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Langmuir
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Liquid chromatographic analysis of the interaction
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between amino acids and aromatic surfaces using
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single-wall carbon nanotubes
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Kazuki Iwashita †, Kentaro Shiraki †, Rieko Ishii ‡, Takeshi Tanaka ‡, and Atsushi Hirano ‡,*
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†
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Ibaraki 305-8573, Japan
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‡
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Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan.
Faculty of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba,
Nanomaterials Research Institute, National Institute of Advanced Industrial Science and
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* To whom correspondence should be addressed Tel: +81-29-849-1064, Fax:
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+81-29-861-2786, E-mail:
[email protected] 13
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Abstract
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Proteins have nonspecific adsorption capacities for solid surfaces. Although the nonspecific
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adsorption capacities are generally understood to be related to the hydrophobicity or charge
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density of the surfaces, little is known at the amino acid level about the interaction between
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proteins and polyaromatic surfaces such as carbon nanotubes, which have recently been used
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for biotechnology applications. In this study, we investigated the interaction between
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proteinogenic amino acids and carbon nanotubes using high-performance liquid
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chromatography on silica matrices coated by single-wall carbon nanotubes (SWCNTs).
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Among the amino acids used in this study, tryptophan, tyrosine and phenylalanine showed
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exceptional affinity for the matrices. The characteristic affinities of these amino acids were
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ascribed to their unique interactions with the large polyaromatic surfaces of the SWCNTs.
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These results are useful for understanding and controlling protein adsorption onto aromatic
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surfaces.
27
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Langmuir
Introduction
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The nonspecific adsorption of proteins for solid surfaces of materials affects protein
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structure, stability and activity.1–3 This adsorption of proteins is driven by noncovalent
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interactions such as hydrogen bonding, electrostatic interactions, hydrophobic interactions
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and van der Waals forces, all of which are derived from the material surface structure and
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chemistry and the amino acid residues of the protein. Because protein surfaces are generally
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heterogeneous, it is useful to investigate the interactions between amino acids and solid
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surfaces to attain a basic understanding of the adsorption reaction.
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A number of studies have reported on the interaction of amino acids with solid surfaces
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such as metal,4,5 inorganic oxide,6,7 and activated carbons.8 Such interactions have been
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investigated by various experimental techniques9 such as chromatography,10 solid-state
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NMR,11 and attenuated total reflectance infrared spectroscopy12 as well as by density
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functional theory studies13 and molecular dynamics simulations.14 For example, an affinity
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scale for the interaction of amino acids with the silica surface via hydroxyl groups, which was
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determined by a density functional theory calculation, indicates favorable interactions of the
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hydrophilic amino acids with the surface through hydrogen bonding.15 In addition,
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interactions of amino acids with hydrophobic and aromatic surfaces of solids were described
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by adsorption isotherms.16 However, little is known about the interaction of amino acids with
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polyaromatic surfaces.17
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Recently, interactions between proteins and carbon nanotubes, which have polyaromatic
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surfaces, have been shown to affect protein structure, stability and activity.18–20 To date,
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interactions between carbon nanotubes and amino acids have primarily been studied using
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computer simulations.21–24 Molecular dynamics simulations showed that aromatic amino
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acids have a high affinity among proteinogenic amino acids.24 However, few experimental 3
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studies have been performed to investigate this interaction.25,26 In addition, SWCNTs as the
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chromatographic stationary phases have been used to demonstrate their affinities for various
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substances such as peptides and aromatic compounds.27 In this study, the interaction between
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carbon nanotubes and amino acids was investigated using high-performance liquid
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chromatography (HPLC) on silica matrices with carbon nanotubes as ligands; single-wall
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carbon nanotubes (SWCNTs) were used as ligands, and the silica matrices were modified by
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amino groups, which play a role in the noncovalent immobilization of SWCNTs on the silica
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matrices;28 noncovalent modifications are generally useful to investigate the unique
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properties of the SWCNTs because covalent modifications disrupt their structures and affect
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such properties. The retention time was used as an index to evaluate the affinity. To the best
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of our knowledge, this is the first report of using a chromatographic approach to examine the
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interaction between SWCNTs and amino acids. The results obtained from these
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measurements should be useful for understanding protein adsorption onto polyaromatic
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surfaces at the amino acid level and hence controlling the protein adsorption capacity,
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structure, stability and activity on them.
