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Multiple fold increase in activity of ferroxidase–apoferritin complex by silver and gold nanoparticles
BASIC SCIENCE Nanomedicine: Nanotechnology, Biology, and Medicine 9 (2013) 185 – 193
Research Article
nanomedjournal.com
Multiple fold increase in activity of ferroxidase–apoferritin complex by silver and gold nanoparticles Afolake Sennuga, PhD, Jacqueline van Marwijk, PhD, Chris G. Whiteley, PhD⁎ Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, Grahamstown, South Africa Received 12 December 2011; accepted 25 May 2012
Abstract The effect of silver (Ag) and gold (Au) nanoparticles on the ferroxidase activity of apoferritin showed a 110-fold increase in specific activity and a 9-fold increase over the control at the respective molar ratios of Au-apoferritin and Ag-apoferritin nanoparticles (NPs) of 500:1 and 1000:1. Typical color change, from pale yellow to brown, occurred when apoferritin was mixed with AgNO3 or AuCl3 followed by sodium borohydride to afford respective metal-apoferritin NP complexes in a ratio of between 250:1 and 4000:1. These complexes were characterized by ultraviolet-visible inductively coupled plasma–optical emission spectroscopy, Fourier transform infrared spectroscopy, transmission electron microscopy, and energy-dispersive x-ray spectroscopy. Transmission electron microscopy revealed that the size of NPs increased as the molar ratio of metal to apoferritin increased, with an average size of 3–6 nm generated with Au-to-apoferritin and/or Ag-toapoferritin molar ratios of 250:1 to 4000:1. Fourier transform infrared spectrometry showed no structural changes of apoferritin when the NPs were attached to the protein. From the Clinical Editor: In this paper the utility of gold and silver nanoparticles in augmenting the activity of the ferroxidase-apoferritin complex is described. Both NPs dramatically increased the ferroxidase activity. © 2013 Elsevier Inc. All rights reserved. Key words: Silver nanoparticles; Gold nanoparticles; Apoferritin; Ferroxidase activity
The multidisciplinary field of nanotechnology, which refers to characterization, synthesis, and applications of functional particles on a nanoscale (10 –9 m), has engulfed a myriad of new opportunities in health care, environmental biology, chemical and material sciences, and communications. 1 One of the fundamental principles that set this type of technology apart from traditional standard protocols is that as the particle itself becomes smaller and smaller, its association with biomacromolecules changes, inevitably leading these molecules to behave differently than their native counterparts. Though biological syntheses of noble metal nanoparticles (NPs) through the bioreduction of metal salts by both prokaryotic and eukaryotic organisms 2–8 are more cost-effective, simple, and
The authors thank the National Research Foundation (South Africa), Rhodes University Research Council, and Third World Organisation for Women in Science (TWOWS) for financial assistance. ⁎Corresponding author: Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, P.O. Box 94, Grahamstown 6140, South Africa. E-mail address: [email protected] (C.G. Whiteley).
more eco-friendly than the more hazardous chemical approach, a limitation remains in the form of the ability to control the mechanism that determines particle size and shape. 9 The use of a protein cavity or cage as a limited-growth field for NPs is not novel 10 and serves as an ideal template to confine particle growth in a homogeneous distribution as well as a stabilizer against particle aggregation. Apoferritin, a globular protein of approximately 440 kDa in 24 identical subunits that has ferroxidase activity and catalyzes the oxidation of Fe 2+ to Fe 3+ in the presence of molecular oxygen as an electron acceptor, is one such cage. The subunits form a spherical protein shell of 12 nm with an internal aqueous cavity of 8 nm. 11 Apart from Fe 3+, several other zero-valent apoferritin-encapsulated metal NPs have been prepared within this protein cage, and these include palladium, 12 platinum, 13 copper, 14 cobalt and nickel, 11 and cadmium. 15 Recently we reported 16 on enhanced activity of ferroxidase in apoferritin in the presence of platinum NPs of a defined size. To the best of our knowledge, this was the first time that such an increased activity of this enzyme in the presence of NPs had been reported, and consequently we were interested in extending this idea by considering the effect of gold (Au) and/or silver (Ag) on this enzyme's activity. We anticipated that apoferritin associated
1549-9634/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nano.2012.05.020 Please cite this article as: Sennuga A., van Marwijk J., Whiteley C.G., Multiple fold increase in activity of ferroxidase–apoferritin complex by silver and gold nanoparticles. Nanomedicine: NBM 2013;9:185-193, http://dx.doi.org/10.1016/j.nano.2012.05.020
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with the Ag-NP and/or Au-NP would have a wide range of affinities and activities dependent on the size of the NP.
Methods Materials Horse spleen apoferritin (HSAF), gold chloride (AuCl3), silver nitrate (AgNO3), and ferrous ammonium sulfate [(NH4)2Fe(SO4)2⋅6H2O] were obtained from Sigma-Aldrich (Johannesburg, South Africa), and sodium borohydride (NaBH4) was purchased from Merck (Johannesburg, South Africa). All other reagents were of analytical grade. Synthesis of metal-HSAF NP complex Metal (M) NPs of Ag and Au were synthesized within HSAD as described. 17 Briefly, HSAF (10 μM) in Tris-buffered saline (50 mM Tris, 150 mM NaCl, pH 8) was incubated (60 minutes, 4°C) with varying volumes of AuCl3 or AgNO3 (0.1 M) to give a theoretical loading of M/HSAF ratio 1:250–1:4000. While maintaining the pH at pH 8 with 0.1 M NaOH, NaBH4 at five times the metal concentration was added and the solution stirred for a further 60 minutes until the sample color turned black (Au) or deep orange (Ag). The solution was then dialyzed (24 hours) against Tris buffer (10 mM, pH 8), changing every 6 hours, centrifuged (2 minutes, 5000 rpm), and filtered through a 0.22-μm filter. The sample (250 μL) was purified by size exclusion chromatography (Sephadex G-50, 1.8 × 15 cm) and the elution of the M-HSAF NP complex monitored at 280 nm. Protein content was determined by Bradford assay, and the purity and stability of the complex was assessed by native polyacrylamide gel electrophoresis.
