Adsorption of amino acids by fullerenes and fullerene nanowhiskers

C₆₀ (99.5%) was purchased from MTR Ltd, USA. D-tyrosin (Tyr), D-triptphane (Trp), D-phenylalanine (Phe), D-proline (Pro), D-leusine (Leu), D-aspartic acid (Asp), D-valine (Val) and D-argenine (Arg) were obtained from Wako Chemical, Japan and D-alanine, (Ala) D-alaninyl-D-anlanyl-D-alanine (Tri-Ala) were purchased from Sigma USA. We used the D-enanthiomer of the amino acids to minimize external contamination and detrimental effects on microorganisms

Solutions of amino acids were prepared with concentrations of up to 2 mmol dm⁻³. In the case of Trp, since solubility is relatively low, we used concentration prepared solution of up to 1 mmol dm⁻³. The solution was adjusted around the isoelectric point of each amino acid to use 0.1 mol dm⁻³ HCl and/or Na(OH). Several diluted solutions were used in an adsorption treatment.

The FNWs were made according to Miayzawa et al [22]. C₆₀ molecules formed aggregates with diameters of approximately 1.8 μm. The FNWs were 645 ± 170 nm wide and 6.6 ± 2.5 μm long. A scanning electron miscroscopy (SEM) image and an illustration of the FNWs are shown in figure 1.

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Around 0.02 g of C₆₀ or FNWs was weighed by an analytical scale, as an adsorbent. The correct weight was used in estimating adsorption. The weighed adsorbent and 6.5 cm³ of an amino acid solution were put in a sample bottle with a stopper. After the stopper was tightly closed, the sample bottle was shaken for 3 h by a rotational shaker (RKVSE, ATR, USA). The rotational speed was 60 rpm. After shaking the sample bottle, the equilibrium pH was measured (pH meter TPX-999, Toko chemical Laboratories Co. LTD). PHs of the suspension after the adsorption treatments were different from the pH of the initial solution. The pHs of the suspension are shown in table 1. The suspension was filtered using a 0.2 μm disposal filter. The initial solution and treated solution were measured by a total organic carbon analyzer (TOC-5000A, Shimadzu, Japan). The TOC measured the infrared absorption of a C = O bond. The solution is sprayed into a silica tube with catalysis in a furnace at 680 °C in which the highly purified air is flown. Organic compounds are burned, the CO₂ gas produced was carried to the CO₂ detector with the flowing gas, and the concentration of carbon dioxide was measured. We used glycine as a standard material. One sample was measured seven or eight times. We chose five to eight raw data for which the CV value was the smallest or less than 0.01. A CV value is the ratio of (standard deviation)/(average value). Concentrations of the initial and treated solution were obtained by the average of the chosen data. Adsorption was evaluated by A = V(C-C₀)/W, where A is adsorption, V is volume of solution, C is the concentration of the treated solution, C₀ is the concentration of the initial solution and W is the weight of adsorbent.

The amino acids used are classified by the side chain. Ala, Val and Leu have an alkyl chain, Asp has a carboxyl group, Arg has an amino group and Phe, Tyr, Trp and Pro have an aromatic and an indole group, and a cyclic structure, respectively.

Isotherms for the adsorption of amino acids by C₆₀ and FNWs are shown in figures 2–5. The side chains of Ala, Val and Leu were hydrophobic. Ala and Val were not adsorbed by C₆₀ or FNWs. However, Leu could be adsorbed slightly by FNWs (max. 0.0020 mmol dm⁻³) and C₆₀ (max. 0.0018 mmol dm⁻³) as shown in figure 2. Leu might be adsorbed by FNWs and C₆₀ in effect on the amino acid's side chain. However these extents of adsorption might be within the various errors. Ala, Val and Leu were seldom adsorbed by them. Amino acids, of which the side chain is an aromatic, an indole or of a cyclic structure, (e.g. Phe, Tyr, Trp and Pro) were also hardly adsorbed by C₆₀ and FNWs. Phe might be adsorbed slightly by FNWs (figure 3(a)). In the adsorption of Trp and Pro, one experimental point shows the extent of adsorption. The point has some highly experimental uncertainties though the pH range is not so wide in the adsorption treatment (table 1). FNWs might adsorb Tyr. And Asp and Arg, which have a positive or a negative charge, respectively, were also not adsorbed by the C₆₀ and FNWs (figures 4 and 5). In the adsorption treatment of Asp, the pH varied greatly (table 1). C₆₀ preferentially adsorbs Asp in concentrated solutions with pH 2–8. Arg is adsorbed by FNWs and C₆₀. Arg is adsorbed more strongly than other amino acids (see figures 2–5).

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The isotherms for the adsorption of Tri-Ala are shown in figure 6. Tri-Ala can be adsorbed by C₆₀ and FNWs. The charge of carboxyl and the amino group will not have a strong effect on the interaction of the side chain (methyl group) and C₆₀ and FNWs.