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Materials and Methods
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Materials
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Glycine (Gly), (His),
L-alanine
(Ala),
L-phenylalanine
L-valine
(Phe),
(Val),
L-tyrosine
L-leucine
(Tyr),
(Leu),
L-isoleucine
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L-histidine
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deoxycholate, urea, sodium phosphate, citric acid, sodium hydroxide, hydrochloric acid and
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N-methyl-2-pyrrolidone (NMP) were obtained from Wako Pure Chemical Inc. Ltd. (Osaka,
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Japan). Ammonium sulfate ((NH4)2SO4) was obtained from Nacalai Tesque, Inc. (Kyoto,
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Japan). Raw SWCNTs produced by high-pressure catalytic CO decomposition were obtained 4
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(Ile),
(Trp), sodium
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from NanoIntegris Inc. (Menlo Park, CA, USA). Amino-modified silica with a 3.5-µm
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diameter was was obtained from Chemco Plus Scientific Co., Ltd (Osaka, Japan). A C18
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column (4.6 mm I.D. × 250 mm column, TSKgel ODS-80Ts) was obtained from Tosoh Corp.
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(Tokyo, Japan).
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Dispersion of SWCNTs
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Aliquots of 30 mg of the SWCNTs were predispersed at 1 mg/mL in 30 mL of NMP using
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an ultrasonic processor (Nanoruptor NR-350, Cosmo Bio, Tokyo, Japan) for 1 min at a power
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of 350 W. The solutions were dispersed using an ultrasonic homogenizer (Sonifier 250D,
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Branson Ultrasonics Corp., Danbury, CT, USA) equipped with a 0.5 in. flat tip for 1 h at a
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power density of 23 W cm−2. To prevent heating during sonication, the bottle containing the
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sample solution was immersed in a water bath maintained at 18 °C. The dispersed sample
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solution was centrifuged at 210,000 × g for 10 min using an ultracentrifuge (S80AT3 rotor,
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Hitachi Koki Co, Ltd, Tokyo, Japan) to remove residual catalytic metal particles, nanotube
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bundles, and other impurities. The upper 70% of the supernatant was collected as the
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debundled SWCNT solution. Finally, the supernatant was diluted to 0.2 mg/mL.
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Measurement of absorption spectra of SWCNTs
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Absorption spectra of the SWCNTs were measured to estimate the mean diameter of the
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SWCNTs used in this study. The SWCNTs were dispersed by the same methods described
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above but using 1 wt % sodium deoxycholate aqueous solution instead of NMP. The
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dispersed sample solution was centrifuged at 210,000 × g for 1 h using the ultracentrifuge,
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and then the upper 70% of the supernatant was collected as the debundled SWCNT solution.
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Absorption spectra of the 20-fold diluted supernatant with 1 wt % sodium deoxycholate were
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measured using a UV–vis–NIR spectrophotometer (UV-3600, Shimadzu, Kyoto, Japan) with
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a quartz cell with a path length of 10 mm. 5
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Preparation of SWCNT-coated silica column
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A SWCNT-coated silica column was prepared by the method described below. First, 0.9 g
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of amino-modified silica was added to aliquots of 20 mL of the SWCNT solution dispersed
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by NMP. The solution was then rotated for 1 h at room temperature to coat the
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amino-modified silica with the SWCNTs using a rotator (MTR-103, As One, Osaka, Japan).
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Subsequently, the suspension was centrifuged at 3000 × g for 3 min using a centrifuge
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(5415R, Eppendorf, Hamburg, Germany), and then 16 mL of supernatant was replaced with
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chloroform/diethylene glycol (1:4, v/v). These centrifugation and supernatant-exchange
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processes were repeated three times. Finally, 4 mL of slurry was collected and then packed in
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a 2.1 mm I.D. × 125 mm empty column (Chemco Plus Scientific Co., Ltd., Osaka, Japan) by
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flowing chloroform/hexane (1:1, v/v) at 22 MPa. A similar method was also used without the
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SWCNTs to prepare an amino-modified silica column as a control.
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Retention time analysis of amino acids in the liquid chromatographic columns.
