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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Amyloid fibril formation by -lactoglobulin is inhibited by gold nanoparticles
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Subrata Sardar, Sampa Pal, Sanhita Maity, Jishnu Chakraborty, Umesh Chandra Halder ∗
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Q2 Organic Chemistry Section, Department of Chemistry, Jadavpur University, Kolkata 700032, India
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Article history: Received 25 January 2014 Received in revised form 29 April 2014 Accepted 2 May 2014 Available online xxx
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Keywords: -Lactoglobulin (-lg) Amyloid fibril Gold nanoparticles (AuNP) Inhibition
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1. Introduction
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The endogenous deposition of protein fibrillar aggregates in the tissues is the cause of several neurodisorders. In the present study the native -lactoglobulin (-lg) has been driven towards amyloid fibrillar aggregates when it was exposed to heat at 75 ◦ C for 1 h at pH 7.5. The citrate stabilized gold nanoparticle (AuNPs) of 20 nm diameter is shown to inhibit the thermal aggregation of -lg. The stability of the -lg against heat stress was assessed by studying its aggregation at 75 ◦ C, either in presence or in absence of AuNPs. AuNPs stabilizes the monomeric and dimeric forms of the -lg inhibiting the nucleation and elongation for the formation of higher aggregates. Incubation of -lg with AuNPs at 75 ◦ C results in the formation of smaller ragged aggregates. Results show that AuNPs possess the capability of inhibiting the formation of amyloid fibrillar aggregates of -lg in a concentration-dependent manner and may thus facilitate the refolding into native like structure. AuNPs thus serve as nano-chaperon to inhibit the protein aggregation. Thus chaperon like activity of AuNP may be exploited in the design of rational therapeutics for the neurodegenerative diseases. © 2014 Published by Elsevier B.V.
Nanomaterials and nanotechnology have received enormous popularity in the last decade due to their wider application in industry, technology, biological science and in the field of fundamental research [1–3]. Owing to their small size and penetration ability nanoparticles can be used as biosensors, drug carriers, cancer therapeutic agents and bio labelling materials [4–7]. However the interactions of the nanoparticles [NPs] with the biomolecules, especially proteins, are very important for understanding the biocompatibility in biomedical applications. It is commonly known that the surface adsorption can lead the unfolding and hence structural changes of proteins [8,9]. Upon adsorption on silica nanoparticles, lysozyme was found to undergo a reduction in both alpha-helical content and enzymatic activity, with greater loss on larger NPs [10]. Such absorption of proteins, on the other hand, is also effective for surface coating to stabilize nanoparticle and reduce their cytotoxicity. Recently it has been shown that when exposed to biomolecules, the bare NPs form a ‘nanoparticle protein corona’ [11]. Relevance of such protein corona on the biological impacts of nanoparticle has also been discussed [12]. The change in protein structure and function are strongly dependent on both
∗ Corresponding author. Tel.: +91 33 2414 6223; fax: +91 33 2413 7902. E-mail address: [email protected] (U.C. Halder).
nature of adsorbed protein and the physicochemical properties of the solid surfaces. Hence ribonuclease A unfolds and loses stability on adsorption on silica nanoparticle surfaces [13], but no significant change in structure and stability of cytochrome-C has been observed on interaction with zinc oxide nanoparticle [14]. Recent reports show that some nanomaterials can induce the formation of protein-based aggregates or catalyze the formation of protein fibrils by modifying the protein structure and leading to the growth of extended assemblies [15,16]. Proteins at the nanoparticle surface are observed to be partially unfolded. Such nanoparticle induced unfolded proteins likely catalyze the observed aggregate formation and growth. Incorrect folding or misfolding of proteins resulting in the formation of protein aggregates has been considered as a wide ranging phenomenon and is of importance in different areas like food industry, disease pathology, biopharmaceutical industry, etc. In vivo tissue deposition of fibrillar protein aggregates called amyloid is the cause of several degenerative diseases. Thus the reverse process, namely the folding of proteins by the synthetic nanoparticle has received much attention and only a few have been reported [17,18]. As with other nanoparticles, gold nanoparticles show a size dependent behaviour up to a diameter of about 100 nm; they are characterized by their intensive light absorption which is caused by excitation of collective oscillations of valence bond electrons, the so-called ‘plasmon resonance’. In most cases, functionalized gold nanoparticles are used for different biomedical applications of AuNPs [19,20].
http://dx.doi.org/10.1016/j.ijbiomac.2014.05.006 0141-8130/© 2014 Published by Elsevier B.V.
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Bovine beta lactoglobulin (-lg) is a well known globular whey protein (18,000 Da, pI 5.2). It comprises 162 amino acid residues and is predominantly a -sheet protein. -lg is believed to function as transporters of some hydrophobic molecules such as retinol and long chain fatty acid molecules across the intestinal membrane. Upon heating above 60 ◦ C, the native structure of -lg begins to change, with a decrease in amount of ordered zones and an increase in the exposure of the tryptophan and free thiol group [21,22]. It has been found to be converted readily to amyloid fibrils upon heating at acidic pH [23,24]. At pH 7.0 the protein exists as a reversible dimer and extent of dimerization depends on pH, temperature, protein concentration and ionic strength of the medium. Furthermore, -lg forms soluble aggregates of polymerized protein after heating 80 ◦ C for 1 hr at pH ∼7.0. Reducing and nonreducing SDS-PAGE results indicated that both covalent and non-covalent interactions are responsible for aggregate formation [25,26]. In the present study, we have synthesized and used AuNPs of a particular size instead of any functionalized gold nanoparticles as reported by other workers. In this paper we demonstrated the interaction of gold nanoparticles with the model amyloidogenic protein -lg and studied the self assembly of thermally perturbed unfolded -lg in the presence and absence of gold nanoparticle of 20 nm diameter with various concentrations. We have characterized the aggregates, size distribution and the morphology of the species formed during thermal exposure of -lg at in the absence and presence of gold nanoparticle with various concentrations at 75 ◦ C for 1 h at pH ∼7.5 by Rayleigh Scattering, thioflavin T (Th T) assay, dynamic light scattering (DLS) and transmission electron microscopy (TEM). Our results showed that AuNPs are capable of retarding the formation of self assembly of -lg.
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2. Materials and methods
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2.1. Reagents and chemicals
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-lg sample using Shimadzu-TCC 240A UV-Vis spectrophotometer. AuNPs samples showed a characteristic signal at 520 ± 2 nm which confirms the formation of AuNPs of 20 nm diameter (AuNP20). The size was further confirmed by TEM image. This absorption band of AuNP20 was shifted in presence of native and heat treated -lg. 2.4. Thermal aggregation of ˇ-lg Beta lactoglobulin undergoes aggregation readily upon thermal exposure at 80 ◦ C for 1 h and produces spherical protein aggregates at pH 7.5 in 10 mM Na-phosphate buffer [25,26]. Thus the aggregation of -lg was then measured in absence and presence of AuNPs taking protein concentration 2.5 mg/ml and exposing it to 75 ◦ C for 1 h. 2.5. Electrophoresis measurements Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed under nonreducing condition using 15% acrylamide resolving gel according to Laemmli’s method [29]. Samples of -lg solution (2.5 mg/ml) in 10 mM sodium phosphate buffer, pH 7.5 were heated to 75 ◦ C for 1 h in absence and in presence of AuNPs (100 M) separately. The resulting solutions were filtered with a syringe filter with 0.2 m membrane filter. The sample solutions were mixed with equal amount of SDS-gel loading solutions and boiled for 95–100 ◦ C before they were analyzed with SDS-PAGE. Aliquots (20 l) of heat treated -lg solution in the presence and absence of AuNPs were loaded in the wells. Similarly 30 l of native -lg solution (2.5 mg/ml) were loaded in another wells. Electrophoretic separations were done applying a maximum voltage of 100 volts for 1 h. The gel was stained with Coomassie Brillant Blue R-250 and destained in the usual way using the solution containing methanol and acetic acid. 2.6. Rayleigh light scattering
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Bovine -lactoglobulin (-lg) was isolated and purified from cow milk as described earlier [27]. Since the extinction coefficient of -lg (0.96 mg−1 ml−1 cm−1 at 280 nm) is known, different concentrations of protein samples were prepared by dissolving -lg samples in milli-Q-water and then measuring the O.D. at 280 nm. Chloroauric acid (HAuCl4 ) was purchased from Sisco Research Laboratoty (Mumbai, India). Tri-sodium citrate dehydrate, methanol, acetic acid, sodium sulphate, sodium dihydrogen phosphate (all AR grade) were purchased from Merck (Mumbai, India). Acrylamide, bis acryl amide, N,N,N ,N -tetramethylethylenediamine (TEMED), ammonium per sulphate, SDS, bromophenol blue and Coomassie briliant blue were obtained from Sigma-Aldrich. The other chemicals used were of highest purity available.
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2.2. Preparation of gold nanoparticles (AuNPs)
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AuNPs were prepared by the reduction of hydrogen tetra chloroaurate (III) (HAuCl4 ) trihydrate with sodium citrate following the methods of Frens [28]. Glasswares used in this preparation were thoroughly cleaned in aqua regia (nitric acid: sulphuric acid 1:3), rinsed in milli-Q-water and then dried in an oven. AuNPs were synthesized by adding rapidly trisodium citrate (5 ml, 38.8 mM) to a boiling solution of HAuCl4 (50 ml, 1 mM), then solution was kept continuously boiling for another 30 min to give a wine red solution. After filtering the solution through 25 mm syringe filter with 0.2 m membrane, the filtrate was stored at 4 ◦ C.
