Bioconjugate Chem. 2005, 16, 1058−1062
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ARTICLES Formation and Characterization of Stable Human Serum Albumin-Tris-malonic Acid [C60]Fullerene Complex Bogdan Belgorodsky,§ Ludmila Fadeev,§ Varda Ittah,† Hadar Benyamini,‡ Stanislav Zelner,§ Dan Huppert,§ Alexander B. Kotlyar,⊥ and Michael Gozin*,§ School of Chemistry, Faculty of Exact Sciences, and Department of Biochemistry and Bioinformatics Unit, Faculty of Life Sciences, Tel Aviv University, Tel Aviv, 69978, Israel, and School of Chemistry, Faculty of Exact Sciences, Bar Ilan University, Ramat Gan, 52900, Israel. Received April 3, 2005; Revised Manuscript Received June 2, 2005
The preparation and characterization of the stable human serum albumin (HSA)-C3 isomer of trismalonic acid [C60]fullerene complex is reported. Other than the anti-fullerene antibody, a stable protein-fullerene complex with a native protein has never been observed. This study may provide valuable answers to the growing concern regarding the effects of carbonaceous nanomaterials on human health on one hand and, on the other, may lead to the development of novel antioxidant therapeutic agents, radiopharmaceuticals, and components for bioelectronic devices.
INTRODUCTION
Since the discovery of fullerenes (1, 2), a broad range of bioactivity was found for various fullerene-derived materials. This includes antiviral and antibacterial properties, antioxidant and neuroprotective activities, cell signaling and apoptosis, and compounds with the potential to be developed as anticancer drugs and diagnostic agents (3-6). A number of fullerene derivatives were reported to interact with proteins (7-11) and even function as enzyme inhibitors (3-6, 12). However, other than the anti-fullerene antibody (13), a stable and welldefined protein-fullerene complex with a native protein has never been observed. Here we report the preparation and structural and functional characterization of a complex formed between human serum albumin (HSA) and the eee-isomer of tris-malonic acid [C60]fullerene (CF) (14). The study of such complexes may provide valuable answers to the growing concern regarding the effects of carbonaceous nanomaterials on human health (15-16) on one hand and, on the other, may lead to the development of novel functional biomaterials. EXPERIMENTAL PROCEDURES
Complex Preparation. CF was synthesized according to the previously reported methods (17-19). Fatty acid and globulin free HSA, fine chemicals and solvents were purchased from Sigma-Aldrich. A solution of 100.0 mg * To whom correspondence should be addressed. Tel: (+972) 3-640-5878, Fax: (+972) 3-640-5879, E-mail: cogozin@ mgchem.tau.ac.il. § School of Chemistry, Faculty of Exact Sciences, Tel Aviv University. ‡ Bioinformatics Unit, Faculty of Life Sciences, Tel Aviv University. † School of Chemistry, Faculty of Exact Sciences, Bar Ilan University. ⊥ Department of Biochemistry, Faculty of Life Sciences, Tel Aviv University.
of HSA and 3.1 mg of CF (3.0 µmole) in 1.5 mL of trisacetate buffer (100 mM, pH 7.2) was incubated for 12 h at RT. Then, the complexation solution was loaded on a Sephadex G-25 gel-permeation column (Pharmacia Biotech) or on HPLC TSK-GEL column (as described below) and noncomplexed CF was removed by elution with a tris-acetate buffer (1.0 M, pH 7.2). The buffer concentration was reduced by reloading the complex-containing fraction on the second Sephadex G-25 column and eluting the complex with a tris-acetate buffer (1.0 mM, pH 7.2). The resulting solution of the complex was lyophilized for storage and further experiments. After reconstitution in water, the complex concentration in solution was determined by UV-vis spectroscopy and a BioRad protein assay (Bio-Rad Lab). All UV-vis spectra were recorded at RT on a S-3150 diode array spectrophotometer (Scinco). Size-Exclusion Chromatography. Size-exclusion high performance liquid chromatography of a monomeric HSA, CF and HSA-CF complex was performed on a 300 × 7.8 mm G3000SWXL TSK-GEL column (Tosoh Biosep) using a tris-acetate buffer (100 mM, pH 7.2) as the isocratic eluent. All HPLC analyses and separations were performed on the SpectraSYSTEM equipped with a UV6000-LP diode array detector (ThermoFinnigan). Time-Resolved FRET Spectroscopy. Time-resolved fluorescence was measured using the time-correlated single-photon counting (TCSPC) technique. As an excitation source, we used a CW mode-locked Nd:YAG-pumped dye laser (Coherent Nd:YAG Antares and a 702 dye laser), providing a high repetition rate (>1 MHz) of short pulses (2 ps at full width half-maximum, fwhm). The (TCSPC) detection system is based on a Hamamatsu 3809U, photomultiplier, Tennelec 864 TAC, Tennelec 454 discriminator and a computer-based multichannel analyzer (nucleus PCA-II). The overall instrumental response was about 50 ps (fwhm). Measurements were taken at 10 nm spectral width.