(Perkin-Elmer, Boston, Massachusetts). Apoferritin without NPs was used as the control. Transmission electron microscopy (TEM) Samples for TEM analysis were prepared by placing a drop of the M-HSAF NP sample onto carbon-coated copper grids; excess sample was removed after a minute using blotting paper and the grids air-dried before analysis. Duplicate samples were prepared and negatively stained using 1% uranyl acetate. Mean particle size and standard deviations were determined by the analysis of 200 particles using the computer software Scandium (Olympus, Melville, New York). Electron micrographs were taken using a JEOL JEM-1210 transmission electron microscope (JEOL, Tokyo, Japan) operating at 80 keV. Energy-dispersive x-ray spectroscopy (EDX) Freeze-dried samples were placed on graphite tape, which was in turn placed on an aluminium stub. Elemental analysis was performed with a TESCAN scanning electron microscope with an EDX scanner (INCAPentalFeTx3; Oxford Instruments, Brno, Czech Republic) operating at 20 keV. Assay of HSAF-ferroxidase activity HSAF-ferroxidase activity was determined at 420 nm according to a published procedure 18 with a slight modification. HSAF (1 μM) in sodium borate–cacodylate buffer (50 mM, pH 5.5) was incubated at 22°C with ferrous ammonium sulfate [(NH4)2Fe(SO4)2⋅6H2O] (50 mM) and the change in absorbance monitored at 420 nm for 4 minutes. A control containing only ferrous ammonium sulfate was used as the blank. The amount of ferritin (Fe 3+) produced was estimated from a standard curve. Effect of Ag-NPs and/or Au-NPs on ferroxidase activity of HSAF
Characterization of M-HSAF NP complex Ultraviolet-visible (UV-vis) spectroscopy UV-vis absorption spectra (250–700 nm) of the M-HSAF NP complexes were obtained using a UV spectrophotometer (Spectroquant Pharo 300; Merck) to evaluate the plasmon bands associated with the Ag-NPs and/or Au-NPs.
This assay was performed in exactly the same way as that described above for HSAF-ferroxidase activity except that each of the Ag-HSAF or Au-HSAF NP complexes (1 μM) was used. The negative control contained only HSAF and substrate, whereas the positive control contained HSAF attached to Ag-NPs or Au-NPs. Protein determination
Inductively coupled plasma–optical emission spectroscopy (ICP-OES) M-HSAF NP complexes (250 μL) were dissolved in aqua regia (1:3 nitric to hydrochloric acid; 350 μL for Au; 500 μL for Ag) and digested with hydrogen peroxide (1.0 mL, 60°C, 1 hour). Metal quantification was carried out using an inductively coupled optical emission spectrophotometer (iCAP 6000 series; Thermoelectron, Cambridge, United Kingdom) after suitable standard curves were constructed with varying concentrations of respective metal salt standards (Sigma-Aldrich).
The protein concentration for all experiments was routinely determined, in triplicate, according to the method of Bradford. 19 Into a 96-well titer plate was placed either protein-NP sample (5 μL) followed by Bradford reagent (245 μL). The mixture was incubated (22°C, 10 minutes), the absorbance of the solution measured at 595 nm, and the concentration of the unknown sample determined using a bovine serum albumin standard curve.
Fourier transform infrared (FT-IR) spectrometry Freeze-dried samples of each M-HSAF NP complex were analyzed from 600 to 4000 cm −1 with a scan resolution of 4 cm −1 using a Perkin-Elmer 100 FT-IR spectrophotometer equipped with a universal attenuated total reflectance sampling accessory
All analyses were carried out in triplicate and values reported as the means with standard deviation P b 0.05 vs. controls. Where necessary, analysis of variance was conducted using Statistica for Windows, version 8 (Statsoft Inc., Tulsa, Oklahoma) and Microsoft Excel 2010.
Statistical analyses
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Figure 1. Color differentiation of (A) Au-NPs and (B) Ag-NPs synthesized with varying molar concentrations of metal salts to a fixed concentration of HSAF (250:1–4000:1 M/HSAF). Control, HSAF alone.
Results Synthesis of M-HSAF NP complex M-HSAF NP complexes of Ag and/or Au, as seen under TEM imaging were formed after HSAF was incubated with varying concentrations of AuCl3 or AgNO3 (60 minutes, 4°C, pH 8) followed by treatment with NaBH4 (5 equivalents). Excess metal was removed by dialysis, and any aggregated metal NPs that were not stabilized by HSAF were removed by centrifugation (5000 rpm). The preparation of the complex was carried out at pH 8 so as to increase the electronegativity of the interior of HSAF, thereby improving electrostatic attraction between positive metallic cations and the core of HSAF. Different molar concentrations of metal salts were incubated with a fixed concentration of HSAF so as to vary the particle size. 20,21 The change in NP size was determined by TEM analysis, and a variation in molar ratios of MHSAF was used to determine the saturation point of HSAF in terms of the amount of Ag and/or Au that the protein was capable of encapsulating. NP formation was monitored visibly by color change from light yellow to orange (Ag) or light to a dark-brown/black (Au) (Figure 1), which was consistent with other studies for the synthesis of Ag-NPs and/or Au-NPs that were stabilized by enzymes or proteins. 17,22 Color change has been reported to be the initial evidence of NP formation. 23 The intensity of color increased with an increased amount of metal salt added to the aqueous solution of HSAF. A control experiment containing an equal amount of HSAF was set up under the same conditions, and no color change was noticed after addition of NaBH4. In an earlier study involving platinum NPs, 16 more particles were
Figure 2. Size exclusion chromatography elution profiles of (A) Au-HSAF NPs and (B) Ag- HSAF NPs synthesized with different molar concentrations of metal salt to a fixed concentration of HSAF. Control, HSAF only.
stabilized as the precursor Pt/HSAF molar ratio increased from 250:1 to 4000:1, suggesting an increase in the encapsulation of metal atoms with increasing metal salt concentration in solution. With Ag and/or Au, however, each M-NP complex precipitated at an M/HSAF ratio greater than 1000:1 with a decrease in NP complex concentration, a decrease in color intensity, and an indication that the saturation point of HSAF had been reached (Figure 2). The M-HSAF complexes for Ag and Au were further purified by size exclusion chromatography, and the elution profile (Figure 2) indicated a single peak coincidental with a peak of pure apoferritin. Analysis with native polyacrylamide gel electrophoresis (5%) showed a co-migration of HSAF alone and of M-HSAF NP complexes (results not shown). The bands suspected of containing NPs of Ag and/or Au were excised and digested; elemental analysis by ICP-OES confirmed the presence of the respective metals. All these results implied that both Ag-NPs and Au-NPs were independently attached to HSAF. The protein content of HSAF was determined for each of the Ag/HSAF and Au/HSAF ratios, before and after NP synthesis, and a certain degree of stability was retained by HSAF, as nearly 76% of the protein was accounted for by all Au/HSAF ratios except for 54% (1000:1); 13% (2000:1), and 7% (4000:1) (Table 1). With Ag-HSAF complexes 95% of the protein was accounted for up to a ratio of 1000:1, decreasing to 38% (2000:1)
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Table 1 Estimation of Au-HSAF and Ag-HSAF nanoparticle complex concentrations Theoretical M/HSAF ratio Au Control 250:1 500:1 1000:1 2000:1 4000:1 Ag Control 250:1 500:1 1000:1 2000:1 4000:1
Starting [HSAF] (μM)⁎
[HSAF] after NP [M] M/ synthesis (μM) (μM) † HSAF ratio ‡,¶
M/ HSAF ratio ║,¶
10 10 10 10 10 10
7.6 7.6 6.2 5.4 1.3 0.7
– 1082.2 2369.8 3571.7 209 94
– 108 237 357 21 9
– 142 382 661 160 13
10 10 10 10 10 10
9.5 9.6 10.1 9.6 3.8 0.8
– 497.7 642.5 1251.5 767.2 63.6
– 50 64 125 77 6
– 52 64 130 202 80
Ag and Au nanoparticle (NP) and protein (HSAF, horse spleen apoferritin) concentrations were determined by ICP-OES and Bradford method, respectively. ⁎ Based on estimation by Bradford method. † Molar concentration of M-NPs based on M atom concentration as estimated by ICP-OES. ‡ Calculated ratio of M atoms to HSAF based on the starting concentration of HSAF (i.e., 10 μM). ║ Estimated ratio of M atoms to HSAF based on HSAF concentration after synthesis. ¶ Values represent mean of triplicate samples with standard deviation b10%.