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2.3. UV–visible spectroscopic characterization of AuNP samples
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At room temperature optical absorption spectra in the wavelength range 350–700 nm were taken in presence and absence of
The effect of citrate coated AuNPs on the aggregation of heat exposed -lg was monitored by Rayleigh Light Scattering (RLS) measurement by observing emission at 350 nm after exciting the solution at 350 nm using 10 mM phosphate buffer at pH 7.5 in a Shimadzu spectrofluorimeter (Shimadzu 5301 PC). The fluorescence spectra were collected at 25 ◦ C using 1 cm path-length cell and protein concentration 0.25 mg/ml. The excitation and emission slits were set at 5 nm. 2.7. Thoiflavin T (ThT) assay Th T is a dye which shows enhanced fluorescence at 480 nm when bound to amyloid fibrils [30]. Thus to investigate and compare the aggregates formed by heat exposed -lg in absence and presence of AuNPs, the following assay was employed. Briefly 250 l of -lg samples (native and heat treated) having concentration 1 mg/ml was taken. It was then added to 1.75 ml of 50 M Th T solution (stock 1 mM Th T in 20 mM sodium phosphate buffer, pH 7.5) mixed thoroughly and incubated for 5 min. The assay solution was excited at 450 nm and the emissions were measured over the range 460–600 nm. Slit widths for both excitation and emission were kept at 5 nm. Three replicates were performed and the data were averaged. 2.8. ANS fluorescence studies to monitor the hydrophobicity Exposure of hydrophobic patches in protein during the aggregation process was monitored using polarity sensitive fluorescent probe 1-Anilinonapthalene-8-sulfonate (ANS) [31]. A stock solution of ANS was added to each aliquot of native and heat treated
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-lg (both in presence and absence of citrate coated AuNPs) so that the final ANS concentration in each aliquot was 30 M. Typically, ANS concentration was 50 molar excess of protein concentration. The ANS-fluorescence intensities were measured using Shimadzu RF-5301 PC with excitation at 370 nm and scanning the emission wavelength from 450 nm to 550 nm. Slit widths were set at 5 nm for both excitation and emission. Each spectrum was blank corrected. Data points were the average of triplicate measurements.
2.9. Analysis of secondary structures by CD spectroscopy To trace the structural or conformational changes that gold nanoparticles might induced on -lg, circular dichroism measurements were carried out on a Jasco Spectropolarimeter (J-815) at 20◦ C in the far-UV (200–250 nm) and near-UV regions (250–300 nm) using the rectangular cells of 1 nm and 10 mm pathlength, respectively. Heat treated -lg solutions (in presence and absence of AuNP) having concentrations 0.25 mg/ml and 1.0 mg/ml were used for far-UV CD and near-UV CD measurements, respectively. All the spectra are average of three scans. The final spectrum was obtained after the subtraction of corresponding solvent spectrum. The far UV-CD curves were fitted into a curve-fitting program CDNN 2.1 to determine the percent of secondary structures present in -lg under different conditions.
2.10. Monitoring of secondary structures of ˇ-lg during thermal incubation with AuNPs by FT-IR spectroscopy
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For FT-IR measurements, native and heat treated -lg solutions (50 l) (in presence and absence of AuNPs) having concentrations 20 mg/ml were taken in a microcon filter device and diluted with 200 l of D2 O. It was then quickly centrifuged at 4000 × g for 8 min until the volume reached ∼50 l. After that 200 l of D2 O was added again and centrifuged for another 8–10 min. This process of D2 O exchange was repeated 3–4 times [32]. Finally, the D2 O exchanged -lg samples were placed between two CaF2 windows separated by a 50 m thick teflon spacer. FT-IR scans were collected in the range of 1400–1800 cm−1 at a resolution of 2 cm−1 in N2 environment using a Spectrum 100 FT-IR spectrometer (Perkin Elmer). Spectrum of D2 O at pD 7.5 was collected and subtracted from sample spectrum.
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2.11. Dynamic light scattering (DLS) measurements
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The diffusion of nanoparticles in solution induces fluctuations in the intensity of the scattered light. DLS detects these fluctuations using an auto correlator on a microsecond time scale and is used to analyze the distribution of the molecules and supramolecular aggregates as it is very sensitive to particle size [33]. Different sizes of molecules in the solution can be observed in different peaks provided their sizes vary sufficiently. In our experiment, DLS measurements were performed with heat treated -lg in presence and absence of citrate coated AuNPs employing Zetasizer Nanos (Malvern Instrument, U.K.) equipped with 633 nm laser and using 2 ml rectangular cuvette (path length 10 mm). Measurements were done at 20 ◦ C taking 250 l of heat-treated -lg sample in 1.75 ml Na-phosphate buffer. Then varying amounts of AuNP (25–100 M) were mixed thoroughly and kept for 10 min. Similar measurements were done separately with heat treated -lg in absence of AuNPs and with only AuNP samples. The time-dependent auto correlation function was acquired with twelve acquisitions for each run. Each data is an average of five such acquisitions.
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2.12. Transmission Electron Microscopy (TEM) The morphological studies of AuNPs and heat treated -lg aggregates (in absence and presence of AuNPs) were performed by using high resolution transmission electron microscope (JEOL-HRTEM2011, Tokyo, Japan) with an accelerating voltage of 80–85 kV in different magnifications. The sample solutions were sonicated for 60 s and diluted 50–150 times. A droplet of the diluted sample was put on a carbon coated copper grid of mesh size 300 C (Pro Sci Tech). After 20 s the droplet was removed with a filter paper followed by a droplet of 2% uranyl acetate (Sigma, Steinheim Germany) solution put on the grid and finally removed after 15 s and left for air dry and used for imaging purpose.
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3. Results and discussion
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3.1. Synthesis and characterization of citrate stabilized AuNPs
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The reduction of HAuCl4 (Au3+ ) to yield colloidal gold particles occurred via the transfer of electrons from the citrate to the Au3+ ions leading to the formation of Au0 . The metallic gold then nucleates and grows to form AuNPs and are subsequently capped and stabilized by surrounding citrate ions. Formation of AuNPs was confirmed by the appearance of dark cherry-red colour of the colloidal solutions. The inset shows a picture of glass vials containing AuNP solution. The UV–vis absorption characteristics of the colloidal solution are shown in Fig. 1. The UV–vis spectra of AuNPs and AuNP + -lg (heat stressed) showed the typical surface plasmon resonance (SPR) bands, whereas no such band was observed in the wavelength region 350–700 nm of spectrum of -lg (alone). The SPR band for AuNPs (alone) appeared at 520 nm (due to the strong red colour of AuNPs) and a broad absorption band at 250 nm. This characteristic plasmon band supported the formation of AuNPs of size 20 ± 2 nm. The decrease in intensity (arbitrary units) and mild red shift (520–525 nm) in absorption wavelength were observed in presence of -lg at room temperature. The red shift in wavelength of SPR band of Au is due to conjugation of AuNP with -lg. Fig. 1, inset plot shows a gradual increase of absorption wavelength with increasing
Fig. 1. UV–vis absorption spectra in the wavelength range of 350–750 nm of citrate stabilized 1 mM AuNPs (line a) (max = 520 nm), AuNPs solution in presence of -lg (line b) (max = 525 nm) and of -lg alone (line c). Inset picture show the actual colour of AuNP solution and the inset plot represents the change in absorption wavelength with AuNP concentrations.
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Fig. 2. SDS-PAGE (15%) patterns of native -lg (lane 2 and 3), heat stressed -lg at 75 ◦ C for 1 h in absence (lane 5 and 6) and in presence of 100 M of AuNPs (lane 4 and 7). Lane 1 represents the SDS PAGE pattern of marker protein of known molecular weight (PageRulerTM, Prestained Protein Ladder, 10, 17, 26, 34, 43, 55, 72, 95, 130, and 170 kDa, respectively; Fermentus Life Science, #SM0671) run parallely.
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-lg concentrations. Similar conclusion was made by other group of researchers [34]. When proteins interact with AuNPs, the apparent size of the particles increases due to adsorption of protein and the resultant change in dielectric constant. 3.2. SDS-polyacrylamide gel electrophoresis study
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In the SDS-PAGE analysis under non-reducing condition (Fig. 2) the native -lg appears as single band (lane 2–3) corresponding to the momoneric state of -lg. But the heat treated -lg solution (heating at 75 ◦ C for 1 h) appeared as series of protein bands in SDSPAGE indicating the formations of oligomers of -lg (lane 5–6). This technique dissociates all the non-covalently bonded aggregates into monomers while the disulphide-linked aggregates remain intact. The cys121 of -lg forms S-S linkage when heated 75–80 ◦ C leading to the formation of dimers and other covalently bound “intermediates” during aggregations [25,35,36] by scrambling of disulphide bonds (creation of new intramolecular and intermolecular disulphide bridges and rearrangement of old intramolecular disulphide bridges) [37]. Heating at 75 ◦ C decreases the intensity of the band corresponding to the monomer (band I) and subsequent increase in the intensities of the bands (II–VIII) corresponding to different oligomers and higher aggregates. But the SDS-PAGE analysis of heat treated -lg in presence of AuNPs under non-reducing condition shows a quite different result. The band (III–VII) corresponding to -lg oligomers having lower electrophoretic mobilities and the band (VIII) corresponding to larger aggregate which do not enter into the gel are absent (lane 4 and 7). Thus citrate coated AuNPs are capable of inhibiting the aggregation -lg under heat stress. Immobilization of -lg on AuNP surface mediated by free thiol (cys121 ) on -lg is the main reason to attenuate the propensity of random protein–protein interactions resulting inhibition of protein aggregation.). Lane 1 represents the SDS PAGE pattern of marker protein of known molecular weight (PageRulerTM, Prestained Protein Ladder, 10, 17, 26, 34, 43, 55, 72, 95, 130, and 170 kDa, respectively; Fermentus Life Science, #SM0671) run parallely.
