5], which are known to bind various drug substances andfatty acids [6, 7].
Proteins in general can bind ions, organic compounds,
(B)

(A)

(C)

Fig. 1 Chemical structures of additives. (A) Caprylic acid; Cap. (B) Nacetyltryptophan; AT. (C) Arginine hydrochloride; ArgHCl.

*Correspondence author: Kentaro Shiraki. Phone: +81-29-853-5306 Fax:
+81-29-853-5215 E-mail: Shiraki@bk.tsukuba.ac.jp

and detergents [8, 9]. Binding of these compounds canalter solution properties of the proteins, e.g., stability,aggregation tendency, and solubility. In this short note,
we examined the commonality of the effects of AT andCap on the aggregation of five proteins that have different
chemical and physical properties: human and bovine
serum albumin, hen egg-white lysozyme, ovalbumin, andbovine γ-globulin. The effects of AT and Cap on thethermal stability were also examined, since it is related toprotein aggregation [10]. In addition, we investigatedwhether the synergetic effects exist with arginine (seeFig.1 for chemical structure), which is a well-known
aggregation suppressor of proteins [11–13].
2. Materials and method
2.1 Materials
Human serum albumin (HSA), bovine serum albumin(BSA), hen egg-white lysozyme (lysozyme), ovalbumin,
γ-globulin from bovine blood (BGG), N-acetyl-DLtryptophan (AT), and caprylic acid (Cap) were obtained
from Sigma-Aldrich Corp. (St Louis, Mo., USA) Sodiumhydroxide (NaOH), sodium dihydrogenphosphate, andL–Arginine hydrochloride (ArgHCl) were obtained fromWako Pure Chemical Industries, Ltd. (Osaka, Japan). Allchemicals used were of reagent grade and used asreceived.
2.2 Circular dichroism spectroscopy
Denaturation temperatures (Tm) were determinedfollowing signal changes in the far-UV circulardichroism (CD) on a Jasco CD spectropolarimeter modelJ-720W with a Peltier cell holder model PTC-348W(Japan Spectroscopic Co., Ltd., Tokyo, Japan). Sample
solutions containing 0.4 mg/ml protein (HSA, BSA, andlysozyme) or 0.8 mg/ml protein (ovalbumin) in 20 mM
sodium phosphate, pH 7.0, in the absence and presenceof additives, were prepared and adjusted to pH 7.0 by theaddition of NaOH. CD signals were monitored at 224 nm
for Cap-containing samples and at 238 nm for AT- or
arginine hydrochloride (ArgHCl)-containing samples
with a heating rate of 1°C/min using a 0.1-cm path-length
cell. A lower wavelength could not be used for AT- or

4The effects of HSA stabilizer on protein aggregation

ArgHCl-containing samples due to high absorbance. The
data obtained were fitted to a conventional two-state
equation, from which the apparent Tm was determined
denaturation temperatures were not determined for BGG,
as it is a mixture of many kinds of antibody moleculesthat generate a broad transition pattern for thermal
unfolding.
2.3 Turbidity measurements
Thermal aggregation of proteins was measured asfollows. Protein solutions at 1.0 mg/ml in 20 mM sodium
phosphate, pH 7.0, in the absence and presence ofadditives, were prepared and adjusted to pH 7.0 by theaddition of NaOH. A 2.0 mL aliquot of the solutions wasintroduced into a 1-cm path-length glass cell. The sample
solutions were incubated at 70°C, 80°C, or 90°C for BGG
and at 75°C or 90°C for lysozyme, and the turbidity at
400 nm was monitored using a Jasco spectrophotometermodel V-550 (Japan Spectroscopic Co., Ltd., Tokyo,Japan).
3. Results and discussion
3.1 Influence of AT and Cap on thermal denaturationof proteins
The effects of AT and Cap on thermal stability wereexamined using far-UV CD signal at 238 nm for AT andat 224 nm for Cap. These wavelengths were selected as
the lowest possible wavelength due to strong UV
absorbance of AT and Cap. In addition, the concentrationof AT and Cap above 3 mM could not be tested for thesame reason, i.e., strong UV absorbance. CD intensity of0.4 mg/ml protein solution was followed with increasing
temperature from 45°C to 90°C, from which the midtransition temperature, Tm, was determined by curve
fitting to a two-state transition model. The results aresummarized in Table 1. Control experiments, i.e., in theabsence of additives, showed that the melting curves gave
an identical Tm values within experimental errors,
whether it was followed at 238 or 224 nm for HSA, BSA,
lysozyme, and ovalbumin. As shown in Table 1, 3 mM
AT increased the Tm of HSA and BSA by approximately
8°C and approximately 6°C, while 3 mM Cap increased itcontrary, both 3 mM AT and Cap exhibited negligible
effects on the Tm of lysozyme and ovalbumin. There may
be reduction of Tm (−2.8°C) for lysozyme by 3 mM AT. It
is thus evident that AT and Cap have no stabilization
effects on lysozyme and ovalbumin, unlike their effects
on HSA and BSA. This in turn suggests that AT and Cap
do not bind to these two proteins or bind to them in boththe native and denatured states. That is, unlike HSA andBSA, they may bind to lysozyme and ovalbumin, but notpreferentially to the native structure.
3.2 Influence of AT and Cap on thermal aggregationof proteins
Then, we examined the effects of heating on
aggregation of the above four proteins and BGG. Thermal
aggregation was examined by following turbidity at 400
nm. HSA, BSA, and ovalbumin showed no apparent
increase in turbidity upon heating up to 90°C, indicatingthat these proteins do not generate large aggregates, thesizes sufficiently large to cause light scattering at 400 nm:
it is possible that they may form small oligomers. No
apparent aggregation was also observed upon heating forthese proteins in the presence of 20 mM AT and Cap,
implying that these additives do not enhance aggregation:note that 20 mM AT and Cap could be used in thisexperiment, which used 400 nm for turbidity
measurements.
The effects of 20 mM AT and Cap were examined onthermal aggregation of lysozyme. Fig. 2 shows theturbidity increase when incubated at 75°C (A) and 90°C
(B). At 75°C, 20 mM AT (squares) and Cap (triangles)
accelerated turbidity increase, more strongly for Cap
relative to their absence (circles). The onset of turbidity
increase occurred at shorter time, and the turbidity
increase was sharper in the presence of 20 mM AT and
more with 20 mM Cap. Turbidity increase occurred much
more rapidly at 90°C (circles). At this temperature as well,
20 mM AT and Cap shortened the onset of turbidity
increase and enhanced the rate of aggregation. It is thus
evident that AT and Cap enhanced the aggregation oflysozyme. In thermal denaturation experiments, 3 mM
AT and Cap showed marginal effects on lysozyme