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We used typical amino acids, which had aromatic, alkyl, carboxyl and amino groups with no sulfur or hydroxide groups in the side chains. Most amino acids will not be adsorbed by C₆₀ and FNWs by virtue of physical and chemical interactions such as the electrostatic force, hydrogen bonding, and chemical reactions etc. Since Phe, Trp, Tyr and Pro have aromatic or cyclic side chains, we expected that the surfaces of C₆₀ and FNWs would interact with the side chain of those amino acids. The amino or carboxyl group of the amino acid frame probably has a charge like –NH³⁺ or –COO⁻ in the solution. The charge of the amino acids would prevent interaction between amino acids and C₆₀ or FNWs.

Although there have been hardly any experimental investigations into the adsorption of amino acids by C₆₀ and FNWs, theoretical simulation groups have investigated the interaction between amino acids and C₆₀ or C₈₀ fullerene. De Leon et al showed the interaction between 20 bio-amino acids and C₆₀ using theoretical simulation [5]. When we pay attention to the amino acids we used, the hydrogen of the frame of Ala, Asp, Phe or Pro was combined with C₆₀. The distance is from 0.272 (Asp) to 0.442 (Ala) nm. In the case of Val, Arg and Trp, there is also another weak bond between the hydrogen of the side chain and C₆₀. In addition, Leu and Tyr have three weak bonds with C₆₀ involving the hydrogen of the amino acid frame and the side chain. The oxygen of the carboxyl group of Leu was combined with C₆₀. On the other hand, the hydrogen of the amine group of the Tyr frame was combined with C₆₀ as the weak bond. They also showed the free energy of dissociation to increase in the order Val < Phe < Pro < Asp < Ala < Trp < Tyr < Arg < Leu. In their results, the hydrogen of the frame of amino acids combined with C₆₀ by a weak bond, while the side chain did not have a strong effect on the adsorption of amino acids and C₆₀. The combination of 20 bio-amino acids with C₈₀ fullerene was theoretically investigated by de Leon et al [6]. The free energies of dissociation of C₈₀ were generally small as compared with those of C₆₀. The hydrogen of the amino acid frame was connected with C₈₀ as the hydrogen bond. Other hydrogen and/or oxygen did not affect the adsorption behavior of C₈₀.

We did not observe significant adsorption of amino acids by C₆₀ and FNWs, contrary to theoretical predictions by de Leon et al [5]. Since Arg and Tyr were adsorbed by C₆₀ and FNWs, the adsorption of Arg and Tyr might affect the bonding. De Leon et al used an idealized model and neglected the charge of the amino and carboxyl groups of the frame such as –NH³⁺ and –COO⁻. When an amino acid dissolves in water, the amino group and/or carboxyl group has a positive and/or a negative charge. The reason why C₆₀ did not adsorb amino acids in our experiment is mainly related to the charge. C₆₀ prefers the hydrophobic interaction and will not prefer the hydrophilic materials in the adsorption behavior, that is to say that C₆₀ dissolves in toluene but does not dissolve in water, generally. An organic molecule with a hydrophilic group such as the amino and carboxyl groups will hardly be adsorbed by the C₆₀ surface.

In the interaction between the side chains of the amino acids with C₆₀ and FNWs, carboxyl and amino groups in the frame of the amino acid are mainly charged positively and/or negatively. If the charged carboxyl or amino group affects adsorption by C₆₀ and FNWs, the carboxyl and amino groups of peptides might have less effect on the interaction of the side chain with C₆₀ and FNWs, because the middle of the side chain of the peptide will not be close to the charged carboxyl or amino group. Figure 6 shows that Tri-Ala was adsorbed more by C₆₀ and FNWs than the Ala monomer.

The adsorption of proteins by C₆₀ was investigated [1, 23, 24]. It was demonstrated that small C₆₀ aggregates could adsorb human and bovine serum albumins. The results were almost the same, such that the side chain of Tyr or Trp was adsorbed by the C₆₀ aggregate. The structure of the albumin changed due to the adsorption by the C₆₀ aggregate. In particular, an α-helix changed due to the interaction between C₆₀ and the albumin. The side chains of Tyr (indole group) and Tyr (phenyl group) would react to the six-membered ring of C₆₀ by the hydrophobic interaction and the structure around Trp and Tyr would be slightly changes by the C₆₀. The concentrations of C₆₀ and albumin are also important for the adsorption between them. The interaction between C₆₀ and albumin was hindered at high and low concentrations of C₆₀ in the constant albumin solution. In addition, although a C₆₀ molecule did not affect the structure of albumin, a small C₆₀ aggregate affected it [1]. Trp was not adsorbed by C₆₀ and FNWs in this work. The charge of the carboxyl and amino groups have a strong effect on the adsorption of Trp. In the case of Tyr, its concentration might affect the adsorption behavior by C₆₀ and FNW. And also, the concentration of other amino acids solutions might affect the adsorption behavior to C₆₀ and FNW.

The interaction between graphene or single-wall carbon nanotubes (SWCNTs) and a protein was investigated by Zuo et al [8]. Trp and Tyr affected adsorption behavior. They also expected a similar adsorption mechanism, like hydrophobic interaction, between Trp or Tyr and graphene or SWCNTs.