Interaction of Human Serum Album and C60 Aggregates in Solution

An important property of C60 in aquatic ecotoxicology is that it can form stable aggregates with nanoscale dimensions, namely nC60. Aggregation allows fullerenes to remain suspended for a long time, and the reactivity of individual C60 is substantially altered in this aggregate form. Herein, we investigated the interaction of nC60 and human serum album (HSA) using the methods of fluorescence, fluorescence dynamics, circular dichroism (CD), and site marker competitive experiments. We proposed a binding model consistent with the available experimental results for the interactions of nC60 with HSA. During the interaction process, the structure and conformation of HSA were affected, leading to functional changes of drug binding sites of HSA.


Introduction
Since the discovery of fullerene (C 60 ) in 1985 [1], concerns have been raised about the proposed applications of C 60 and its derivatives in biology and pharmacology [2,3]. These potential applications include items as varied as antiviral, anticancer, or antioxidant agents [4], and drug delivery vehicles [5,6]. Despite the broad range of research focused on the application potential of fullerenes, their toxicological and environmental effects are still not well known [7]. With an octanol-water OPEN ACCESS partition coefficient log K OW of 6.67 and a solubility of less than 10 −9 mg/L, pristine fullerene is poorly soluble in water [8]. For C 60 fullerene, its interaction with proteins has been poorly studied due in particular to its low solubility in water, and hence much research has focused on the interactions between its water-soluble derivatives and some proteins such as HIV protease [9,10], a fullerene-specific antibody [11], human serum albumin (HSA) [12] and bovine serum albumin (BSA) [13].
An important property of C 60 in aquatic ecotoxicology is its acquisition of charge and ability to form relatively stable clusters (referred to as nC 60 ) in aqueous systems, by means of natural processes like water flow and mixing, as well as by vigorous stirring in the laboratory [14][15][16]. Aggregation allows fullerenes to remain suspended for weeks or months [17], this aspect of nC 60 may provide a more complete understanding of how it will behave in natural systems or in organisms.
In previous work, BSA was found to adopt a more flexible conformational state on the boundary surface of gold nanoparticles as a result of the conformational changes in the bioconjugates [18]. It was also demonstrated that C 60 nanoparticles can be stabilized by nonspecific adsorption of HSA and remain well dispersed even in the physiological environment [19]. Therefore, for further understanding of the transportation of nC 60 fullerene in the bodies and the resulting related bio-effects, the interaction of nC 60 fullerene with proteins is crucial and must be ascertained. In this study, the interaction between human serum albumin and nC 60 in aqueous solution was investigated by fluorescence, fluorescence dynamics, circular dichroism (CD), and binding site marker competitive experiments.

The Fluorescence Quenching of HSA by nC 60
Quenching refers to any process which decreases the fluorescence intensity of a given substance. A variety of processes can result in quenching, such as excited state reactions, energy transfer, complex-formation and collisional quenching. Figure 1 shows the fluorescence spectra of HSA in the absence and the presence of nC 60 in phosphate buffer with an excitation wavelength of 280 nm. HSA displayed the maximum emission at a wavelength of 343 nm. The fluorescence intensity of HSA decreased with increasing concentration of nC 60 and the maximum emission wavelength was slightly red shifted. It is known that the shift of maximum emission wavelength corresponded to a polarity change around the chromophore residues. Red shift indicates that the microenvironment of fluorophors in HSA is changed after addition of nC 60 . aggregates. HSA concentration was 20 μM. The concentration of C 60 (from A to F) was 0, 1.41, 2.83, 5.66, 8.48, and 11.30 μM, respectively (pH = 7.4; ex = 280 nm).