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3.3. Rayleigh Light Scattering (RLS) measurements
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Rise of RLS data from a protein solution is an indication of aggregation. Thus lowering of RLS data should be a useful parameter in support of disaggregation or disassembly of protein. Fig. 3 shows a change in scattering intensity measured at 350 nm after exciting
Fig. 3. Rayleigh Light Scattering data (turbidity measurements): Effect of increasing amount of AuNPs on heat stressed -lg at 75 ◦ C for 1 h during the incubation of -lg with AuNP at pH 7.5 (square). Control measurements with AuNPs alone (circle). The samples were excited and emitted at 350 nm.
the heat treated -lg solution at 350 nm in absence and presence of varying amount of AuNPs at pH 7.5. It shows a gradual decrease in scattering intensity with the increase amount of AuNP solutions. Highest scattering intensity of the solution (3 fold) is observed in absence of AuNPs, suggesting the probability of aggregate formation with heat treated -lg in absence of AuNPs. But the heating of -lg solutions in presence of citrate coated AuNPs up to 100 M, shows a remarkable decrease in scattering intensity. The control measurements with varying amounts of AuNPs alone were performed and compared. Higher AuNPs concentrations scattered UV light at 350 nm. Hence gradual decrease in scattering intensity of the resultant heat treated -lg solutions in presence of citrate coated AuNPs supports the capping of protein molecules on AuNPs surfaces and disaggregation of -lg in presence of AuNPs. 3.4. Disassembly of ˇ-lg aggregates with AuNPs studied by Th T assay The disassembly of -lg aggregates was further confirmed by Th T assay. Th T is a cationic benzothiazole dye showing enhanced fluorescence upon binding to protein assembly. The native -lg undergoes thermal aggregation in absence of AuNP, showing maximum Th T intensity upon binding with Th T (Fig. 4A). This result is in accordance with other amyloidogenic proteins. Furthermore lowering of Th T fluorescence intensities were observed with the -lg samples heated in presence of varying amount of AuNPs, indicating prominent dimer formation with lesser amount of higher protein assembly in presence of AuNPs (lane 4 and 7). Thus citrate coated AuNPs are capable of suppressing thermal aggregation of -lg in a concentration dependent manner. AuNPs thus appeared to act in a similar fashion to molecular chaperones. The plot of the Th T intensity vs. AuNP concentrations (Fig. 4B) showed that the Th T intensities decreased with the increase of AuNP concentrations (25–100 M). The results indicated that the citrate capped AuNP interferes and suppress the fibrilization pathway of -lg. It has been reported that AuNPs form self-cluster while inducing the aggregation of lysozyme [38] or while showing the size dependent chaperon properties in the aggregation of citrate synthase [17]. 3.5. ANS fluorescence studies to monitor the hydrophobicity during fibrillation of ˇ-lg The aggregates induced by thermal exposure of -lg were further confirmed by ANS fluorescence studies. Molten globule state
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Fig. 5. ANS flouresence of heat stressed -lg (75 ◦ C for 1 h) in the absence and presence of AuNPs in 10 mM phosphate buffer at pH 7.5, excitation was done at 380 nm and emissions were measured in the wavelength range 400–550 nm. Fig. 5 lines a–d corresponds to heat stressed -lg in the presence of 0, 25, 50 and 100 M AuNPs. Protein concentrations during ANS flouresence measurements were 0.25 mg/ml.
3.6. Secondary Structural changes of ˇ-lg with AuNP Studied by CD spectroscopy
Fig. 4. (A) Th T assay to study the aggregation of heat stressed (75 ◦ C for 1 h) -lg in absence and presence of AuNPs where the final -lg and Th T concentrations were 125 g and 50 M, respectively in 10 mM phosphate buffer at pH 7.5. Native -lg was incubated with 0 M (line a), 25 M (line b), 50 M (line c) and 100 M (line d) of AuNPs. Fluorescence emissions were monitored in the wavelength range 460–600 nm after excitation at 450 nm. (B) Bar diagram of the end-point Th T intensity versus AuNP concentrations in Th T assay to study the aggregation of heat stressed (75 ◦ C for 1 h) -lg in 10 mM phosphate buffer at pH 7.5. Fluorescence emissions were monitored in the wavelength range 460–600 nm after excitation at 450 nm. Standard deviations are within the range of ±3.0.
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of proteins can be detected by binding of ANS to the hydrophobic region of protein that ultimately results in an increase of fluorescence intensity [39]. Fig. 5 showed the ANS fluorescence spectra of thermally exposed -lg in absence or presence of AuNPs. In the absence of AuNPs the heat exposed -lg at pH 7.5 showed highest ANS fluorescence intensity at 480 nm. This increase in fluorescence intensity may be attributed to more access of ANS to the hydrophobic patches present in protein upon thermal denaturation. These hydrophobic patches enhance the protein–protein interactions leading to thermal aggregation of -lg [25]. But in the presence of AuNPs during thermal exposure of -lg, significant decrease of ANS fluorescence was observed indicating the loss of ANS biding sites. Gradual decrease of ANS fluorescence intensity with marginal red shifting of emission maximum was observed upon thermal exposure of -lg solutions in presence of varying amount of AuNPs. The lowering of ANS binding in presence of AuNPs suggests the formation of lesser hydrophobic patches by -lg upon thermal stress at 75 ◦ C. AuNPs are thus capable of suppressing -lg aggregation in a concentration dependent fashion.
We investigated the secondary structural changes of heat exposed -lg in absence and presence of AuNPs with respect to native -lg in 10 mM phosphate buffer, pH 7.5. The results of the far UV-CD studies are shown in Fig. 6. The native -lg (Fig. 6, line-a) shows a negative signal around 216 nm which is characteristic for the -sheet structure of the protein. But the heat stressed -lg shows (Fig. 6, line-b) an increase in negative ellipticity value at 216 nm suggesting the retaining of the native secondary structure with the possibility of formation of crossed linked sheet structure. The thermal denaturation opens up the buried cys121 and leading to the formation of disulphide linked oligomers. Higher non-covalent oligomer formation at this high temperature is the cause of such crossed linked -sheet structures. But the CD spectra of heat stressed -lg in presence of varying amounts of
Fig. 6. Far UV-CD spectra of native and heat stressed -lg (75 ◦ C for 1 h) in absence and presence of AuNPs in 10 mM phosphate buffer at pH 7.5. Fig. 6, line a shows native -lg whereas lines b–e corresponds to heat stressed -lg in the presence of 0, 25, 50 and 100 M AuNPs. Line ‘f’ corresponds to the CD spectrum of only AuNPs (100 M). A plot of MRE values at 209 nm against varying AuNPs concentrations is represented (inset, Fig. 6). Concentrations of -lg samples in far UV-CD measurements were 0.25 mg/ml.
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Fig. 7. FT-IR spectra in the region 1400–1750 cm−1 of native (a), heat treated -lg (b) and heat treated -lg in presence of AuNPs within the concentration range of 25 M (c), 50 M (d), 75 M (e), 100 M (f), respectively. Protein concentrations were 20 mg/ml. FT-IR spectra in the amide-I region 1600–1700 cm−1 (inset, Fig. 7). Each spectrum is an average of 32 scans in D2 O solvent at 25 ◦ C.
Fig. 8. Number particle size distribution spectra in DLS studies of (a) only 1 mM AuNPs, (b) heat stressed -lg (75 ◦ C for 1 h, 2.0 mg/ml), (c) heat stressed -lg (75 ◦ C for 1 h, 2.0 mg/ml) in presence of 25 M AuNPs, (d) heat stressed -lg (75 ◦ C for 1 h, 2.0 mg/ml) in presence of 50 M AuNPs. Each of the spectra is an average of 48 scans.
shifts from 1632 cm−1 to 1650 cm−1 (Fig. 7, inset). The curve-g shows a significant broadening and shifting of amide I band to ∼1650 cm−1 , indicating large decrease in -sheet structure and consequent increase in ␣-helical and random coil structural content. The FT-IR studies adequately support the CD results (Table 1). AuNPs are thus inhibiting the aggregation of -lg in a concentration dependent manner.
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citrate coated AuNPs show a significant change in their pattern (Fig. 6, lines c–e). The incubation of -lg at 75 ◦ C in the presence of AuNPs changes the secondary structure as the minimum at 216 nm becomes less pronounced and thus indicating the loss of gross -sheet structure. There was concomitant increase in ␣helical structure as evidenced by an increase in ellipticity at 210 nm and 222 nm in each case. Thus AuNPs induced - to ␣-helical and random coil transitions are observed when -lg sample were thermally stressed at 75 ◦ C in presence of varying amount of AuNPs (Fig. 6, lines c–e). To further assess AuNP-induced - to ␣-helical transitions, the individual secondary structural elements were analyzed with CDNN 2.1 software and the results are shown in Table 1. These observations suggest that AuNPs are capable of inhibiting the formation of cross linked -sheet structure and favouring the formation of ␣-helical structural content.