and 8% (4000:1). This loss in protein may be due to either protein degradation during the synthesis of the M-HSAF NP complexes or the masking of the protein by the NPs, thereby making it unavailable to the Bradford reagent. Characterization of M-HSAF NP complex UV-vis spectroscopy Synthesized NPs were characterized by UV-vis spectroscopy and the absorption maximum analyzed by a spectral scan from 200 to 700 nm. With Au-HSAF NP complexes there were two absorbance maxima at 280 nm, corresponding to pure HSAF, and 520 nm, which represented a characteristic surface plasmon resonance band of a spherical Au nanoparticle b20 nm in diameter (Figure 3, A). 22,24 The intensity of these two absorbance peaks increased with increasing Au-NP concentration up to 1000:1 Au/HSAF, after which there was a sharp decline. As indicated above, this was due to precipitation of the Au-NPs at Au/HSAF ratio greater than 1000:1. With Ag-HSAF NP complexes there was, apart from the obvious protein absorbance peak present at 280 nm, a rather prominent surface plasmon resonance band at 414 nm (Figure 3, B), which was close to that reported in the literature. 17 As with Au-NP complexes, the intensity of this plasmon resonance band increased with increasing Ag-NP concentration up to a 1000:1 molar ratio of Ag/HSAF. The decrease in intensity of this band at Ag/HSAF N 1000:1 was due to precipitation of the NPs during synthesis.
Figure 3. UV-vis absorption spectra of (A) Au-HSAF and (B) Ag-HSAF complexes showing typical surface plasmon resonance bands associated with each M/HSAF ratio. Peaks observed around 280 nm were due to protein absorption band of HSAF.
ICP-OES analysis ICP-OES was used to estimate the number of Au-NPs and/or Ag-NPs stabilized by the HSAF, the metal stoichiometry of AuHSAF and/or Ag-HSAF samples in solution, and the saturation point of HSAF in terms of encapsulated Au and/or Ag atoms. Results showed a decrease in Au-NP and Ag-NP concentration with increase in molar ratio of respective Au and Ag salt to a fixed concentration of HSAF (Table 1, note †). No Au or Ag metal was detected in the control experiment (HSAF only). The Au/HSAF and Ag/HSAF ratios based on protein content of HSAF before and after synthesis, respectively, were also estimated (Table 1, notes ‡,ǁ). The highest number of Au and Ag atoms stabilized was 357 and 125, respectively, based on the starting HSAF concentration, and 661 and 202 based on the final concentration of HSAF after synthesis. The smallest number of Ag-NPs stabilized by HSAF was 77, which reflected the affinity of HSAF for single-valence metal salts. 17 FT-IR analysis The biologically synthesized Au-HSAF and/or Ag-HSAF NP complexes were analyzed by FT-IR spectroscopy to investigate if there were any functional groups on HSAF that were responsible for the stabilization and/or coordination of the NPs
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Figure 4. FT-IR spectra of (A) Au-HSAF NPs and (B) Ag-HSAF NPs. (i) HSAF control. Molar concentration ratios of metal salts to HSAF: (ii) 250:1; (iii) 500:1; (iv) 1000:1; (v) 2000:1; (vi) 4000:1.
as well as to study the structural integrity of HSAF after the synthesis of the NPs. The presence of amide I (1590–1650 cm −1) and amide II (1500–1560 cm −1) bands, which are characteristic of proteins and peptides, 25 in both spectra of HSAF and M-HSAF NP complexes (Figure 4) indicated protein stability. Furthermore, the presence of an amide I peak (1630–1650 cm −1), often ascribed to predominantly α-helical structured protein (like HSAF) in aqueous solutions, suggested that the synthesis of Au-NPs and/or Ag-NPs in the presence of HSAF may not have compromised the overall structure of HSAF. 26 With a more detailed analysis of the FT-IR spectra of the AuHSAF and/or Ag-HSAF NP complexes, it was noticed that additional peaks appeared in the amide I region at 1737–1740 cm −1 (Figure 4, spectra iii–vi), which were not seen in the spectrum of HSAF (Figure 4, spectrum i), suggesting the binding of Au and Ag (before reduction to NPs) to the carboxylate side
chains of acidic amino acids. These residues predominantly line the interior of HSA and give it its net negative charge at physiological pH, 17,27 implying a possibility of M-NP synthesis within the cavity of HSA. TEM analysis Au-HSAF and Ag-HSAF NP complexes were characterized by TEM so as to determine not only particle size and distribution, and position of the NP in the HSAF, but also to confirm the synthesis of the M-NPs in the presence of HSAF. The NPs were predominantly spherical (Figure 5), agreeing with the spherical shape of the HSAF interior. Analysis of 200 particles in each set of experimental samples revealed a general increase in the size of the NPs with an increase in Au/HSAF or Ag/HSAF ratio. This was due to increased nucleation of NPs during synthesis as a result of increased concentration of
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Figure 5. TEM micrographs and size distribution of (A) Au-NPs; (B) Ag-NPs. Molar concentrations of M/HSAF: (I) 250:1; (II) 500:1; (III) 1000:1; (IV) 2000:1; (V) 4000:1. (C) HSAF only (control). (D) Au-NPs stained with 1% uranyl acetate; (E) Ag-NPs stained with 1% uranyl acetate. Scale bar, 100 nm. 200 particles were analyzed. (F) EDX spectra of Au-NPs (I) and Ag-NPs (II).