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Retention time analysis of the proteinogenic amino acids was performed using an HPLC
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system (Shimadzu, Kyoto, Japan) comprising a degasser (DGU-14A), a pump (LC-10ADvp),
115
a column oven (CTO-10ACvp), a UV–vis detector (SPD-10AVvp), a photo-diode array
116
detector (SPD-M10Avp), and a system controller (SCL-10Avp). The isocratic HPLC was
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conducted with a flow rate of 0.2 mL min−1 for the SWCNT-coated and amino-modified
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silica columns or 1.0 mL min−1 for the C18 column at 25 °C using a 10 mM sodium
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phosphate buffer solution (pH 7.0) in the presence or absence of 1 M (NH4)2SO4 or 8 M urea
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or using a glycine–citrate–phosphate buffer solution (pH 2.0–7.0) comprising 10 mM glycine,
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10 mM sodium citrate, and 10 mM sodium phosphate, the pH values of which were adjusted
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by sodium hydroxide and hydrochloric acid. 20 µL amino acid solutions dissolved in the
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buffer solutions were loaded into the columns. The absorbance of the amino acids was 6
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monitored at 210 nm for the sodium phosphate buffer solutions with or without additives,
125
except urea; the absorbance was measured at 225 nm for the sodium phosphate buffer
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solution with urea or for the glycine–citrate–phosphate buffer solutions. The column dead
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time (t0) was determined as the time when water appeared as a peak. The obtained retention
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data were used to derive the values of the retention factor (k), k = (tr − t0)/t0. All retention
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factors were determined as the average of three experiments.
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Measurement of amino acid solubility
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Tryptophan, phenylalanine or histidine was transferred into test tubes containing 10 mM
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sodium phosphate buffer solutions in the presence or absence of 1 M (NH4)2SO4 or 8 M urea
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at pH 7.0 to measure their solubility in the solutions. The suspension was maintained at 25 °C
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for 48 h with mixing using the rotator (MTR-103) to saturate the solutions with the amino
135
acids. The suspension was subsequently centrifuged at 25 °C and 15,000 × g for 3 min to
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obtain supernatants saturated with the amino acids. After either a 50-fold (for tryptophan and
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phenylalanine) or a 3-fold (for tyrosine) dilution of the supernatants with 10 mM sodium
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phosphate buffer solution (pH 7.0), the absorbance of the supernatants was measured at 280
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nm for tryptophan and tyrosine or at 260 nm for phenylalanine using an UV–vis
140
spectrophotometer (ND-2000, NanoDrop Technologies, Wilmington, DE, USA). The
141
absorbance was converted to the concentration based on the standard curve determined for
142
each amino acid. The solubility was determined as the average of three experiments.
143 144
Results and Discussion
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Characterization of single-wall carbon nanotubes
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SWCNTs produced by high-pressure catalytic CO decomposition were used as ligands of
147
silica matrices. Prior to the column chromatography experiments, absorption spectra of the 7
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SWCNTs dispersed in 1 wt % sodium deoxycholate were measured to estimate their
149
diameters from absorbance peak positions (Figure 1); the dispersing effect of sodium
150
deoxycholate on the SWCNTs is so pronounced that it enables one to determine absorbance
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peak positions.29 Spectral peaks were observed at 400–1350 nm. The peaks at approximately
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940–1350 and 620–940 nm correspond to first- and second-order optical transitions for
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semiconducting SWCNTs, whereas those at approximately 400–620 nm are assigned to
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first-order optical transitions for metallic SWCNTs. Such peaks indicate that the diameters of
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the SWCNTs used in this study were approximately 0.8–1.1 nm30 The silica matrices used in
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this study to fabricate SWCNT-coated silica matrices are modified by amino groups to confer
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their high affinity for SWCNTs.31 The matrices of the SWCNT-coated silica, hereafter
158
designated as SWCNT-NH2-silica, were made by mixing the SWCNTs dispersed by NMP
159
with the amino-modified silica matrices. A chromatographic column filled with the matrices
160
was then connected to the HPLC system. Another column filled with matrices of the original
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amino-modified silica, hereafter designated as NH2-silica, was also used as a control.
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Chromatography of proteinogenic amino acids in the SWCNT-NH2-silica column
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The retention time of amino acids in the columns was measured to assess their affinity for
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the matrices. Figure 2 shows chromatograms of the proteinogenic amino acids in the
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SWCNT-NH2-silica column as well as the NH2-silica column, which were examined in 10
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mM sodium phosphate buffer at pH 7.0. The chromatograms were monitored by measuring
167
their absorbance at 210 nm. The column dead time was determined to be 1.681 ± 0.012 min
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for SWCNT-NH2-silica column and 1.675 ± 0.022 min for the NH2-silica column, which
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were defined as the time that peak appears when water was loaded. The column dead time
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was used to define the adjusted retention time, as shown in Figure 2 (dashed lines). The
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retention times for each amino acid are listed in Table 1. 8
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In the SWCNT-NH2-silica column, the retention time for tryptophan was 4.444 ± 0.230
173
min, which was the longest among the amino acids. This result appears to be reasonable
174
because tryptophan is commonly known as the most hydrophobic proteinogenic amino acid;
175
Table 1 also indicates the partition free energies of the amino acid side chains from octanol to
176
a neutral aqueous solution32; the more negative the transfer free energy is, the more
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hydrophobic the amino acid is. Interestingly, the second retained amino acid in the
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SWCNT-NH2-silica column was histidine, which is a basic amino acid; however, its side
179
chain is not sufficiently charged at the pH values used because the pKa of its side chain is 6.0.