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3.7. Analysis of secondary structures of ˇ-lg with AuNPs by FT-IR
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The FT-IR spectra of -lg samples (native and heat exposed in absence and presence of AuNPs) in D2 O-buffer are shown in Fig. 7. The resulting spectrum for native -lg (Curve-a) shows the amide I band at around 1632 cm−1 and amide II band at 1480 cm−1 . These features are characteristic for the protein having predominant sheet structures [40,41]. Most of the information on the protein’s secondary structure is contained in the amide I band in the region 1700–1600 cm−1 . Heat exposed -lg (in absence of AuNP) showed a broadening of peak centred around 1634 cm−1 with the appearance of a shoulder in around 1620 cm−1 region and thus identifying the cross-linked -sheet structure similar to those found in the aggregates [42]. When -lg solutions are incubated at 75 ◦ C with gradual increasing amount of AuNPs, the amide I band gradually
3.8. Dynamic Light Scattering (DLS) measurement
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To confirm our previous results, we employed DLS to study the particle size distributions (Fig. 8). DLS is frequently used to examine the hydrodynamic radius of spherical particles in solution and the intensity of the scattered light is dominated by large particles in solution. Although the -lg aggregates/fibrils are heterogeneous and with long rod-like shapes, DLS analysis can provide a quantitative estimation of aggregates distribution. In order to understand the nature of association occurring during thermal incubation of -lg at 75 ◦ C in absence and presence of AuNPs, we attempted to assess the oligomeric status of -lg during aggregation and disaggregation process. DLS analysis can provide a qualitative estimation of the aggregate distribution. The data were plotted as scattered light intensity versus size in radius. Here we first examined the size distribution of AuNPs alone (Fig. 8, curve a). The hydrodynamic diameter of bare AuNP was ∼20 ± 2 nm which was in good agreement with the size obtained from TEM measurement. The size distribution curves (Fig. 8b–d) of heat stressed (75 ◦ C) -lg in absence and presence of AuNPs (25 and 50 M) showed the formation of aggregates of different sizes. In absence of AuNP, DLS analysis showed the wider distribution of large aggregates of -lg with the diameters at ranging from ∼40 nm to 400 nm
Table 1
Q5 Structural integrity of native and heat treated structural -lg at 75 ◦ C for 1 h (in absence and presence of AuNPs) as determined by circular dichroism (CD)a . -lg Samples
% of ␣-Helix
% of -Sheet
% of -Turn
% of Random coil
Native (a) Heat stressed in absence of AuNp (b) Heat stressed in presence of 25 M AuNP (c) Heat stressed in presence of 50 M AuNP (d) Heat stressed in presence of 75 M AuNP
11.9 10.2 14.7 15.8 17.2
45.2 46.3 34.4 30.2 26.4
11.0 12.0 15.2 12.3 11.5
31.9 31.5 35.7 41.7 44.9
a
419
Calculated by CDNN 2.1 software.
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Fig. 9. Selected TEM images of AuNPs at different magnifications (a–c) and heat stressed (75 ◦ C for 1 h) -lg in absence (d–f) and in presence (g–i) of AuNPs in 10 mM phosphate buffer at pH 7.5. During incubations concentrations of -lg and AuNPs were 2.5 mg/ml and 25 M, respectively. TEM images (j–k) represent the prominent presence of AuNPs on the -lg fibrillar aggregates at higher concentrations (100 M).
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(Fig. 8, curve b). In presence of 25 M AuNP, the size of the aggregates decreased largely and species with smaller diameter became prominent (Fig. 8, curve c). Further increasing the concentration of AuNPs up to 50 M shows smaller change in the size of the protein molecules (Fig. 8, curve d). Thus upon incubation with AuNPs, size distribution of large -lg particles (aggregates) decreased and the scattering was dominated by particles with smaller size. Our results thus showed that AuNPs can suppress the formation of larger aggregates of -lg during its thermal stress at 75 ◦ C.
3.9. Characterization of the aggregates by TEM To confirm the results obtained from previous sections, the morphological studies of the end-point products formed in the heat induced aggregation of -lg in the absence and presence of AuNPs were done by TEM. The bare AuNPs appear as spherical black particles with a diameter of about 20 nm in the images (Fig. 9a–c). In the absence of AuNPs, -lg forms long, smooth fibrillar aggregates (Fig. 9d–f) upon incubation at 75 ◦ C for 1 h. However incubation
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with 25 M AuNP, -lg forms aggregates having different morphological characters (Fig. 9g–i). The number of well-defined fibril is reduced. Some fibrils became fragmented. Moreover some spherical oligomers along with formation of amorphous aggregate were observed. Such species are observed either attached to the fibrils or in association with the AuNPs even at higher concentrations (100 M) (Fig. 9j and k).
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4. Discussion
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The native -lg forms covalently linked intermediates during its aggregation at pH 7.5 upon thermal exposure heating at 75 ◦ C [35,36]. These intermediates correspond to the formation of dimers, trimers and tetramers of -lg, respectively with involvement of free –SH group of cys121 of the monomer. The results of SDS-PAGE (Fig. 2) support the formation of such covalently linked oligomers along with much larger aggregates that could not penetrate the gel (lane 3–5). Aggregation may not be limited to the formation of linear aggregates but branched aggregates appear at longer times, as shown in DLS and TEM images. It has been shown that disulfidelinked intermediates further react to yield larger size aggregates via non-covalent interactions [25,43]. Understanding the nanoparticle–protein interaction is crucial for the development of nanomedicine, since the nanoparticles are capable of entering in the biological system to act as delivery, imaging and diagnostic tools for different illness. In our studies here we have focused on the understanding the effect of the most biocompatible nanoparticle, AuNP on the aggregation of heat exposed -lg at 75 ◦ C in physiological pH using 20 nm AuNPs. Firstly SDS-PAGE experiment shows unambiguously that incubation of -lg at 75 ◦ C in presence of AuNPs resists the formation of oligomeric species (like trimer, tetramer, etc.) and higher aggregates (lane 5 and 6) whereas incubation of -lg in absence of AuNPs can produce all the oligomeric and larger aggregates which can not enter into the gel (lane 2–4). AuNPs thus stabilize the monomeric and dimeric forms of the protein inhibiting the nucleation and elongation for the formation of higher aggregates. Similarly the results of ANS-fluorescence measurement and Th T experiments confirmed our observations in the SDS-PAGE study. Both ANS and Th T experiments showed that citrate coated AuNPs were able to inhibit the aggregation of -lg as evidenced from the low end point Th T fluorescence. This lowering of ThT fluorescence intensity and hence extent of inhibition is dependent on AuNP concentrations (Fig. 4B). The result may be confirmed by the morphological studies with TEM, showing the generation of smaller, fragmented fibril and some spherical oligomers in the presence of AuNPs. Thermal incubation of -lg in presence of higher AuNP concentration (100 M) generates only the smaller spherical aggregates. In the DLS analysis, the larger fibrillar aggregates generated from thermal incubation of -lg in absence of AuNP were dominating in scattering the light while smaller ragged aggregates including the dimmers were dominating in presence of AuNPs. Results of CD and FT-IR support the secondary structural changes with decrease of -sheet structural contents when the -lg solutions were treated with AuNPs at 75 ◦ C. Other results suggest that the bare AuNPs in nanomolar range are capable of retarding protein aggregation and redirecting it into off-pathway intermediate formation. Similar effect has been observed when organic molecules like poly phenol derivative (−) epi-gallocatechine gallate retards and redirects A and ␣-synuclein in Parkinson’s disease forming off-pathway oligomers [44]. Only a few examples are available in the literature where bare AuNP can prevent the aggregation of proteins. Very recently it has been observed that cysteine coated AuNPs are capable of suppressing the heat induced aggregation of BSA and citrate synthase [45]. On
Scheme 1. Schematic representation of coupled mechanism of heat stressed -lg amyloid fibrillation in absence and presence of AuNPs of ∼20 nm diameter.