Table 1 Denaturation temperature of proteins in the presence and absence of 3 mM AT or 3 mM Cap
protein

HSABSAlysozyme

ovalbumin

denaturation temperature

Tm (°C)

ΔTm (°C)

Tm (°C)

ΔTm (°C)

Tm (°C)

ΔTm (°C)

Tm (°C)

ΔTm (°C)

no additive(measured at 238 nm)

61.2

－

60.3

－

73.7

－

74.4

－

3 mM AT

(at 238 nm)

69.3

8.1

66.2

5.9

70.9

-2.8

75.1

3.9

no additive(at 224 nm)

62.0－61.6－73.0－

73.8

－

3 mM Cap(at 224 nm)

77.4

15.4

77.8

16.1

72.1

-0.9

73.5

-0.3

Tm shows the denaturation temperature, while ΔTm shows the difference in the denaturation temperature in the absence and presenceof additive.

by approximately 15°C–16°C, demonstrating stronger
stabilization effects of Cap. These results indicate
preferential binding of AT and Cap to the native structure,
leading to stabilization of the native proteins. On thedenaturation. The observed enhancement of aggregationsuggests that AT and Cap most likely bind to protein andenhance aggregation.

Oki, Arakawa and Shiraki

Fig. 2 Thermal aggregation of lysozyme with no additives (circles), 20mM AT (squares), and 20 mM Cap (triangles). Samples containing
1.0 mg/ml lysozyme and 20 mM phosphate buffer prepared at pH
7.0 and heated to 75°C (A) and 90°C (B). A and A0 show themeasured and the starting absorbance at 400 nm, respectively.

Fig. 3 shows turbidity increase of BGG at 70°C (A),80°C (B), and 90°C (C). At 70°C, the turbidity increase
occurred at ~200 s followed by a sharp increase in theabsence of the additives (circles). In the presence of 20mM AT (squares), the turbidity increase was significantlyreduced, although the onset time appeared to beunchanged. Cap at 20 mM further reduced the rate of

5turbidity increase (triangles). These results demonstratethat both AT and Cap can suppress the aggregation ofBGG, Cap being more effective. At 80°C (Fig. 3B), thereappeared to be a small delay in the onset of turbidity
increase by 20 mM AT (squares) and 20 mM Cap(triangles) compared to their absence, although the rates
appeared to be similar with and without AT or Cap. At
90°C (Fig. 3C), three curves overlapped on top of eachother. It thus appears that AT and Cap can exert
suppressive effects on aggregation of BGG at lower
temperatures, at which aggregation is slower, but lose
their effectiveness when aggregation becomes faster at90°C. Because BGG is a mixture of widely different
antibodies, its melting properties could not be measured.
The observed aggregation suppression of BGG by ATand Cap may be due to their binding to BGG in different
way than they bind to lysozyme, whose thermal
aggregation was enhanced. They may bind to BGG, sothat they mask the hydrophobic regions of BGG andthereby suppress its aggregation. Since the effects at 20mM are rather weak in particular at higher temperatures,
it would be of great interest to see if higher AT and Cap
concentrations can increase the aggregation suppressiveeffects. This could not be done, however, due to thesolubility limit of AT. In any case, the results suggestthat binding of AT and Cap to BGG is weak, in the orderof mM range.
3.3 Protein stabilizing or destabilizing effect of ATand Cap in the presence of ArgHCl
We demonstrated above that AT and Cap can enhance
the stability of HSA and BSA and can suppress theaggregation of BGG. Synergistic effects of AT and Cap
with ArgHCl, a well-known aggregation suppressor [11–13], were then examined. Table 2 shows the effects of
100 mM ArgHCl on the Tm of HSA and BSA. It slightly
increased the Tm by about 4°C for both proteins, far lessthan the increase observed for 3 mM AT and Cap. WhenFig. 3 Thermal aggregation of BGG with no additives (circles), 20 mM AT (squares), and 20 mM Cap (triangles). Samples containing 1.0 mg/ml BGG and20 mM phosphate buffer prepared at pH 7.0 and heated at 70°C (A), 80°C (B), and 90°C (C). A and A0 show the measured and starting absorbancesat 400 nm, respectively.