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As observed from the partition free energy, histidine is relatively hydrophilic (Table 1);
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therefore, the observed retention of histidine was ascribed to its interaction with NH2-silica
182
matrices rather than with the SWCNTs. To support this suggestion, the relationship between
183
the logarithm of the retention factor (log k) for the NH2-silica column and that for the
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SWCNT-NH2-silica column was calculated, as shown in Figure 3; the retention factor (k)
185
values were calculated as k = (tr − t0)/t0 using the retention time (tr) and the column dead time
186
(t0).33 The result showed a strong correlation, except for tryptophan, tyrosine and
187
phenylalanine. As expected, histidine was also included in the correlation. This result
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indicates that the proteinogenic amino acids, except for tryptophan, tyrosine and
189
phenylalanine, have marginal affinity for SWCNTs but instead interact with NH2-silica
190
matrices. By contrast, the exceptional results of the three aromatic amino acids, i.e.,
191
tryptophan, tyrosine and phenylalanine, indicate their unique affinity for the SWCNTs but not
192
for the NH2-silica matrices. In fact, tryptophan, tyrosine, phenylalanine and peptides
193
containing residues of these amino acids have been reported to have affinities for
194
SWCNTs.23,34–36
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Effects of a kosmotrope and a chaotrope on the retention time 9
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To investigate the origin of the high affinity of the aromatic amino acids, tryptophan,
197
tyrosine and phenylalanine, their retention times in the SWCNT-NH2-silica column were
198
measured in the presence of kosmotropes or chaotropes. Kosmotropes are known to enhance
199
hydrophobic interactions because they increase the surface tension of solutions37–39. A
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representative kosmotrope is (NH4)2SO4, which is generally used for protein crystallization or
201
precipitation on the basis of the enhancing effect of hydrophobic interaction. If the affinity of
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the amino acids for the SWCNTs is due to hydrophobic interactions, then their retention
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times should be delayed by the kosmotrope because of the enhancement of hydrophobic
204
interactions. In contrast to kosmotropes, chaotropes are known to reduce hydrophobic
205
interactions. Urea, a chaotrope, has been previously suggested to favor hydrophobic amino
206
acids and SWCNT surfaces using molecular dynamics simulation.40,41 In addition, we have
207
previously reported that urea induces the desorption of SWCNTs from hydrogel surfaces.42
208
(NH4)2SO4 and urea were thus used as a kosmotrope and a chaotrope, respectively, to
209
investigate the affinity of the aromatic amino acids for the SWCNTs.
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Figure 4 shows the representative chromatograms for tryptophan, tyrosine and
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phenylalanine in the presence or absence of 1 M (NH4)2SO4 or 8 M urea. Tryptophan was the
212
most sensitive among the amino acids to the additives. The retention times and adjusted
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retention times for the amino acids are listed in Table 2. The fact that tryptophan exhibited a
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significantly longer time delay than the other amino acids in the presence of (NH4)2SO4
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indicates that it has a larger hydrophobic interaction area on the SWCNTs compared with the
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other amino acids because the enhancement of the hydrophobic interaction by the increase in
217
surface tension is theoretically proportional to the hydrophobic area.37 By contrast, tyrosine
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was less affected than tryptophan and phenylalanine, suggesting that tyrosine has a smaller
219
hydrophobic interaction area on the SWCNTs. In contrast to (NH4)2SO4, the retention time 10
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was significantly reduced by urea, independent of the nature of the amino acids. The
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favorable attractions of urea with the hydrophobic amino acids and with the SWCNT surfaces,
222
as mentioned above, probably leads to a decreased affinity of the amino acids for the
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SWCNTs. The attractive interactions will also be useful for reducing the adsorption capacity
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of proteins onto SWCNT surfaces.
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Effects of a kosmotrope and a chaotrope on the solubility of amino acids
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The additive effects on the interaction between the SWCNTs and tryptophan, tyrosine and
227
phenylalanine, as shown above, can be discussed in terms of the solubility of the amino acids.