the other hand the bare AuNPs and the copolymeric nanoparticles have been used as rescuing molecules in the retardation of A-40 fibrilization [46,47]. Based on this study, it appears that AuNPs may process the capability of suppressing the aggregation of proteins and facilitate the formation of dimeric and native-like monomeric structure. Results of previous studies and our current observations show that this aggregation inhibitive property of AuNPs is not only dependent on their size and charge on their surface but also depends on their concentration. The Scheme 1 described a simple mechanism that can explain nicely our experimental observations and protein aggregation inhibitory activity of AuNPs. Heating at 75 ◦ C in absence of AuNPs, the Cys121 of -lg becomes exposed on thermal denaturation and forms covalently linked (s-s bond) ‘intermediates’ like dimer, trimer and tetramer, etc. The presence of monomer along with other oligomeric forms can be explained in the view of intra and intermolecular disulfide bond exchange mechanism. These disulfide linked intermediates undergo further aggregation via non-covalent interactions like hydrophobic interactions to yield larger aggregate. Aggregation would not be limited to the formation of linear aggregates but branched aggregates appear after a longer period of time. But incubation of -lg at 75 ◦ C in presence of AuNP helps (leads to the formation of dimer and smaller aggregates of -lg). At high temperature and concentration, AuNPs may tend to form nano-cages (appears in the TEM image, Fig. 9). Gold nano-cages likely to have higher heat capacities, knowing as nano-fluids [48]. The smaller nanoparticle having greater surface area, enhance the formation of nano-cages. The cage-trapped protein may be thermally shielded, as some excess heat is likely to be adsorbed by nano surface leading to the refolding of -lg into its native-like structure. Moreover the interaction of the protein molecules, on the surface of nano-cluster reduces the hydrophobic interaction between the protein molecules and thereby inhibiting aggregation. 5. Conclusion In this study we have examined the effects of citrate stabilized AuNP on heat induced aggregation of an important globular protein beta lactoglobulin (-lg). It was observed that AuNPs are able to inhibit the formation of amyloid fibrillar aggregates of lg under thermal condition in a concentration dependent manner. Such inhibition may be due to the stabilization of -lg on the nanoparticle surface. In the earlier reports, the bare nanoparticles showed similar activities against amyloidosis [46]. Previous studies
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also showed that these smaller gold nanoparticles (
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Amyloid fibril formation by -lactoglobulin is inhibited by gold nanoparticles
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Subrata Sardar, Sampa Pal, Sanhita Maity, Jishnu Chakraborty, Umesh Chandra Halder ∗
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Q2 Organic Chemistry Section, Department of Chemistry, Jadavpur University, Kolkata 700032, India
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Article history: Received 25 January 2014 Received in revised form 29 April 2014 Accepted 2 May 2014 Available online xxx
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Keywords: -Lactoglobulin (-lg) Amyloid fibril Gold nanoparticles (AuNP) Inhibition
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1. Introduction
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The endogenous deposition of protein fibrillar aggregates in the tissues is the cause of several neurodisorders. In the present study the native -lactoglobulin (-lg) has been driven towards amyloid fibrillar aggregates when it was exposed to heat at 75 ◦ C for 1 h at pH 7.5. The citrate stabilized gold nanoparticle (AuNPs) of 20 nm diameter is shown to inhibit the thermal aggregation of -lg. The stability of the -lg against heat stress was assessed by studying its aggregation at 75 ◦ C, either in presence or in absence of AuNPs. AuNPs stabilizes the monomeric and dimeric forms of the -lg inhibiting the nucleation and elongation for the formation of higher aggregates. Incubation of -lg with AuNPs at 75 ◦ C results in the formation of smaller ragged aggregates. Results show that AuNPs possess the capability of inhibiting the formation of amyloid fibrillar aggregates of -lg in a concentration-dependent manner and may thus facilitate the refolding into native like structure. AuNPs thus serve as nano-chaperon to inhibit the protein aggregation. Thus chaperon like activity of AuNP may be exploited in the design of rational therapeutics for the neurodegenerative diseases. © 2014 Published by Elsevier B.V.
Nanomaterials and nanotechnology have received enormous popularity in the last decade due to their wider application in industry, technology, biological science and in the field of fundamental research [1–3]. Owing to their small size and penetration ability nanoparticles can be used as biosensors, drug carriers, cancer therapeutic agents and bio labelling materials [4–7]. However the interactions of the nanoparticles [NPs] with the biomolecules, especially proteins, are very important for understanding the biocompatibility in biomedical applications. It is commonly known that the surface adsorption can lead the unfolding and hence structural changes of proteins [8,9]. Upon adsorption on silica nanoparticles, lysozyme was found to undergo a reduction in both alpha-helical content and enzymatic activity, with greater loss on larger NPs [10]. Such absorption of proteins, on the other hand, is also effective for surface coating to stabilize nanoparticle and reduce their cytotoxicity. Recently it has been shown that when exposed to biomolecules, the bare NPs form a ‘nanoparticle protein corona’ [11]. Relevance of such protein corona on the biological impacts of nanoparticle has also been discussed [12]. The change in protein structure and function are strongly dependent on both
∗ Corresponding author. Tel.: +91 33 2414 6223; fax: +91 33 2413 7902. E-mail address: [email protected] (U.C. Halder).
nature of adsorbed protein and the physicochemical properties of the solid surfaces. Hence ribonuclease A unfolds and loses stability on adsorption on silica nanoparticle surfaces [13], but no significant change in structure and stability of cytochrome-C has been observed on interaction with zinc oxide nanoparticle [14]. Recent reports show that some nanomaterials can induce the formation of protein-based aggregates or catalyze the formation of protein fibrils by modifying the protein structure and leading to the growth of extended assemblies [15,16]. Proteins at the nanoparticle surface are observed to be partially unfolded. Such nanoparticle induced unfolded proteins likely catalyze the observed aggregate formation and growth. Incorrect folding or misfolding of proteins resulting in the formation of protein aggregates has been considered as a wide ranging phenomenon and is of importance in different areas like food industry, disease pathology, biopharmaceutical industry, etc. In vivo tissue deposition of fibrillar protein aggregates called amyloid is the cause of several degenerative diseases. Thus the reverse process, namely the folding of proteins by the synthetic nanoparticle has received much attention and only a few have been reported [17,18]. As with other nanoparticles, gold nanoparticles show a size dependent behaviour up to a diameter of about 100 nm; they are characterized by their intensive light absorption which is caused by excitation of collective oscillations of valence bond electrons, the so-called ‘plasmon resonance’. In most cases, functionalized gold nanoparticles are used for different biomedical applications of AuNPs [19,20].
http://dx.doi.org/10.1016/j.ijbiomac.2014.05.006 0141-8130/© 2014 Published by Elsevier B.V.
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Bovine beta lactoglobulin (-lg) is a well known globular whey protein (18,000 Da, pI 5.2). It comprises 162 amino acid residues and is predominantly a -sheet protein. -lg is believed to function as transporters of some hydrophobic molecules such as retinol and long chain fatty acid molecules across the intestinal membrane. Upon heating above 60 ◦ C, the native structure of -lg begins to change, with a decrease in amount of ordered zones and an increase in the exposure of the tryptophan and free thiol group [21,22]. It has been found to be converted readily to amyloid fibrils upon heating at acidic pH [23,24]. At pH 7.0 the protein exists as a reversible dimer and extent of dimerization depends on pH, temperature, protein concentration and ionic strength of the medium. Furthermore, -lg forms soluble aggregates of polymerized protein after heating 80 ◦ C for 1 hr at pH ∼7.0. Reducing and nonreducing SDS-PAGE results indicated that both covalent and non-covalent interactions are responsible for aggregate formation [25,26]. In the present study, we have synthesized and used AuNPs of a particular size instead of any functionalized gold nanoparticles as reported by other workers. In this paper we demonstrated the interaction of gold nanoparticles with the model amyloidogenic protein -lg and studied the self assembly of thermally perturbed unfolded -lg in the presence and absence of gold nanoparticle of 20 nm diameter with various concentrations. We have characterized the aggregates, size distribution and the morphology of the species formed during thermal exposure of -lg at in the absence and presence of gold nanoparticle with various concentrations at 75 ◦ C for 1 h at pH ∼7.5 by Rayleigh Scattering, thioflavin T (Th T) assay, dynamic light scattering (DLS) and transmission electron microscopy (TEM). Our results showed that AuNPs are capable of retarding the formation of self assembly of -lg.
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2. Materials and methods
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2.1. Reagents and chemicals
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-lg sample using Shimadzu-TCC 240A UV-Vis spectrophotometer. AuNPs samples showed a characteristic signal at 520 ± 2 nm which confirms the formation of AuNPs of 20 nm diameter (AuNP20). The size was further confirmed by TEM image. This absorption band of AuNP20 was shifted in presence of native and heat treated -lg. 2.4. Thermal aggregation of ˇ-lg Beta lactoglobulin undergoes aggregation readily upon thermal exposure at 80 ◦ C for 1 h and produces spherical protein aggregates at pH 7.5 in 10 mM Na-phosphate buffer [25,26]. Thus the aggregation of -lg was then measured in absence and presence of AuNPs taking protein concentration 2.5 mg/ml and exposing it to 75 ◦ C for 1 h. 2.5. Electrophoresis measurements Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed under nonreducing condition using 15% acrylamide resolving gel according to Laemmli’s method [29]. Samples of -lg solution (2.5 mg/ml) in 10 mM sodium phosphate buffer, pH 7.5 were heated to 75 ◦ C for 1 h in absence and in presence of AuNPs (100 M) separately. The resulting solutions were filtered with a syringe filter with 0.2 m membrane filter. The sample solutions were mixed with equal amount of SDS-gel loading solutions and boiled for 95–100 ◦ C before they were analyzed with SDS-PAGE. Aliquots (20 l) of heat treated -lg solution in the presence and absence of AuNPs were loaded in the wells. Similarly 30 l of native -lg solution (2.5 mg/ml) were loaded in another wells. Electrophoretic separations were done applying a maximum voltage of 100 volts for 1 h. The gel was stained with Coomassie Brillant Blue R-250 and destained in the usual way using the solution containing methanol and acetic acid. 2.6. Rayleigh light scattering
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Bovine -lactoglobulin (-lg) was isolated and purified from cow milk as described earlier [27]. Since the extinction coefficient of -lg (0.96 mg−1 ml−1 cm−1 at 280 nm) is known, different concentrations of protein samples were prepared by dissolving -lg samples in milli-Q-water and then measuring the O.D. at 280 nm. Chloroauric acid (HAuCl4 ) was purchased from Sisco Research Laboratoty (Mumbai, India). Tri-sodium citrate dehydrate, methanol, acetic acid, sodium sulphate, sodium dihydrogen phosphate (all AR grade) were purchased from Merck (Mumbai, India). Acrylamide, bis acryl amide, N,N,N ,N -tetramethylethylenediamine (TEMED), ammonium per sulphate, SDS, bromophenol blue and Coomassie briliant blue were obtained from Sigma-Aldrich. The other chemicals used were of highest purity available.