10.1021/bc050103c CCC: $30.25 © 2005 American Chemical Society Published on Web 07/21/2005
HSA−Tris-malonic Acid [C60]Fullerene Complex
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Figure 1. (a) Size-exclusion chromatogram of a sample containing HSA-CF complex (tR ) 11.45 min) and an unbound CF ligand (tR ) 38.57 min). (b) Size-exclusion chromatogram of a monomeric HSA reference standard. Both chromatograms a and b were monitored by diode array detector (200-800 nm) and reported here at 240 nm. (c) UV-vis spectrum obtained for an HSA-CF complex peak in chromatogram a. (d) UV-vis spectra obtained for a CF’s peak in chromatogram a. (e) UV-vis spectrum obtained for an HSA reference standard in chromatogram b.
Figure 2. Deconvoluted ESI-mass spectra of (a) HSA-CF complex and (b) monomeric HSA. RESULT AND DISCUSSION
Complex Formation. The HSA-CF complex formation was obtanied at physiological pH, by incubation of the fatty acid free native HSA solution with a 2-fold
excess of CF. The resulting HSA-CF complex was found to be stable enough to be purified by size-exclusion liquid chromatography, and sustained variations in pH, lyophylization, and reconstitution. The size-exclusion chromatorgaphic conditions, suitable for separation of HSA protein monomers and dimers, were used for analysis and purification of the complex from an excess of an unbound CF ligand. Under these conditions the retention time (RT) of the HSA-CF complex elution (Figure 1a) closely resembled the RT of a ligand-free monomeric HSA (Figure 1b), while the complex’s UV-vis spectrum clearly indicated incorporation of fullerene chromophore (Figure 1c). The excess of the noncomplexed CF was eluted much later, with the RT and UV-vis spectrum (Figure 1d) matching a separately analyzed CF reference standard. UV-vis analysis and standard BioRad assay of the purified HSA-CF complex revealed that only a single CF molecule is bound by a monomeric HSA host. These findings were confirmed by electrospray mass spectroscopy analysis, unambiguously showing that the molecular mass of the complex (Figure 2a) corresponds to a sum of masses of monomeric HSA (Figure 2b) and one CF ligand (see Supporting Information, Figure S1). Binding Constant by Fluorescence Titration Studies. To quantitatively determine the stability of the
Figure 3. (a) Reduction in HSA’s Trp214 fluorescence during a titration with CF. To 1 µM of HSA solution in 10 mM tris acetate buffer (pH 7.2) were added (solution concentration of CF was increased from 0 to 2.8 µM) aliquots of CF until no substantial changes in the emission intensity were detected. The excitation was performed at λ ) 280 nm, and emission was measured at λmax ) 345 nm. (b) Graph describing the intensity of the HSA’s fluorescence emission (at 345 nm) as a function of CF concentration ([) and calculated binding isotherm (intermittent line).
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Figure 4. (a) Experimental fluorescence decay curves of monomeric HSA (blue) and HSA-CF complex (black). The excitation was performed at λ ) 280 nm and emission was measured at λmax ) 345 nm. (b) The distance distribution between the HSA’s Trp214 and the CF.
complex, a binding study was performed by gradual addition of CF to HSA. The HSA-CF complex formation was measured by monitoring the HSA's Trp214 fluorescence quenching in solution, upon addition of the CF aliquots (Figure 3a). Fitting the titration results to a theo-
Belgorodsky et al.
retical rectangular hyperbola curve (binding isotherm), calculated by nonlinear least-mean-square algorithm (20), produced a binding constant for CF of 1.2 × 107 M-1 (Figure 3b). tr-FRET Experiments and CF Location Assessment. To assess the intramolecular distance between HSA’s Trp214 donor and the CF acceptor in the complex, time-resolved fluorescence decay (tr-FRET) experiments of ligand-free HSA protein and HSA-CF complex were performed (Figure 4a). Theoretical decay curves were calculated by numerical solution of the second-order differential equation for each tr-FRET experiment and fitted to the corresponding experimental curves (21, 22). The distance distribution function was obtained from simultaneous global analysis of two experimental fluorescence decay curves, using the Marquardt nonlinear least-squares method (Figure 4b, for calculation details see Supporting Information) (22,23). It was found that in HSA-CF complex the mean distance between the Trp214 donor and CF acceptor is 7.1 Å (with fluctuations of (3.4 Å). Calculations and Docking Studies. An additional attempt to estimate the location of CF in HSA’s threedimensional structure was based on the molecular docking program. Coordinates of the known structure of the HSA protein (24) and a Hartree-Fock energy-minimized structure of the CF ligand (see Supporting Information) were used for building plausible docking models of the complex. The PatchDock program was applied without using any a priori data indicating a CF putative binding site (25). An examination of generated docking models revealed that the eight highest ranked solutions posi-
Figure 5. (a) The overall view of the highest score docking model of the HSA-CF complex, in which the HSA is presented by its van der Waals surface and a space-filling model of CF (in red) is located in its binding pocket. (b) Close-up view of the CF-binding pocket in HSA’s subdomain IIA, showing close proximity between the HSA’s Trp214 (in blue) and the CF ligand (in red).