Quenching Mechanism of nC 60 to HSA
For dynamic quenching, the quenching is an additional process that deactivates the excited state besides radiative emission through the collision between the quencher and fluorophore. As for HSA, the dependence of the emission intensity on quencher concentration ] [Q is given by the Stern-Volmer equation: where F and F 0 are the fluorescence intensity in the absence and in the presence of quencher, respectively; 0 τ is the lifetime of HSA; K q is the bimolecular rate constant for the dynamic reaction of the quencher with the fluorophore; K sv is called the Stern-Volmer constant.
For static quenching, the binding of a quencher ( Q ) to a protein ( M ) can be described with the following reaction: Then the binding constant ( K ) is given by The relation between the fluorescence intensity and concentration of quencher [ Q ] is given by The quenching data were presented as a plot of F F 0 versus ] [Q , and the Stern-Volmer plot is an upward curvature, concave towards the y-axis, as shown in Figure 2A. This result indicates that HSA can be quenched both by collisions and by complex formation with C 60 nanoparticles. Then the fractional fluorescence remaining ( where f is the fraction not complexed, and ]) [ is the fraction not quenched by collisional encounters. According to the definition, f can be obtained from Equation (5), and then rearrangement of Equation (6) yields: The nonlinear fitting analysis was performed by plotting ) Figure 2B). From the Stern-Volmer plot, the value of K SV was 6.56 × 10 4 L·mol −1 . The q K was easily calculated according to Equation (1), 6.56 × 10 12 L·mol −1 ·s −1 , which is much higher than the maximal collided dynamic quenching constant (2.0 × 10 10 L·mol −1 ·s −1 ) [20]. This result indicates that the fluorescence quenching of HSA by the addition of nC 60 is mainly caused by static quenching. There is non-linearity obtained from Stern-Volmer when the nC 60 concentration is lower than 11.30 μM. When nC 60 concentration is higher than 11.30 μM, the intrinsic fluorescence of HSA is significantly quenched. The plot appears to be an upward curvature with increasing nC 60 concentration, which is a characteristic feature of mixed quenching. This suggests that it is not a single quenching mechanism that exists in the binding process. The mechanism of HSA quenching caused by water-soluble pristine nC 60 is different to that caused by water-soluble nC 60 derivative [12].

The Conformation Study of HSA
Synchronous fluorescence spectroscopy is a common method used to provide information about conformational changes of protein since the possible shift of maximum emission wavelength is related to the polarity of the environment. The synchronous fluorescence spectra of HSA-nC 60 system are shown in Figure 3. The maximum emission wavelength of tyrosine residues has a small red shift (from 283 nm to 285 nm) when Δλ = 15 nm, indicating that there are some changes in the environment of the tyrosine residues. The maximum emission wavelength of tryptophan residues red shifts from 279.5 nm to 281.5 nm when Δλ = 60 nm ( Figure 3B). This suggests that there is a less hydrophobic or more polar environment change around the tyrosine residues and tryptophan residues. This may be ascribed to the fact that the hydrophobic amino acid structure surrounding tryptophan residues in HSA tends to collapse slightly, resulting in tyrosine residues and tryptophan residues being more exposed to the aqueous phase. Circular dichroism spectra can sensitively monitor conformation changes in the protein upon interaction with the ligand. In this experiment, the calculated α-helicity content of native HSA solution is 54.9%, and with the addition of nC 60 to the native HSA solution, the α-helicity content of HSA increased to 59.8%, 61.2% and 62.0% (Figure 4). Apparently, the higher the added nC 60 concentration, the more the α-helicity content. The increase of α-helicity content indicated that the binding of HSA and nC 60 induces protein folding, which results in some hydrophobic regions becoming more compact.

Interaction between HSA and nC 60
It is well known that HSA has two major drug binding sites, site I and site II, which are located in the hydrophobic pocket of sub-domain IIA and sub-domain IIIA, respectively. In order to identify the nC 60 -binding site on HSA, two probes were used. One probe of HSA is dansylamide (DNSA), whose binding site is located in the region of sub-domain IIA (sudlow site I); another probe, dansylproline (DP) is bound to HSA in the sub-domain IIIA (sudlow site II) [21,22]. During the experiment, nC 60 was gradually added to the solution of HSA with site probes held in equimolar concentrations (20 μM). No absorption of these two probes to nanoparticles of C 60 was observed in capillary electrophoresis analysis (data not shown). As shown in Figure 5, only the probes or HSA alone have no fluorescence signal within the wavelengths 400-600 nm. When the probes and HSA are mixed together, the DNSA-HSA complex has a maximum emission wavelength at 487 nm (excitation wavelength 350 nm) and DP-HSA has a maximum emission wavelength at 480 nm (excitation wavelength 375 nm). With the addition of nC 60 to the HSA-probe solution, the fluorescence intensity of the HSA-probe complex was significantly lower than that without nC 60 , indicating that the binding of DNSA/DP to HSA was affected by adding nC 60 . The fluorescence intensity of DNSA-HSA was lower than that of DP-HSA, indicating that nC 60 could more strongly influence the binding of DNSA to HSA than that of NP to HSA.