100 mM ArgHCl was combined with 3 mM AT, the Tm
was slightly changed from the value at 3 mM AT,indicating no effects of ArgHCl. An identical trend wasobserved for Cap. That is, the addition of 100 mM

6The effects of HSA stabilizer on protein aggregation

Table 2 Denaturation temperature of serum albumin in the presence and absenceof 3 mM AT, 3 mM Cap, or 0.1 M ArgHClProtein

HSA

BSA

Denaturation temperature

Tm (°C)

ΔTm (°C)

Tm (°C)

ΔTm (°C)

No additive

61.2

－

60.3

－

100 mM ArgHCl

65.6

4.4

64.6

4.3

3 mM AT

69.3

8.1

66.2

5.9

3 mM Cap

81.1

19.9

79.3

19.0

100 mM ArgHCl + 3 mM AT

70.1

8.9

67.5

7.2

100 mM ArgHCl + 3 mM Cap

79.0

17.8

78.4

18.1

Tm shows the denaturation temperature, while ΔTm shows the difference in thedenaturation temperature in the absence and presence of additive.

Fig. 4 Thermal aggregation of lysozyme heated at 90°C (A) and BGG heated at 70°C (B) with no additives (closed circles), 20 mM AT (closed squares), 20mM Cap (closed triangles), 100 mM ArgHCl (open circles), 100 mM ArgHCl + 20 mM AT (open squares), and 100 mM ArgHCl + 20 mM Cap(open triangles). Samples containing 1.0 mg/ml lysozyme or BGG and 20 mM phosphate buffer prepared at pH 7.0. A and A0 show the measured andstarting absorbance at 400 nm, respectively.

ArgHCl slightly changed the Tm of BGG in the presenceof 3 mM Cap.
Then, the synergistic effects of 100 mM ArgHCl onlysozyme and BGG aggregation were examined. Fig. 4Ashows the results for lysozyme. When heated at 90°C,
100 mM ArgHCl greatly delayed the onset of turbidity
increase (open circles vs closed circles). The rate ofturbidity increase was also slower in 100 mM ArgHCl.
This suppressive effect of ArgHCl was reduced by 20mM AT (open squares) and 20 mM Cap (open triangles),reflecting the effects of AT or Cap by itself, which was toenhance the aggregation of lysozyme.
Similarly, 100 mM ArgHCl significantly reduced
turbidity increase of BGG when heated at 70°C, as shownin Fig. 4B. That is, the onset of turbidity increase wasdelayed by 100 mM ArgHCl (open circles) relative to thecontrol (closed circles). AT (closed squares) at 20 mM
also similarly suppressed the turbidity increase. However,when these two additives were combined, the aggregation
suppression was similar to 100 mM ArgHCl alone or 20mM AT alone (see open squares). Thus, it is clear that
there is no synergy between ArgHCl and AT. On thecontrary, the suppressive effects of 100 mM ArgHCl or
20 mM Cap were largely lost when they were combined.
As described earlier, 20 mM Cap showed strong effects
on BGG aggregation (see closed triangles). When

combined with 100 mM ArgHCl, the turbidity increase
was close to that of the control (see open triangles). It
thus appears that ArgHCl and Cap suppress the effect of
the other additives. Although this observation cannot bereadily explained, it might be that ArgHCl influences thebinding of Cap to BGG or alters the way Cap binds to theprotein.
4. Conclusion
This study showed the effect of AT and Cap as
additives on the thermal stability of proteins. We
successfully concluded as follows: 1) AT and Cap highly
increased the thermal stability of human and bovine
albumins; 2) AT and Cap suppressed the heat-induced
aggregation of bovine γ-globulin as well as albumins; 3)
In contrast, AT and Cap rather accelerated the hen eggwhite lysozyme; and 4) The synergistic effect of AT andCap with ArgHCl, the well-known aggregation
suppressor, was not observed toward heat-induced
aggregation of γ-globulin and lysozyme. These findingsof AT and Cap as additives in protein solution improve
the protein stability toward the thermal stresses, which
may use the storage additive in ambient temperature.
Acknowledgements

Oki, Arakawa and Shiraki

This study was supported in part by University ofTsukuba and JSPS KAKENHI (Grant Numbers
15K13812 and 15H03583) from the Ministry ofEducation, Culture, Sports, Science, and Technology(MEXT), Japan.
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Communicated by Ohmae Eiji