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2.2. Preparation of gold nanoparticles (AuNPs)
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AuNPs were prepared by the reduction of hydrogen tetra chloroaurate (III) (HAuCl4 ) trihydrate with sodium citrate following the methods of Frens [28]. Glasswares used in this preparation were thoroughly cleaned in aqua regia (nitric acid: sulphuric acid 1:3), rinsed in milli-Q-water and then dried in an oven. AuNPs were synthesized by adding rapidly trisodium citrate (5 ml, 38.8 mM) to a boiling solution of HAuCl4 (50 ml, 1 mM), then solution was kept continuously boiling for another 30 min to give a wine red solution. After filtering the solution through 25 mm syringe filter with 0.2 m membrane, the filtrate was stored at 4 ◦ C.
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2.3. UV–visible spectroscopic characterization of AuNP samples
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At room temperature optical absorption spectra in the wavelength range 350–700 nm were taken in presence and absence of
The effect of citrate coated AuNPs on the aggregation of heat exposed -lg was monitored by Rayleigh Light Scattering (RLS) measurement by observing emission at 350 nm after exciting the solution at 350 nm using 10 mM phosphate buffer at pH 7.5 in a Shimadzu spectrofluorimeter (Shimadzu 5301 PC). The fluorescence spectra were collected at 25 ◦ C using 1 cm path-length cell and protein concentration 0.25 mg/ml. The excitation and emission slits were set at 5 nm. 2.7. Thoiflavin T (ThT) assay Th T is a dye which shows enhanced fluorescence at 480 nm when bound to amyloid fibrils [30]. Thus to investigate and compare the aggregates formed by heat exposed -lg in absence and presence of AuNPs, the following assay was employed. Briefly 250 l of -lg samples (native and heat treated) having concentration 1 mg/ml was taken. It was then added to 1.75 ml of 50 M Th T solution (stock 1 mM Th T in 20 mM sodium phosphate buffer, pH 7.5) mixed thoroughly and incubated for 5 min. The assay solution was excited at 450 nm and the emissions were measured over the range 460–600 nm. Slit widths for both excitation and emission were kept at 5 nm. Three replicates were performed and the data were averaged. 2.8. ANS fluorescence studies to monitor the hydrophobicity Exposure of hydrophobic patches in protein during the aggregation process was monitored using polarity sensitive fluorescent probe 1-Anilinonapthalene-8-sulfonate (ANS) [31]. A stock solution of ANS was added to each aliquot of native and heat treated
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-lg (both in presence and absence of citrate coated AuNPs) so that the final ANS concentration in each aliquot was 30 M. Typically, ANS concentration was 50 molar excess of protein concentration. The ANS-fluorescence intensities were measured using Shimadzu RF-5301 PC with excitation at 370 nm and scanning the emission wavelength from 450 nm to 550 nm. Slit widths were set at 5 nm for both excitation and emission. Each spectrum was blank corrected. Data points were the average of triplicate measurements.
2.9. Analysis of secondary structures by CD spectroscopy To trace the structural or conformational changes that gold nanoparticles might induced on -lg, circular dichroism measurements were carried out on a Jasco Spectropolarimeter (J-815) at 20◦ C in the far-UV (200–250 nm) and near-UV regions (250–300 nm) using the rectangular cells of 1 nm and 10 mm pathlength, respectively. Heat treated -lg solutions (in presence and absence of AuNP) having concentrations 0.25 mg/ml and 1.0 mg/ml were used for far-UV CD and near-UV CD measurements, respectively. All the spectra are average of three scans. The final spectrum was obtained after the subtraction of corresponding solvent spectrum. The far UV-CD curves were fitted into a curve-fitting program CDNN 2.1 to determine the percent of secondary structures present in -lg under different conditions.
2.10. Monitoring of secondary structures of ˇ-lg during thermal incubation with AuNPs by FT-IR spectroscopy
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For FT-IR measurements, native and heat treated -lg solutions (50 l) (in presence and absence of AuNPs) having concentrations 20 mg/ml were taken in a microcon filter device and diluted with 200 l of D2 O. It was then quickly centrifuged at 4000 × g for 8 min until the volume reached ∼50 l. After that 200 l of D2 O was added again and centrifuged for another 8–10 min. This process of D2 O exchange was repeated 3–4 times [32]. Finally, the D2 O exchanged -lg samples were placed between two CaF2 windows separated by a 50 m thick teflon spacer. FT-IR scans were collected in the range of 1400–1800 cm−1 at a resolution of 2 cm−1 in N2 environment using a Spectrum 100 FT-IR spectrometer (Perkin Elmer). Spectrum of D2 O at pD 7.5 was collected and subtracted from sample spectrum.
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2.11. Dynamic light scattering (DLS) measurements
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The diffusion of nanoparticles in solution induces fluctuations in the intensity of the scattered light. DLS detects these fluctuations using an auto correlator on a microsecond time scale and is used to analyze the distribution of the molecules and supramolecular aggregates as it is very sensitive to particle size [33]. Different sizes of molecules in the solution can be observed in different peaks provided their sizes vary sufficiently. In our experiment, DLS measurements were performed with heat treated -lg in presence and absence of citrate coated AuNPs employing Zetasizer Nanos (Malvern Instrument, U.K.) equipped with 633 nm laser and using 2 ml rectangular cuvette (path length 10 mm). Measurements were done at 20 ◦ C taking 250 l of heat-treated -lg sample in 1.75 ml Na-phosphate buffer. Then varying amounts of AuNP (25–100 M) were mixed thoroughly and kept for 10 min. Similar measurements were done separately with heat treated -lg in absence of AuNPs and with only AuNP samples. The time-dependent auto correlation function was acquired with twelve acquisitions for each run. Each data is an average of five such acquisitions.
3
2.12. Transmission Electron Microscopy (TEM) The morphological studies of AuNPs and heat treated -lg aggregates (in absence and presence of AuNPs) were performed by using high resolution transmission electron microscope (JEOL-HRTEM2011, Tokyo, Japan) with an accelerating voltage of 80–85 kV in different magnifications. The sample solutions were sonicated for 60 s and diluted 50–150 times. A droplet of the diluted sample was put on a carbon coated copper grid of mesh size 300 C (Pro Sci Tech). After 20 s the droplet was removed with a filter paper followed by a droplet of 2% uranyl acetate (Sigma, Steinheim Germany) solution put on the grid and finally removed after 15 s and left for air dry and used for imaging purpose.
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3. Results and discussion
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3.1. Synthesis and characterization of citrate stabilized AuNPs
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The reduction of HAuCl4 (Au3+ ) to yield colloidal gold particles occurred via the transfer of electrons from the citrate to the Au3+ ions leading to the formation of Au0 . The metallic gold then nucleates and grows to form AuNPs and are subsequently capped and stabilized by surrounding citrate ions. Formation of AuNPs was confirmed by the appearance of dark cherry-red colour of the colloidal solutions. The inset shows a picture of glass vials containing AuNP solution. The UV–vis absorption characteristics of the colloidal solution are shown in Fig. 1. The UV–vis spectra of AuNPs and AuNP + -lg (heat stressed) showed the typical surface plasmon resonance (SPR) bands, whereas no such band was observed in the wavelength region 350–700 nm of spectrum of -lg (alone). The SPR band for AuNPs (alone) appeared at 520 nm (due to the strong red colour of AuNPs) and a broad absorption band at 250 nm. This characteristic plasmon band supported the formation of AuNPs of size 20 ± 2 nm. The decrease in intensity (arbitrary units) and mild red shift (520–525 nm) in absorption wavelength were observed in presence of -lg at room temperature. The red shift in wavelength of SPR band of Au is due to conjugation of AuNP with -lg. Fig. 1, inset plot shows a gradual increase of absorption wavelength with increasing
Fig. 1. UV–vis absorption spectra in the wavelength range of 350–750 nm of citrate stabilized 1 mM AuNPs (line a) (max = 520 nm), AuNPs solution in presence of -lg (line b) (max = 525 nm) and of -lg alone (line c). Inset picture show the actual colour of AuNP solution and the inset plot represents the change in absorption wavelength with AuNP concentrations.
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Fig. 2. SDS-PAGE (15%) patterns of native -lg (lane 2 and 3), heat stressed -lg at 75 ◦ C for 1 h in absence (lane 5 and 6) and in presence of 100 M of AuNPs (lane 4 and 7). Lane 1 represents the SDS PAGE pattern of marker protein of known molecular weight (PageRulerTM, Prestained Protein Ladder, 10, 17, 26, 34, 43, 55, 72, 95, 130, and 170 kDa, respectively; Fermentus Life Science, #SM0671) run parallely.