Figure 6. (a) Graph presenting changes in concentrations of Cytochrome c (FeII) at pH 7.4 (monitored at 550 nm), due to superoxide scavenging activity of HSA-CF complex added at various concentrations (0-1.0 mM of the complex). (b) Graph comparing the scavenging activity of the free HSA (b), the HSA-CF complex (red 2), and the noncomplexed CF ([) at various concentrations. Scavenging activity is defined as ([V - b] × 100/V), where V is the rate of cytochrome c (FeII) formation during the linear phase (0-7 min) of a control experiment (without antioxidants); and b is the rate of cytochrome c (FeII) formation during the linear phase in the presence of tested antioxidants in various concentrations. Scavenging activity of the noncomplexed CF, at concentrations above 0.6 mM (]), was found to be unusually high, possibly due to assay readings interference by CF’s absorbance or CF clusters formation.
HSA−Tris-malonic Acid [C60]Fullerene Complex
tioned CF in the same cavity, with differences mostly in the ligand’s orientation. In all these models, the CF was found located at a distance of approximately 5 Å from the Trp214 residue (Figure 5), which is in excellent agreement with our tr-FRET experiments’ global analysis. Determination of SOD-like Activity for Complex by in Vitro Xanthineoxidase/Cytochrome C Assay. CF was extensively investigated as a free radical scavenger in vitro (17-19) and in vivo (26) and was recently described as a superoxide dismutase mimetic (27). Free HSA protein has also been reported to have antioxidant activity (28). To examine the antioxidant properties of the new HSA-CF complex, a standard xanthine oxidase/ cytochrome c assay was utilized (Figure 6a) (29, 30). It was found that the ability of HSA-CF complex to quench superoxide radicals was slightly lower than that of the free HSA protein and was measurably higher than of the free CF ligand (Figure 6b). Although no additive antioxidant activity was observed for the HSA-CF complex, the free radical scavenging capabilities of the HSA protein were almost unaffected. Our experimental data and docking calculations lead to the conclusion that an amphiphilic, negatively charged CF ligand is bound in the HSA’s subdomain IIA (a binding site formed by hydrophobic side chains with the entrance to the pocket surrounded by positively charged residues) (31, 32). The binding constant of the CF was found to be comparable with published values for other organic molecules that strongly bind to same site of HSA, such as bilirubin (9.5 × 107 M-1), iodipamide (9.9 × 106 M-1), and 3-carboxy-4-methyl-5-propyl-2-furanpropanoate (1.3 × 107 M-1) (32). We believe that, in addition to improved water solubility and biocompatibility, stable protein-fullerene derivative complexes have the potential to be developed as antioxidant therapeutic agents, target-specific radiopharmaceuticals, or components for future bioelectronic devices. ACKNOWLEDGMENT
This work was supported by the Israel Science Foundation and the Multiple Sclerosis Center Foundation of Brigham & Women’s Hospital (Massachusetts General Hospital). Supporting Information Available: Materials, characterization of HSA-CF complex by mass spectroscopy, determination of CF binding constant by fluorescence spectroscopy, and determination of distance between HSA’s Trp214 and CF in HSA-CF complex by global analysis of time-resolved FRET spectroscopy. This material is available free of charge via the Internet at http:// pubs.acs.org. LITERATURE CITED (1) Kroto, H. W., Heath, J. R., O’Brien, S. C., Curl, R. F., and Smalley, R. E. (1985) C60: buckminsterfullerene. Nature 318, 162-163. (2) Kraetschmer, W., Lamb, L. D., Fostiropoulos, K., and Huffman, D. R. (1990) Solid C60: a new form of carbon. Nature 347, 354-358. (3) Bosi, S., Da Ros, T., Spalluto, G., and Prato, M. (2003) Fullerene derivatives: an attractive tool for biological applications. Eur. J. Med. Chem. 38, 913-923. (4) Pantarotto, D., Tagmatarchis, N., Bianco, A., and Prato, M. (2004) Synthesis and biological properties of fullerenecontaining amino acids and peptides. Mini-Rev. Med. Chem. 4, 805-814.
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