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Effect of Au-NPs and Ag-NPs on ferroxidase activity of HSAF Results showed an increase in ferroxidase activity with an increase in the molar ratio of Au/HSAF NPs (Figure 6), with a specific activity of 225 ρmol min −1 mg −1 (ninefold increase over the control) seen at a molar ratio of 500:1 (Au/HSAF). At Au/ HSAF ratios of 250:1 and 1000:1 the specific activities were, respectively, 160 ρmol min −1 mg −1 (sixfold) and 200 ρmol min −1 mg −1 (eightfold), whereas at a ratio of 2000:1 the specific activity was 70 ρmol min −1 mg −1 (2.7-fold). Any increase in Au/HSAF above 2000 led to NP aggregation, with the ferroxidase activity reverting to that of the control. As far as Ag-NPs were concerned, there was an increase in ferroxidase activity with an increase in the molar ratio of Ag/HSAF NPs (Figure 6), with a specific activity of 2800 ρmol min −1 mg −1 (110-fold increase over the control) seen at a molar ratio of 1000:1 Ag/HSAF. At an Ag/HSAF ratio of 500:1, the specific activity was 2350 ρmol min −1 mg −1 (90-fold), whereas at a ratio of 2000:1 the specific activity was 1750 ρmol min −1 mg −1 (67-fold); at a ratio of 250:1 the specific activity was 750 ρmol min −1 mg −1 (29-fold). Any increase in Ag/HSAF above 2000 led to NP aggregation and precipitation of the particles from the interior of the HSAF after the uptake of Fe 2+ ions from solution had occurred, with the ferroxidase activity reverting to that of the control. Discussion
Figure 6. Effect of (A) Au-HASF NPs and (B) Ag-HASF NPs on the ferroxidase activity of HSAF. Results presented are the mean of three independent assays, each done in triplicate.
respective metal salt in solution. It was noticed, however, that there was no significant increase in the size of the NPs with the Au/HSAF ratio of 1000:1, supporting our earlier finding that the Au-NPs had precipitated. The average size of the Au-NPs obtained with different molar concentrations of AuCl3 salt was between 3 and 5 nm (Figure 5), which was indeed generally smaller than that reported in the literature. 22 The size was below that of the interior diameter of HSAF (8 nm), 17 and though literature had reported that it was challenging to synthesize trivalent M-NPs within the apoferritin cage, 22 we believe there was evidence (Figure 5) that these Au-NPs had formed within the core of HSAF. The average size of Ag-NPs synthesized was 3–6 nm (Figure 5), which increased as the Ag/HSAF ratio increased up to 1000:1, after which no significant change was realized. Negative staining with uranyl acetate confirmed the formation of Au-NPs or Ag-NPs within the core of HSAF (Figure 5, D and E, respectively), as can be seen by light protein rings (i.e., HSAF) surrounding the black-dotted M-NP crystals, supporting evidence from literature. 20 EDX analysis The presence and stabilization of Au and Ag in M-HSAF NP complexes was confirmed by EDX analysis (Figure 5, F, panels I and II).
In the present study we have shown evidence that Au-NPs and Ag-NPs, when enclosed within a cagelike apoferritin structure (HSAF), were not only stabilized against aggregation but had an influential effect on HSAF's ferroxidase activity. A maximum increase in ferroxidase activity, facilitated by HSAF, of ninefold above the control was realized with an Au/HSAF molar ratio of 500:1. A lesser influence was noted when this ratio decreased to 250:1 or increased to 1000:1, whereas when the ratio increased (N1000) the activity of the ferroxidase reverted to that of the control. A 110-fold increase in ferroxidase activity, facilitated by HSAF, was realized with an Ag/HSAF molar ratio of 1000:1. A lesser influence was noted when this ratio decreased to 500:1 (90-fold) or 250:1 (29-fold), or increased to 2000:1 (67-fold), whereas when the ratio increased (N2000) the activity of the ferroxidase reverted to that of the control. Au-NPs and Ag-NPs have shown extensive popularity in nanomedicine for their anticancer, antitumor, and antimicrobial properties. 28–31 From a structural and mechanistic point of view, it is not clear why there is this considerable increase in activity of ferroxidase in the present study. Though it may be considered speculation to propose tentative suggestions, it is probable that one or more of the following may occur: 1. Noble metallic NPs, especially Au and Ag, have catalytic properties. 32,33 2. The distances between the glutamic acid, aspartic acid, glutamine, and water molecules within the active site of ferroxidase are about 8–10 Å—well within the range for the Au-NPs or Ag-NPs to bind. 34 This would, in turn, enhance the negativity of the amino acids in the core, increasing the binding of Fe 2+.
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3. The Au-NPs or Ag-NPs interact with the diferrous [Fe-Fe] complex in the enzyme reactive core, increasing the rate of removal of electrons and oxidation to [Fe 3+-Fe 3+]. 4. The NPs facilitate the addition of a water molecule to enhance the formation of the hydrolysis product (FeOOH) and in so doing interact with the released protons to drive the reaction. 35 5. The NPs interact with polar amino acids at the reactive site, changing the conformation of the reactive core and increasing the rate of addition of oxidant.
An in-depth molecular modeling and docking study of the interaction and binding of the NPs with ferroxidase is currently under way and will be reported elsewhere. Studies on metal NPs stabilized by HASF 17 have suggested that divalent precursor metal salts were preferably accommodated within the core of HSAF. This may be because apoferritin in its normal biological function takes up iron in its Fe 2+ state, which is later converted to Fe 3+ within its core. Thus, HSAF may have a greater affinity for divalent metal ions within its cavity. Relative uniform size and shape of Ag-NPs and/or Au-NPs obtained in this study confirmed that HSAF was able to control NP growth within its interior. Furthermore, the formed particles possessed physical (excluding particle morphology) and chemical properties similar to those prepared by chemical means. The encapsulation of the NPs within the cavity of HSAF did not affect its overall protein integrity and structure but would improve an in vivo iron uptake as indicated by an enhanced ferroxidase activity. This may prove beneficial in clinical applications in the treatment and management of diseases associated with poor iron absorption (anemia) and possible decrease in oxidative stress associated with the toxic levels of iron (hemochromatosis) in biological systems. The co-migration of HSAF and M-HSAF indicated that HSAF remained intact after incubation with the metal salts and that both Au-NPs and Ag-NPs remained bound to HSAF. Because it was known that acidic amino acids were present within the core of the HSAF, giving the protein a net negative charge at physiological pH, the shift in amide maxima suggested that the metal NPs interacted with the carbonyl group of the carboxylic side chains of acidic amino acids. 11,27 This was only noticed with Au-NPs in the Au/HSAF ratio of 1000:1–4000:1. It also supported our finding that these M-NPs were present within the core of the HSAF. The release of M-NPs after the uptake of iron further confirmed that these NPs were initially present within the core of the HSAF. It may also suggest the ability of the interior of HSAF to accommodate more than one type of metal atom (Fe and Ag or Au) within its cavity—especially at M/HSAF ratio less than 1000:1. The functional groups responsible for the coordination of both Ag or Au and iron within the core of HSAF may not be involved in the storage and oxidation of iron in the HSAF core. A different mechanism of Fe 2+ uptake and oxidation by apoferritin proposed that the basic amino acids residues within the core of apoferritin were responsible for its ferroxidase activity. 36,37 This site was different from that containing acidic amino residues reported to be implicated in the uptake, nucleation, and stabilization of NPs.