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-lg concentrations. Similar conclusion was made by other group of researchers [34]. When proteins interact with AuNPs, the apparent size of the particles increases due to adsorption of protein and the resultant change in dielectric constant. 3.2. SDS-polyacrylamide gel electrophoresis study
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In the SDS-PAGE analysis under non-reducing condition (Fig. 2) the native -lg appears as single band (lane 2–3) corresponding to the momoneric state of -lg. But the heat treated -lg solution (heating at 75 ◦ C for 1 h) appeared as series of protein bands in SDSPAGE indicating the formations of oligomers of -lg (lane 5–6). This technique dissociates all the non-covalently bonded aggregates into monomers while the disulphide-linked aggregates remain intact. The cys121 of -lg forms S-S linkage when heated 75–80 ◦ C leading to the formation of dimers and other covalently bound “intermediates” during aggregations [25,35,36] by scrambling of disulphide bonds (creation of new intramolecular and intermolecular disulphide bridges and rearrangement of old intramolecular disulphide bridges) [37]. Heating at 75 ◦ C decreases the intensity of the band corresponding to the monomer (band I) and subsequent increase in the intensities of the bands (II–VIII) corresponding to different oligomers and higher aggregates. But the SDS-PAGE analysis of heat treated -lg in presence of AuNPs under non-reducing condition shows a quite different result. The band (III–VII) corresponding to -lg oligomers having lower electrophoretic mobilities and the band (VIII) corresponding to larger aggregate which do not enter into the gel are absent (lane 4 and 7). Thus citrate coated AuNPs are capable of inhibiting the aggregation -lg under heat stress. Immobilization of -lg on AuNP surface mediated by free thiol (cys121 ) on -lg is the main reason to attenuate the propensity of random protein–protein interactions resulting inhibition of protein aggregation.). Lane 1 represents the SDS PAGE pattern of marker protein of known molecular weight (PageRulerTM, Prestained Protein Ladder, 10, 17, 26, 34, 43, 55, 72, 95, 130, and 170 kDa, respectively; Fermentus Life Science, #SM0671) run parallely.
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3.3. Rayleigh Light Scattering (RLS) measurements
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Rise of RLS data from a protein solution is an indication of aggregation. Thus lowering of RLS data should be a useful parameter in support of disaggregation or disassembly of protein. Fig. 3 shows a change in scattering intensity measured at 350 nm after exciting
Fig. 3. Rayleigh Light Scattering data (turbidity measurements): Effect of increasing amount of AuNPs on heat stressed -lg at 75 ◦ C for 1 h during the incubation of -lg with AuNP at pH 7.5 (square). Control measurements with AuNPs alone (circle). The samples were excited and emitted at 350 nm.
the heat treated -lg solution at 350 nm in absence and presence of varying amount of AuNPs at pH 7.5. It shows a gradual decrease in scattering intensity with the increase amount of AuNP solutions. Highest scattering intensity of the solution (3 fold) is observed in absence of AuNPs, suggesting the probability of aggregate formation with heat treated -lg in absence of AuNPs. But the heating of -lg solutions in presence of citrate coated AuNPs up to 100 M, shows a remarkable decrease in scattering intensity. The control measurements with varying amounts of AuNPs alone were performed and compared. Higher AuNPs concentrations scattered UV light at 350 nm. Hence gradual decrease in scattering intensity of the resultant heat treated -lg solutions in presence of citrate coated AuNPs supports the capping of protein molecules on AuNPs surfaces and disaggregation of -lg in presence of AuNPs. 3.4. Disassembly of ˇ-lg aggregates with AuNPs studied by Th T assay The disassembly of -lg aggregates was further confirmed by Th T assay. Th T is a cationic benzothiazole dye showing enhanced fluorescence upon binding to protein assembly. The native -lg undergoes thermal aggregation in absence of AuNP, showing maximum Th T intensity upon binding with Th T (Fig. 4A). This result is in accordance with other amyloidogenic proteins. Furthermore lowering of Th T fluorescence intensities were observed with the -lg samples heated in presence of varying amount of AuNPs, indicating prominent dimer formation with lesser amount of higher protein assembly in presence of AuNPs (lane 4 and 7). Thus citrate coated AuNPs are capable of suppressing thermal aggregation of -lg in a concentration dependent manner. AuNPs thus appeared to act in a similar fashion to molecular chaperones. The plot of the Th T intensity vs. AuNP concentrations (Fig. 4B) showed that the Th T intensities decreased with the increase of AuNP concentrations (25–100 M). The results indicated that the citrate capped AuNP interferes and suppress the fibrilization pathway of -lg. It has been reported that AuNPs form self-cluster while inducing the aggregation of lysozyme [38] or while showing the size dependent chaperon properties in the aggregation of citrate synthase [17]. 3.5. ANS fluorescence studies to monitor the hydrophobicity during fibrillation of ˇ-lg The aggregates induced by thermal exposure of -lg were further confirmed by ANS fluorescence studies. Molten globule state
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Fig. 5. ANS flouresence of heat stressed -lg (75 ◦ C for 1 h) in the absence and presence of AuNPs in 10 mM phosphate buffer at pH 7.5, excitation was done at 380 nm and emissions were measured in the wavelength range 400–550 nm. Fig. 5 lines a–d corresponds to heat stressed -lg in the presence of 0, 25, 50 and 100 M AuNPs. Protein concentrations during ANS flouresence measurements were 0.25 mg/ml.
3.6. Secondary Structural changes of ˇ-lg with AuNP Studied by CD spectroscopy
Fig. 4. (A) Th T assay to study the aggregation of heat stressed (75 ◦ C for 1 h) -lg in absence and presence of AuNPs where the final -lg and Th T concentrations were 125 g and 50 M, respectively in 10 mM phosphate buffer at pH 7.5. Native -lg was incubated with 0 M (line a), 25 M (line b), 50 M (line c) and 100 M (line d) of AuNPs. Fluorescence emissions were monitored in the wavelength range 460–600 nm after excitation at 450 nm. (B) Bar diagram of the end-point Th T intensity versus AuNP concentrations in Th T assay to study the aggregation of heat stressed (75 ◦ C for 1 h) -lg in 10 mM phosphate buffer at pH 7.5. Fluorescence emissions were monitored in the wavelength range 460–600 nm after excitation at 450 nm. Standard deviations are within the range of ±3.0.
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of proteins can be detected by binding of ANS to the hydrophobic region of protein that ultimately results in an increase of fluorescence intensity [39]. Fig. 5 showed the ANS fluorescence spectra of thermally exposed -lg in absence or presence of AuNPs. In the absence of AuNPs the heat exposed -lg at pH 7.5 showed highest ANS fluorescence intensity at 480 nm. This increase in fluorescence intensity may be attributed to more access of ANS to the hydrophobic patches present in protein upon thermal denaturation. These hydrophobic patches enhance the protein–protein interactions leading to thermal aggregation of -lg [25]. But in the presence of AuNPs during thermal exposure of -lg, significant decrease of ANS fluorescence was observed indicating the loss of ANS biding sites. Gradual decrease of ANS fluorescence intensity with marginal red shifting of emission maximum was observed upon thermal exposure of -lg solutions in presence of varying amount of AuNPs. The lowering of ANS binding in presence of AuNPs suggests the formation of lesser hydrophobic patches by -lg upon thermal stress at 75 ◦ C. AuNPs are thus capable of suppressing -lg aggregation in a concentration dependent fashion.
We investigated the secondary structural changes of heat exposed -lg in absence and presence of AuNPs with respect to native -lg in 10 mM phosphate buffer, pH 7.5. The results of the far UV-CD studies are shown in Fig. 6. The native -lg (Fig. 6, line-a) shows a negative signal around 216 nm which is characteristic for the -sheet structure of the protein. But the heat stressed -lg shows (Fig. 6, line-b) an increase in negative ellipticity value at 216 nm suggesting the retaining of the native secondary structure with the possibility of formation of crossed linked sheet structure. The thermal denaturation opens up the buried cys121 and leading to the formation of disulphide linked oligomers. Higher non-covalent oligomer formation at this high temperature is the cause of such crossed linked -sheet structures. But the CD spectra of heat stressed -lg in presence of varying amounts of
Fig. 6. Far UV-CD spectra of native and heat stressed -lg (75 ◦ C for 1 h) in absence and presence of AuNPs in 10 mM phosphate buffer at pH 7.5. Fig. 6, line a shows native -lg whereas lines b–e corresponds to heat stressed -lg in the presence of 0, 25, 50 and 100 M AuNPs. Line ‘f’ corresponds to the CD spectrum of only AuNPs (100 M). A plot of MRE values at 209 nm against varying AuNPs concentrations is represented (inset, Fig. 6). Concentrations of -lg samples in far UV-CD measurements were 0.25 mg/ml.
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Fig. 7. FT-IR spectra in the region 1400–1750 cm−1 of native (a), heat treated -lg (b) and heat treated -lg in presence of AuNPs within the concentration range of 25 M (c), 50 M (d), 75 M (e), 100 M (f), respectively. Protein concentrations were 20 mg/ml. FT-IR spectra in the amide-I region 1600–1700 cm−1 (inset, Fig. 7). Each spectrum is an average of 32 scans in D2 O solvent at 25 ◦ C.