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Table 3 depicts the solubilities of tryptophan, tyrosine and phenylalanine in the presence and
229
absence of (NH4)2SO4 or urea. In addition, their transfer free energy from the buffer solution
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to each additive solutions was estimated, as listed in Table 3; the estimated transfer free
231
energy was defined as ∆G = −RT ln(S/Sw), where R and T are the universal gas constant and
232
the absolute temperature, respectively, and S and Sw are the solubilities in the presence and
233
absence of the additives, respectively.43 The solubility of tryptophan was decreased by the
234
addition of 1 M (NH4)2SO4, which is reasonable because of the high hydrophobicity of
235
tryptophan, as mentioned above (Table 1). However, the estimated transfer free energy of
236
tryptophan was less than that of phenylalanine, despite the longer retention time of
237
tryptophan in the SWCNT-NH2-silica column, suggesting that tryptophan has a larger
238
hydrophobic interaction area with the SWCNTs than that of self-association. By contrast, the
239
solubility of tyrosine was decreased to a lesser extent by 1 M (NH4)2SO4, indicating its lower
240
hydrophobicity. All amino acid solubilities were dramatically increased by the addition of 8
241
M urea, which is consistent with the reduction of retention time by urea, as shown in Figure 4.
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These results support the suggestion that the unique affinity of tryptophan for the SWCNTs
243
was at least partly due to hydrophobic interactions; however, some other interactions, which 11
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are unexplainable by hydrophobic interactions, are involved in the affinity of tryptophan,
245
tyrosine and phenylalanine for the SWCNTs.
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Comparison with conventional hydrophobic columns
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To further understand the SWCNT-NH2-silica column chromatography, a conventional
248
hydrophobic column, i.e., a C18 column, was used as a control column. Figure 5A shows the
249
relationship between retention time logarithms in the C18 column and those in the NH2-silica
250
column, which exhibited relatively strong correlations if histidine was omitted. This result
251
indicates that the NH2-silica column essentially works as though it is a C18 column for the
252
hydrophobic amino acids, which may be due to the alkyl chain moiety on the matrices. The
253
exceptionally strong affinity of histidine for the NH2-silica matrices can be explained by the
254
interaction of its imidazole group with the NH2 group of the matrices, e.g., a cation–π
255
interaction.44
256
Tryptophan, tyrosine, and phenylalanine have increased affinity for the SWCNT-coated
257
matrices, as already described (Figure 3). This affinity of the amino acids for the SWCNTs
258
might be explained by aromatic–aromatic interactions. It is thus useful to describe the
259
interaction of tryptophan, tyrosine or phenylalanine with the phenyl group to understand the
260
effect of the aromatic-aromatic interaction on affinity. Figure 5B shows the relationships of
261
retention factor logarithms between the data for the C18 column and the data for a phenyl
262
column; the latter data were obtained from a previous study.33 The results showed a strong
263
correlation for all amino acids except for tryptophan, tyrosine and histidine. Importantly,
264
tryptophan and tyrosine appear to have a lower affinity for the phenyl group than expected
265
from the correlation, although they have an aromatic side chains. It was thus proposed that
266
tryptophan and tyrosine favor the polyaromatic surfaces specific to the SWCNTs and not the
267
phenyl group. This unique affinity of the amino acids will be useful for the understanding and 12
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control of protein adsorption onto polyaromatic surfaces. In addition, the higher affinities of
269
histidine for the phenyl column, despite its hydrophilicity, clearly indicates the characteristic
270
interaction of histidine with the phenyl groups of the matrices; these affinities may be
271
attributable to cation–π, polar hydrogen–π, or π–π interactions.44–46
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The pH-dependent retention time of amino acids in the SWCNT-NH2-silica column
273
Notably, the silica matrices are modified by the amino groups; therefore, the matrices
274
should be positively charged at the pH value used above, leading to electrostatic interactions
275
between the matrices and amino acids. Such interactions probably affect the retention times
276
of the amino acids in the columns. pH dependence was thus examined for tryptophan and
277
histidine in the pH range of 2.0–7.0 in the presence of glycine–citrate–phosphate buffer to
278
verify the effect of electrostatic interactions. The retention time of tryptophan was shorter at
279
lower pH values, especially pH values lower than 3 (Figure 6). However, the shortened
280
retention time of histidine appeared at pH values lower than 6. These results indicate that
281
electrostatic repulsion occurred between the matrices and the amino acids with a positive net
282
charge because the amino groups of the amino acid backbones are typically protonated at pH
283
values less than approximately 2 and because the histidine side chain is protonated at pH
284
values less than approximately 6.
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Conclusion
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The order of the affinity of proteinogenic, hydrophobic amino acids for the
288
SWCNT-NH2-silica matrices was as follows: glycine ≈ alanine ≈ valine ≈ leucine ≈
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isoleucine < phenylalanine < tyrosine