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30. Eby DM, Luckarift HR, Johnson GR. Hybrid antimicrobial enzyme and silver nanoparticle coatings for medical instruments. Appl Mater Interf 2009;1:1553-60. 31. Buu NQ, Chau NH, Dung NTT, Tien NG. Studies on manufacturing of topical wound dressings based on nanosilver produced by aqueous molecular solution method. J Exp Nanosci 2011;6:409-21. 32. Campbell CT, Sharp JC, Yao YX, Karp EM, Silbaugh TL. Insights into catalysis by gold nanoparticles and their support effects through surface science studies of model catalysts. Faraday Discussions 2011;152: 227-39. 33. Li W, Sun C, Hou B, Zhou X. Room temperature synthesis and catalytic properties of surfactant-modified Ag nanoparticles. Int J Spectrosc 2012;2012:1-7. 34. Liu X, Thiel EC. Ferritin reactions: direct identification of the site for the diferric peroxide reaction intermediate. Proc Natl Acad Sci USA 2004;101:8557-62. 35. Baaghil S, Lewin A, Moore GR, Le Brun NE. Core formation in E. coli bacterio-ferritin requires a functional ferroxidase center. Biochemistry 2003;42:14047-56. 36. Niederer W. Ferritin: iron incorporation and iron release. Cell Mol Life Sci 1970;26:218-20. 37. Macara IG, Hoy TG, Harrison PM. The formation of ferritin from apoferritin: kinetics and mechanism of iron uptake. Biochem J 1972;126: 151-62.
Research Article
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Multiple fold increase in activity of ferroxidase–apoferritin complex by silver and gold nanoparticles Afolake Sennuga, PhD, Jacqueline van Marwijk, PhD, Chris G. Whiteley, PhD⁎ Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, Grahamstown, South Africa Received 12 December 2011; accepted 25 May 2012
Abstract The effect of silver (Ag) and gold (Au) nanoparticles on the ferroxidase activity of apoferritin showed a 110-fold increase in specific activity and a 9-fold increase over the control at the respective molar ratios of Au-apoferritin and Ag-apoferritin nanoparticles (NPs) of 500:1 and 1000:1. Typical color change, from pale yellow to brown, occurred when apoferritin was mixed with AgNO3 or AuCl3 followed by sodium borohydride to afford respective metal-apoferritin NP complexes in a ratio of between 250:1 and 4000:1. These complexes were characterized by ultraviolet-visible inductively coupled plasma–optical emission spectroscopy, Fourier transform infrared spectroscopy, transmission electron microscopy, and energy-dispersive x-ray spectroscopy. Transmission electron microscopy revealed that the size of NPs increased as the molar ratio of metal to apoferritin increased, with an average size of 3–6 nm generated with Au-to-apoferritin and/or Ag-toapoferritin molar ratios of 250:1 to 4000:1. Fourier transform infrared spectrometry showed no structural changes of apoferritin when the NPs were attached to the protein. From the Clinical Editor: In this paper the utility of gold and silver nanoparticles in augmenting the activity of the ferroxidase-apoferritin complex is described. Both NPs dramatically increased the ferroxidase activity. © 2013 Elsevier Inc. All rights reserved. Key words: Silver nanoparticles; Gold nanoparticles; Apoferritin; Ferroxidase activity
The multidisciplinary field of nanotechnology, which refers to characterization, synthesis, and applications of functional particles on a nanoscale (10 –9 m), has engulfed a myriad of new opportunities in health care, environmental biology, chemical and material sciences, and communications. 1 One of the fundamental principles that set this type of technology apart from traditional standard protocols is that as the particle itself becomes smaller and smaller, its association with biomacromolecules changes, inevitably leading these molecules to behave differently than their native counterparts. Though biological syntheses of noble metal nanoparticles (NPs) through the bioreduction of metal salts by both prokaryotic and eukaryotic organisms 2–8 are more cost-effective, simple, and
The authors thank the National Research Foundation (South Africa), Rhodes University Research Council, and Third World Organisation for Women in Science (TWOWS) for financial assistance. ⁎Corresponding author: Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, P.O. Box 94, Grahamstown 6140, South Africa. E-mail address: [email protected] (C.G. Whiteley).
more eco-friendly than the more hazardous chemical approach, a limitation remains in the form of the ability to control the mechanism that determines particle size and shape. 9 The use of a protein cavity or cage as a limited-growth field for NPs is not novel 10 and serves as an ideal template to confine particle growth in a homogeneous distribution as well as a stabilizer against particle aggregation. Apoferritin, a globular protein of approximately 440 kDa in 24 identical subunits that has ferroxidase activity and catalyzes the oxidation of Fe 2+ to Fe 3+ in the presence of molecular oxygen as an electron acceptor, is one such cage. The subunits form a spherical protein shell of 12 nm with an internal aqueous cavity of 8 nm. 11 Apart from Fe 3+, several other zero-valent apoferritin-encapsulated metal NPs have been prepared within this protein cage, and these include palladium, 12 platinum, 13 copper, 14 cobalt and nickel, 11 and cadmium. 15 Recently we reported 16 on enhanced activity of ferroxidase in apoferritin in the presence of platinum NPs of a defined size. To the best of our knowledge, this was the first time that such an increased activity of this enzyme in the presence of NPs had been reported, and consequently we were interested in extending this idea by considering the effect of gold (Au) and/or silver (Ag) on this enzyme's activity. We anticipated that apoferritin associated
1549-9634/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nano.2012.05.020 Please cite this article as: Sennuga A., van Marwijk J., Whiteley C.G., Multiple fold increase in activity of ferroxidase–apoferritin complex by silver and gold nanoparticles. Nanomedicine: NBM 2013;9:185-193, http://dx.doi.org/10.1016/j.nano.2012.05.020
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with the Ag-NP and/or Au-NP would have a wide range of affinities and activities dependent on the size of the NP.
Methods Materials Horse spleen apoferritin (HSAF), gold chloride (AuCl3), silver nitrate (AgNO3), and ferrous ammonium sulfate [(NH4)2Fe(SO4)2⋅6H2O] were obtained from Sigma-Aldrich (Johannesburg, South Africa), and sodium borohydride (NaBH4) was purchased from Merck (Johannesburg, South Africa). All other reagents were of analytical grade. Synthesis of metal-HSAF NP complex Metal (M) NPs of Ag and Au were synthesized within HSAD as described. 17 Briefly, HSAF (10 μM) in Tris-buffered saline (50 mM Tris, 150 mM NaCl, pH 8) was incubated (60 minutes, 4°C) with varying volumes of AuCl3 or AgNO3 (0.1 M) to give a theoretical loading of M/HSAF ratio 1:250–1:4000. While maintaining the pH at pH 8 with 0.1 M NaOH, NaBH4 at five times the metal concentration was added and the solution stirred for a further 60 minutes until the sample color turned black (Au) or deep orange (Ag). The solution was then dialyzed (24 hours) against Tris buffer (10 mM, pH 8), changing every 6 hours, centrifuged (2 minutes, 5000 rpm), and filtered through a 0.22-μm filter. The sample (250 μL) was purified by size exclusion chromatography (Sephadex G-50, 1.8 × 15 cm) and the elution of the M-HSAF NP complex monitored at 280 nm. Protein content was determined by Bradford assay, and the purity and stability of the complex was assessed by native polyacrylamide gel electrophoresis.