Representation of Interaction of HSA and nC 60
Our results above indicate that the fluorescence quenching and conformational changes of HSA can be attributed to the interaction between HSA and nC 60 . C 60 fullerene has also been reported to be bound to the subdomain IIA of HSA using time-resolved fluorescence decay experiments, docking calculations, and binding site alignment methods [13,23]. Here we have employed several approaches in order to address the issue of fullerene interactions with proteins. What needs to be especially pointed out is that it is difficult to incorporate nC 60 into the binding pocket because the size of nC 60 is larger than protein molecules. The hydrodynamic diameter of the nC 60 nanoparticles was determined to be from 50 nm to 110 nm by TEM and DLS, respectively ( Figure 6A,B). Ganazzoli et al. reported atomistic computer simulations of some albumin subdomains on a hydrophobic graphite surface [24]. According to these simulations, we proposed a binding model to describe the interaction between HSA and nC 60 ( Figure 6C). In this model, HSA is adsorbed on the surface of nC 60 aggregate which leads to the fluorescence quenching and conformational changes of HSA. The interaction sites were presumed to be near to Site I and Site II of HSA; following the structure and conformation of HSA was affected during adsorption. In our previous study, we have observed that HSA could compete for the surface of nC 60 with functional enzymes [25].

Materials
C 60 (99.5% purity) was obtained from Sigma Chemical Company, St. Louis, USA. HSA (98% purity, essentially fatty acid and globulin free) was purchased from NEB (USA). All solutions were prepared in ultrapure water with the resistivity of 18.2 MΩ⋅cm (ALGA system, UK). Phosphate buffer (10 mM, pH = 7.4) was prepared with chemicals of analytical pure grade, obtained from commercial sources, and filtered with a 0.22-µmm filter (Millipore, Bedford, MA, USA). All other materials were of analytical pure grade.

Preparation and Characterization of Water-Soluble nC 60
C 60 suspensions were prepared according to the method described previously [26]. Ten milligrams of C 60 was completely dissolved in 10 mL toluene. Then 2.5 mL C 60 toluene solution was added into 50 mL water. After ultrasonic treatment for approximately 4 h, toluene was allowed to evaporate under vacuum for at least 1 h. The mean hydrodynamic diameter of the nC 60 nanoparticles was determined by transmission electron microscopy (TEM) and dynamic light scattering (DLS) (BI-20OSM laser light scattering spectrometer, Brookhaven, USA).

Methods
Absorption spectra were recorded on a UV-2102PCS UV-Vis spectrophotometer (UNICO, USA) equipped with a 1.0 cm quartz cuvette at room temperature. The wavelength range was recorded from 200 to 500 nm.
All fluorescence emission spectra were recorded in the range of 300-450 nm on a LS-55 spectrofluorimeter (PE, USA) equipped with 100 µL quartz cells. The bandwidth of excitation and emission (3 nm) and scan speed (300 nm/min) were kept constant within each data set. Fluorescence intensity at 343 nm was used for calculation in Stern-Volmer equation. All measurements were performed at room temperature (~25 °C).
Binding site marker competitive experiments were carried out by the fluorescence titration methods. The concentration of HSA and dansylamide/dansylproline (DNSA/DP) were all kept at 10 μM. Then nC 60 was gradually titrated into the HSA-DNSA/DP systems. Fluorescence spectra were recorded with an excitation wavelength of 370 nm (for DNSA) and 375 nm (for DNP) in the range of 400-600 nm.
Circular dichroism (CD) spectroscopy was performed on a J-810 spectropolarimeter (Jasco Co., Japan) over a wavelength of 200-260 nm. The scanning speed was set at 500 nm/min. Each spectrum was the average of three successive scans and appropriate buffer solutions running under the same conditions were taken as blank and their contributions were subtracted from the experimental spectra.

Conclusions
We observed a quenching of fluorescence of HSA in the presence of nC 60 , and fluorescence quenching mechanism was investigated by the Stern-Volmer equation which showed a characteristic feature for the combined fluorescence quenching. The binding of HSA to nC 60 increases the polar environment of the Trp residue, resulting in a red shift in the fluorescence spectra. The CD results showed that the percentage of α-helicity of HSA increased, revealing that the protein becomes more compact upon association with nC 60 . The results indicate that the fluorescence quenching and conformational changes of HSA can be attributed to the interaction between HSA and nC 60 . In the binding site marker competitive experiment, the binding of DNSA/DP to HSA was affected by adding nC 60 . We propose that HSA interacted with aggregate nC 60 surface, and led to the functional changes of drug binding sites of HSA.