(Perkin-Elmer, Boston, Massachusetts). Apoferritin without NPs was used as the control. Transmission electron microscopy (TEM) Samples for TEM analysis were prepared by placing a drop of the M-HSAF NP sample onto carbon-coated copper grids; excess sample was removed after a minute using blotting paper and the grids air-dried before analysis. Duplicate samples were prepared and negatively stained using 1% uranyl acetate. Mean particle size and standard deviations were determined by the analysis of 200 particles using the computer software Scandium (Olympus, Melville, New York). Electron micrographs were taken using a JEOL JEM-1210 transmission electron microscope (JEOL, Tokyo, Japan) operating at 80 keV. Energy-dispersive x-ray spectroscopy (EDX) Freeze-dried samples were placed on graphite tape, which was in turn placed on an aluminium stub. Elemental analysis was performed with a TESCAN scanning electron microscope with an EDX scanner (INCAPentalFeTx3; Oxford Instruments, Brno, Czech Republic) operating at 20 keV. Assay of HSAF-ferroxidase activity HSAF-ferroxidase activity was determined at 420 nm according to a published procedure 18 with a slight modification. HSAF (1 μM) in sodium borate–cacodylate buffer (50 mM, pH 5.5) was incubated at 22°C with ferrous ammonium sulfate [(NH4)2Fe(SO4)2⋅6H2O] (50 mM) and the change in absorbance monitored at 420 nm for 4 minutes. A control containing only ferrous ammonium sulfate was used as the blank. The amount of ferritin (Fe 3+) produced was estimated from a standard curve. Effect of Ag-NPs and/or Au-NPs on ferroxidase activity of HSAF
Characterization of M-HSAF NP complex Ultraviolet-visible (UV-vis) spectroscopy UV-vis absorption spectra (250–700 nm) of the M-HSAF NP complexes were obtained using a UV spectrophotometer (Spectroquant Pharo 300; Merck) to evaluate the plasmon bands associated with the Ag-NPs and/or Au-NPs.
This assay was performed in exactly the same way as that described above for HSAF-ferroxidase activity except that each of the Ag-HSAF or Au-HSAF NP complexes (1 μM) was used. The negative control contained only HSAF and substrate, whereas the positive control contained HSAF attached to Ag-NPs or Au-NPs. Protein determination
Inductively coupled plasma–optical emission spectroscopy (ICP-OES) M-HSAF NP complexes (250 μL) were dissolved in aqua regia (1:3 nitric to hydrochloric acid; 350 μL for Au; 500 μL for Ag) and digested with hydrogen peroxide (1.0 mL, 60°C, 1 hour). Metal quantification was carried out using an inductively coupled optical emission spectrophotometer (iCAP 6000 series; Thermoelectron, Cambridge, United Kingdom) after suitable standard curves were constructed with varying concentrations of respective metal salt standards (Sigma-Aldrich).
The protein concentration for all experiments was routinely determined, in triplicate, according to the method of Bradford. 19 Into a 96-well titer plate was placed either protein-NP sample (5 μL) followed by Bradford reagent (245 μL). The mixture was incubated (22°C, 10 minutes), the absorbance of the solution measured at 595 nm, and the concentration of the unknown sample determined using a bovine serum albumin standard curve.
Fourier transform infrared (FT-IR) spectrometry Freeze-dried samples of each M-HSAF NP complex were analyzed from 600 to 4000 cm −1 with a scan resolution of 4 cm −1 using a Perkin-Elmer 100 FT-IR spectrophotometer equipped with a universal attenuated total reflectance sampling accessory
All analyses were carried out in triplicate and values reported as the means with standard deviation P b 0.05 vs. controls. Where necessary, analysis of variance was conducted using Statistica for Windows, version 8 (Statsoft Inc., Tulsa, Oklahoma) and Microsoft Excel 2010.
Statistical analyses
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Figure 1. Color differentiation of (A) Au-NPs and (B) Ag-NPs synthesized with varying molar concentrations of metal salts to a fixed concentration of HSAF (250:1–4000:1 M/HSAF). Control, HSAF alone.
Results Synthesis of M-HSAF NP complex M-HSAF NP complexes of Ag and/or Au, as seen under TEM imaging were formed after HSAF was incubated with varying concentrations of AuCl3 or AgNO3 (60 minutes, 4°C, pH 8) followed by treatment with NaBH4 (5 equivalents). Excess metal was removed by dialysis, and any aggregated metal NPs that were not stabilized by HSAF were removed by centrifugation (5000 rpm). The preparation of the complex was carried out at pH 8 so as to increase the electronegativity of the interior of HSAF, thereby improving electrostatic attraction between positive metallic cations and the core of HSAF. Different molar concentrations of metal salts were incubated with a fixed concentration of HSAF so as to vary the particle size. 20,21 The change in NP size was determined by TEM analysis, and a variation in molar ratios of MHSAF was used to determine the saturation point of HSAF in terms of the amount of Ag and/or Au that the protein was capable of encapsulating. NP formation was monitored visibly by color change from light yellow to orange (Ag) or light to a dark-brown/black (Au) (Figure 1), which was consistent with other studies for the synthesis of Ag-NPs and/or Au-NPs that were stabilized by enzymes or proteins. 17,22 Color change has been reported to be the initial evidence of NP formation. 23 The intensity of color increased with an increased amount of metal salt added to the aqueous solution of HSAF. A control experiment containing an equal amount of HSAF was set up under the same conditions, and no color change was noticed after addition of NaBH4. In an earlier study involving platinum NPs, 16 more particles were
Figure 2. Size exclusion chromatography elution profiles of (A) Au-HSAF NPs and (B) Ag- HSAF NPs synthesized with different molar concentrations of metal salt to a fixed concentration of HSAF. Control, HSAF only.
stabilized as the precursor Pt/HSAF molar ratio increased from 250:1 to 4000:1, suggesting an increase in the encapsulation of metal atoms with increasing metal salt concentration in solution. With Ag and/or Au, however, each M-NP complex precipitated at an M/HSAF ratio greater than 1000:1 with a decrease in NP complex concentration, a decrease in color intensity, and an indication that the saturation point of HSAF had been reached (Figure 2). The M-HSAF complexes for Ag and Au were further purified by size exclusion chromatography, and the elution profile (Figure 2) indicated a single peak coincidental with a peak of pure apoferritin. Analysis with native polyacrylamide gel electrophoresis (5%) showed a co-migration of HSAF alone and of M-HSAF NP complexes (results not shown). The bands suspected of containing NPs of Ag and/or Au were excised and digested; elemental analysis by ICP-OES confirmed the presence of the respective metals. All these results implied that both Ag-NPs and Au-NPs were independently attached to HSAF. The protein content of HSAF was determined for each of the Ag/HSAF and Au/HSAF ratios, before and after NP synthesis, and a certain degree of stability was retained by HSAF, as nearly 76% of the protein was accounted for by all Au/HSAF ratios except for 54% (1000:1); 13% (2000:1), and 7% (4000:1) (Table 1). With Ag-HSAF complexes 95% of the protein was accounted for up to a ratio of 1000:1, decreasing to 38% (2000:1)
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Table 1 Estimation of Au-HSAF and Ag-HSAF nanoparticle complex concentrations Theoretical M/HSAF ratio Au Control 250:1 500:1 1000:1 2000:1 4000:1 Ag Control 250:1 500:1 1000:1 2000:1 4000:1
Starting [HSAF] (μM)⁎
[HSAF] after NP [M] M/ synthesis (μM) (μM) † HSAF ratio ‡,¶
M/ HSAF ratio ║,¶
10 10 10 10 10 10
7.6 7.6 6.2 5.4 1.3 0.7
– 1082.2 2369.8 3571.7 209 94
– 108 237 357 21 9
– 142 382 661 160 13
10 10 10 10 10 10
9.5 9.6 10.1 9.6 3.8 0.8
– 497.7 642.5 1251.5 767.2 63.6
– 50 64 125 77 6
– 52 64 130 202 80
Ag and Au nanoparticle (NP) and protein (HSAF, horse spleen apoferritin) concentrations were determined by ICP-OES and Bradford method, respectively. ⁎ Based on estimation by Bradford method. † Molar concentration of M-NPs based on M atom concentration as estimated by ICP-OES. ‡ Calculated ratio of M atoms to HSAF based on the starting concentration of HSAF (i.e., 10 μM). ║ Estimated ratio of M atoms to HSAF based on HSAF concentration after synthesis. ¶ Values represent mean of triplicate samples with standard deviation b10%.
and 8% (4000:1). This loss in protein may be due to either protein degradation during the synthesis of the M-HSAF NP complexes or the masking of the protein by the NPs, thereby making it unavailable to the Bradford reagent. Characterization of M-HSAF NP complex UV-vis spectroscopy Synthesized NPs were characterized by UV-vis spectroscopy and the absorption maximum analyzed by a spectral scan from 200 to 700 nm. With Au-HSAF NP complexes there were two absorbance maxima at 280 nm, corresponding to pure HSAF, and 520 nm, which represented a characteristic surface plasmon resonance band of a spherical Au nanoparticle b20 nm in diameter (Figure 3, A). 22,24 The intensity of these two absorbance peaks increased with increasing Au-NP concentration up to 1000:1 Au/HSAF, after which there was a sharp decline. As indicated above, this was due to precipitation of the Au-NPs at Au/HSAF ratio greater than 1000:1. With Ag-HSAF NP complexes there was, apart from the obvious protein absorbance peak present at 280 nm, a rather prominent surface plasmon resonance band at 414 nm (Figure 3, B), which was close to that reported in the literature. 17 As with Au-NP complexes, the intensity of this plasmon resonance band increased with increasing Ag-NP concentration up to a 1000:1 molar ratio of Ag/HSAF. The decrease in intensity of this band at Ag/HSAF N 1000:1 was due to precipitation of the NPs during synthesis.
Figure 3. UV-vis absorption spectra of (A) Au-HSAF and (B) Ag-HSAF complexes showing typical surface plasmon resonance bands associated with each M/HSAF ratio. Peaks observed around 280 nm were due to protein absorption band of HSAF.
ICP-OES analysis ICP-OES was used to estimate the number of Au-NPs and/or Ag-NPs stabilized by the HSAF, the metal stoichiometry of AuHSAF and/or Ag-HSAF samples in solution, and the saturation point of HSAF in terms of encapsulated Au and/or Ag atoms. Results showed a decrease in Au-NP and Ag-NP concentration with increase in molar ratio of respective Au and Ag salt to a fixed concentration of HSAF (Table 1, note †). No Au or Ag metal was detected in the control experiment (HSAF only). The Au/HSAF and Ag/HSAF ratios based on protein content of HSAF before and after synthesis, respectively, were also estimated (Table 1, notes ‡,ǁ). The highest number of Au and Ag atoms stabilized was 357 and 125, respectively, based on the starting HSAF concentration, and 661 and 202 based on the final concentration of HSAF after synthesis. The smallest number of Ag-NPs stabilized by HSAF was 77, which reflected the affinity of HSAF for single-valence metal salts. 17 FT-IR analysis The biologically synthesized Au-HSAF and/or Ag-HSAF NP complexes were analyzed by FT-IR spectroscopy to investigate if there were any functional groups on HSAF that were responsible for the stabilization and/or coordination of the NPs
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Figure 4. FT-IR spectra of (A) Au-HSAF NPs and (B) Ag-HSAF NPs. (i) HSAF control. Molar concentration ratios of metal salts to HSAF: (ii) 250:1; (iii) 500:1; (iv) 1000:1; (v) 2000:1; (vi) 4000:1.
as well as to study the structural integrity of HSAF after the synthesis of the NPs. The presence of amide I (1590–1650 cm −1) and amide II (1500–1560 cm −1) bands, which are characteristic of proteins and peptides, 25 in both spectra of HSAF and M-HSAF NP complexes (Figure 4) indicated protein stability. Furthermore, the presence of an amide I peak (1630–1650 cm −1), often ascribed to predominantly α-helical structured protein (like HSAF) in aqueous solutions, suggested that the synthesis of Au-NPs and/or Ag-NPs in the presence of HSAF may not have compromised the overall structure of HSAF. 26 With a more detailed analysis of the FT-IR spectra of the AuHSAF and/or Ag-HSAF NP complexes, it was noticed that additional peaks appeared in the amide I region at 1737–1740 cm −1 (Figure 4, spectra iii–vi), which were not seen in the spectrum of HSAF (Figure 4, spectrum i), suggesting the binding of Au and Ag (before reduction to NPs) to the carboxylate side
chains of acidic amino acids. These residues predominantly line the interior of HSA and give it its net negative charge at physiological pH, 17,27 implying a possibility of M-NP synthesis within the cavity of HSA. TEM analysis Au-HSAF and Ag-HSAF NP complexes were characterized by TEM so as to determine not only particle size and distribution, and position of the NP in the HSAF, but also to confirm the synthesis of the M-NPs in the presence of HSAF. The NPs were predominantly spherical (Figure 5), agreeing with the spherical shape of the HSAF interior. Analysis of 200 particles in each set of experimental samples revealed a general increase in the size of the NPs with an increase in Au/HSAF or Ag/HSAF ratio. This was due to increased nucleation of NPs during synthesis as a result of increased concentration of
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Figure 5. TEM micrographs and size distribution of (A) Au-NPs; (B) Ag-NPs. Molar concentrations of M/HSAF: (I) 250:1; (II) 500:1; (III) 1000:1; (IV) 2000:1; (V) 4000:1. (C) HSAF only (control). (D) Au-NPs stained with 1% uranyl acetate; (E) Ag-NPs stained with 1% uranyl acetate. Scale bar, 100 nm. 200 particles were analyzed. (F) EDX spectra of Au-NPs (I) and Ag-NPs (II).
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Effect of Au-NPs and Ag-NPs on ferroxidase activity of HSAF Results showed an increase in ferroxidase activity with an increase in the molar ratio of Au/HSAF NPs (Figure 6), with a specific activity of 225 ρmol min −1 mg −1 (ninefold increase over the control) seen at a molar ratio of 500:1 (Au/HSAF). At Au/ HSAF ratios of 250:1 and 1000:1 the specific activities were, respectively, 160 ρmol min −1 mg −1 (sixfold) and 200 ρmol min −1 mg −1 (eightfold), whereas at a ratio of 2000:1 the specific activity was 70 ρmol min −1 mg −1 (2.7-fold). Any increase in Au/HSAF above 2000 led to NP aggregation, with the ferroxidase activity reverting to that of the control. As far as Ag-NPs were concerned, there was an increase in ferroxidase activity with an increase in the molar ratio of Ag/HSAF NPs (Figure 6), with a specific activity of 2800 ρmol min −1 mg −1 (110-fold increase over the control) seen at a molar ratio of 1000:1 Ag/HSAF. At an Ag/HSAF ratio of 500:1, the specific activity was 2350 ρmol min −1 mg −1 (90-fold), whereas at a ratio of 2000:1 the specific activity was 1750 ρmol min −1 mg −1 (67-fold); at a ratio of 250:1 the specific activity was 750 ρmol min −1 mg −1 (29-fold). Any increase in Ag/HSAF above 2000 led to NP aggregation and precipitation of the particles from the interior of the HSAF after the uptake of Fe 2+ ions from solution had occurred, with the ferroxidase activity reverting to that of the control. Discussion
Figure 6. Effect of (A) Au-HASF NPs and (B) Ag-HASF NPs on the ferroxidase activity of HSAF. Results presented are the mean of three independent assays, each done in triplicate.
respective metal salt in solution. It was noticed, however, that there was no significant increase in the size of the NPs with the Au/HSAF ratio of 1000:1, supporting our earlier finding that the Au-NPs had precipitated. The average size of the Au-NPs obtained with different molar concentrations of AuCl3 salt was between 3 and 5 nm (Figure 5), which was indeed generally smaller than that reported in the literature. 22 The size was below that of the interior diameter of HSAF (8 nm), 17 and though literature had reported that it was challenging to synthesize trivalent M-NPs within the apoferritin cage, 22 we believe there was evidence (Figure 5) that these Au-NPs had formed within the core of HSAF. The average size of Ag-NPs synthesized was 3–6 nm (Figure 5), which increased as the Ag/HSAF ratio increased up to 1000:1, after which no significant change was realized. Negative staining with uranyl acetate confirmed the formation of Au-NPs or Ag-NPs within the core of HSAF (Figure 5, D and E, respectively), as can be seen by light protein rings (i.e., HSAF) surrounding the black-dotted M-NP crystals, supporting evidence from literature. 20 EDX analysis The presence and stabilization of Au and Ag in M-HSAF NP complexes was confirmed by EDX analysis (Figure 5, F, panels I and II).
In the present study we have shown evidence that Au-NPs and Ag-NPs, when enclosed within a cagelike apoferritin structure (HSAF), were not only stabilized against aggregation but had an influential effect on HSAF's ferroxidase activity. A maximum increase in ferroxidase activity, facilitated by HSAF, of ninefold above the control was realized with an Au/HSAF molar ratio of 500:1. A lesser influence was noted when this ratio decreased to 250:1 or increased to 1000:1, whereas when the ratio increased (N1000) the activity of the ferroxidase reverted to that of the control. A 110-fold increase in ferroxidase activity, facilitated by HSAF, was realized with an Ag/HSAF molar ratio of 1000:1. A lesser influence was noted when this ratio decreased to 500:1 (90-fold) or 250:1 (29-fold), or increased to 2000:1 (67-fold), whereas when the ratio increased (N2000) the activity of the ferroxidase reverted to that of the control. Au-NPs and Ag-NPs have shown extensive popularity in nanomedicine for their anticancer, antitumor, and antimicrobial properties. 28–31 From a structural and mechanistic point of view, it is not clear why there is this considerable increase in activity of ferroxidase in the present study. Though it may be considered speculation to propose tentative suggestions, it is probable that one or more of the following may occur: 1. Noble metallic NPs, especially Au and Ag, have catalytic properties. 32,33 2. The distances between the glutamic acid, aspartic acid, glutamine, and water molecules within the active site of ferroxidase are about 8–10 Å—well within the range for the Au-NPs or Ag-NPs to bind. 34 This would, in turn, enhance the negativity of the amino acids in the core, increasing the binding of Fe 2+.
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3. The Au-NPs or Ag-NPs interact with the diferrous [Fe-Fe] complex in the enzyme reactive core, increasing the rate of removal of electrons and oxidation to [Fe 3+-Fe 3+]. 4. The NPs facilitate the addition of a water molecule to enhance the formation of the hydrolysis product (FeOOH) and in so doing interact with the released protons to drive the reaction. 35 5. The NPs interact with polar amino acids at the reactive site, changing the conformation of the reactive core and increasing the rate of addition of oxidant.
An in-depth molecular modeling and docking study of the interaction and binding of the NPs with ferroxidase is currently under way and will be reported elsewhere. Studies on metal NPs stabilized by HASF 17 have suggested that divalent precursor metal salts were preferably accommodated within the core of HSAF. This may be because apoferritin in its normal biological function takes up iron in its Fe 2+ state, which is later converted to Fe 3+ within its core. Thus, HSAF may have a greater affinity for divalent metal ions within its cavity. Relative uniform size and shape of Ag-NPs and/or Au-NPs obtained in this study confirmed that HSAF was able to control NP growth within its interior. Furthermore, the formed particles possessed physical (excluding particle morphology) and chemical properties similar to those prepared by chemical means. The encapsulation of the NPs within the cavity of HSAF did not affect its overall protein integrity and structure but would improve an in vivo iron uptake as indicated by an enhanced ferroxidase activity. This may prove beneficial in clinical applications in the treatment and management of diseases associated with poor iron absorption (anemia) and possible decrease in oxidative stress associated with the toxic levels of iron (hemochromatosis) in biological systems. The co-migration of HSAF and M-HSAF indicated that HSAF remained intact after incubation with the metal salts and that both Au-NPs and Ag-NPs remained bound to HSAF. Because it was known that acidic amino acids were present within the core of the HSAF, giving the protein a net negative charge at physiological pH, the shift in amide maxima suggested that the metal NPs interacted with the carbonyl group of the carboxylic side chains of acidic amino acids. 11,27 This was only noticed with Au-NPs in the Au/HSAF ratio of 1000:1–4000:1. It also supported our finding that these M-NPs were present within the core of the HSAF. The release of M-NPs after the uptake of iron further confirmed that these NPs were initially present within the core of the HSAF. It may also suggest the ability of the interior of HSAF to accommodate more than one type of metal atom (Fe and Ag or Au) within its cavity—especially at M/HSAF ratio less than 1000:1. The functional groups responsible for the coordination of both Ag or Au and iron within the core of HSAF may not be involved in the storage and oxidation of iron in the HSAF core. A different mechanism of Fe 2+ uptake and oxidation by apoferritin proposed that the basic amino acids residues within the core of apoferritin were responsible for its ferroxidase activity. 36,37 This site was different from that containing acidic amino residues reported to be implicated in the uptake, nucleation, and stabilization of NPs.
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