Fig. 8. Number particle size distribution spectra in DLS studies of (a) only 1 mM AuNPs, (b) heat stressed -lg (75 ◦ C for 1 h, 2.0 mg/ml), (c) heat stressed -lg (75 ◦ C for 1 h, 2.0 mg/ml) in presence of 25 M AuNPs, (d) heat stressed -lg (75 ◦ C for 1 h, 2.0 mg/ml) in presence of 50 M AuNPs. Each of the spectra is an average of 48 scans.
shifts from 1632 cm−1 to 1650 cm−1 (Fig. 7, inset). The curve-g shows a significant broadening and shifting of amide I band to ∼1650 cm−1 , indicating large decrease in -sheet structure and consequent increase in ␣-helical and random coil structural content. The FT-IR studies adequately support the CD results (Table 1). AuNPs are thus inhibiting the aggregation of -lg in a concentration dependent manner.
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citrate coated AuNPs show a significant change in their pattern (Fig. 6, lines c–e). The incubation of -lg at 75 ◦ C in the presence of AuNPs changes the secondary structure as the minimum at 216 nm becomes less pronounced and thus indicating the loss of gross -sheet structure. There was concomitant increase in ␣helical structure as evidenced by an increase in ellipticity at 210 nm and 222 nm in each case. Thus AuNPs induced - to ␣-helical and random coil transitions are observed when -lg sample were thermally stressed at 75 ◦ C in presence of varying amount of AuNPs (Fig. 6, lines c–e). To further assess AuNP-induced - to ␣-helical transitions, the individual secondary structural elements were analyzed with CDNN 2.1 software and the results are shown in Table 1. These observations suggest that AuNPs are capable of inhibiting the formation of cross linked -sheet structure and favouring the formation of ␣-helical structural content.
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3.7. Analysis of secondary structures of ˇ-lg with AuNPs by FT-IR
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The FT-IR spectra of -lg samples (native and heat exposed in absence and presence of AuNPs) in D2 O-buffer are shown in Fig. 7. The resulting spectrum for native -lg (Curve-a) shows the amide I band at around 1632 cm−1 and amide II band at 1480 cm−1 . These features are characteristic for the protein having predominant sheet structures [40,41]. Most of the information on the protein’s secondary structure is contained in the amide I band in the region 1700–1600 cm−1 . Heat exposed -lg (in absence of AuNP) showed a broadening of peak centred around 1634 cm−1 with the appearance of a shoulder in around 1620 cm−1 region and thus identifying the cross-linked -sheet structure similar to those found in the aggregates [42]. When -lg solutions are incubated at 75 ◦ C with gradual increasing amount of AuNPs, the amide I band gradually
3.8. Dynamic Light Scattering (DLS) measurement
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To confirm our previous results, we employed DLS to study the particle size distributions (Fig. 8). DLS is frequently used to examine the hydrodynamic radius of spherical particles in solution and the intensity of the scattered light is dominated by large particles in solution. Although the -lg aggregates/fibrils are heterogeneous and with long rod-like shapes, DLS analysis can provide a quantitative estimation of aggregates distribution. In order to understand the nature of association occurring during thermal incubation of -lg at 75 ◦ C in absence and presence of AuNPs, we attempted to assess the oligomeric status of -lg during aggregation and disaggregation process. DLS analysis can provide a qualitative estimation of the aggregate distribution. The data were plotted as scattered light intensity versus size in radius. Here we first examined the size distribution of AuNPs alone (Fig. 8, curve a). The hydrodynamic diameter of bare AuNP was ∼20 ± 2 nm which was in good agreement with the size obtained from TEM measurement. The size distribution curves (Fig. 8b–d) of heat stressed (75 ◦ C) -lg in absence and presence of AuNPs (25 and 50 M) showed the formation of aggregates of different sizes. In absence of AuNP, DLS analysis showed the wider distribution of large aggregates of -lg with the diameters at ranging from ∼40 nm to 400 nm
Table 1
Q5 Structural integrity of native and heat treated structural -lg at 75 ◦ C for 1 h (in absence and presence of AuNPs) as determined by circular dichroism (CD)a . -lg Samples
% of ␣-Helix
% of -Sheet
% of -Turn
% of Random coil
Native (a) Heat stressed in absence of AuNp (b) Heat stressed in presence of 25 M AuNP (c) Heat stressed in presence of 50 M AuNP (d) Heat stressed in presence of 75 M AuNP
11.9 10.2 14.7 15.8 17.2
45.2 46.3 34.4 30.2 26.4
11.0 12.0 15.2 12.3 11.5
31.9 31.5 35.7 41.7 44.9
a
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Calculated by CDNN 2.1 software.
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Fig. 9. Selected TEM images of AuNPs at different magnifications (a–c) and heat stressed (75 ◦ C for 1 h) -lg in absence (d–f) and in presence (g–i) of AuNPs in 10 mM phosphate buffer at pH 7.5. During incubations concentrations of -lg and AuNPs were 2.5 mg/ml and 25 M, respectively. TEM images (j–k) represent the prominent presence of AuNPs on the -lg fibrillar aggregates at higher concentrations (100 M).
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(Fig. 8, curve b). In presence of 25 M AuNP, the size of the aggregates decreased largely and species with smaller diameter became prominent (Fig. 8, curve c). Further increasing the concentration of AuNPs up to 50 M shows smaller change in the size of the protein molecules (Fig. 8, curve d). Thus upon incubation with AuNPs, size distribution of large -lg particles (aggregates) decreased and the scattering was dominated by particles with smaller size. Our results thus showed that AuNPs can suppress the formation of larger aggregates of -lg during its thermal stress at 75 ◦ C.
3.9. Characterization of the aggregates by TEM To confirm the results obtained from previous sections, the morphological studies of the end-point products formed in the heat induced aggregation of -lg in the absence and presence of AuNPs were done by TEM. The bare AuNPs appear as spherical black particles with a diameter of about 20 nm in the images (Fig. 9a–c). In the absence of AuNPs, -lg forms long, smooth fibrillar aggregates (Fig. 9d–f) upon incubation at 75 ◦ C for 1 h. However incubation
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with 25 M AuNP, -lg forms aggregates having different morphological characters (Fig. 9g–i). The number of well-defined fibril is reduced. Some fibrils became fragmented. Moreover some spherical oligomers along with formation of amorphous aggregate were observed. Such species are observed either attached to the fibrils or in association with the AuNPs even at higher concentrations (100 M) (Fig. 9j and k).
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4. Discussion
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The native -lg forms covalently linked intermediates during its aggregation at pH 7.5 upon thermal exposure heating at 75 ◦ C [35,36]. These intermediates correspond to the formation of dimers, trimers and tetramers of -lg, respectively with involvement of free –SH group of cys121 of the monomer. The results of SDS-PAGE (Fig. 2) support the formation of such covalently linked oligomers along with much larger aggregates that could not penetrate the gel (lane 3–5). Aggregation may not be limited to the formation of linear aggregates but branched aggregates appear at longer times, as shown in DLS and TEM images. It has been shown that disulfidelinked intermediates further react to yield larger size aggregates via non-covalent interactions [25,43]. Understanding the nanoparticle–protein interaction is crucial for the development of nanomedicine, since the nanoparticles are capable of entering in the biological system to act as delivery, imaging and diagnostic tools for different illness. In our studies here we have focused on the understanding the effect of the most biocompatible nanoparticle, AuNP on the aggregation of heat exposed -lg at 75 ◦ C in physiological pH using 20 nm AuNPs. Firstly SDS-PAGE experiment shows unambiguously that incubation of -lg at 75 ◦ C in presence of AuNPs resists the formation of oligomeric species (like trimer, tetramer, etc.) and higher aggregates (lane 5 and 6) whereas incubation of -lg in absence of AuNPs can produce all the oligomeric and larger aggregates which can not enter into the gel (lane 2–4). AuNPs thus stabilize the monomeric and dimeric forms of the protein inhibiting the nucleation and elongation for the formation of higher aggregates. Similarly the results of ANS-fluorescence measurement and Th T experiments confirmed our observations in the SDS-PAGE study. Both ANS and Th T experiments showed that citrate coated AuNPs were able to inhibit the aggregation of -lg as evidenced from the low end point Th T fluorescence. This lowering of ThT fluorescence intensity and hence extent of inhibition is dependent on AuNP concentrations (Fig. 4B). The result may be confirmed by the morphological studies with TEM, showing the generation of smaller, fragmented fibril and some spherical oligomers in the presence of AuNPs. Thermal incubation of -lg in presence of higher AuNP concentration (100 M) generates only the smaller spherical aggregates. In the DLS analysis, the larger fibrillar aggregates generated from thermal incubation of -lg in absence of AuNP were dominating in scattering the light while smaller ragged aggregates including the dimmers were dominating in presence of AuNPs. Results of CD and FT-IR support the secondary structural changes with decrease of -sheet structural contents when the -lg solutions were treated with AuNPs at 75 ◦ C. Other results suggest that the bare AuNPs in nanomolar range are capable of retarding protein aggregation and redirecting it into off-pathway intermediate formation. Similar effect has been observed when organic molecules like poly phenol derivative (−) epi-gallocatechine gallate retards and redirects A and ␣-synuclein in Parkinson’s disease forming off-pathway oligomers [44]. Only a few examples are available in the literature where bare AuNP can prevent the aggregation of proteins. Very recently it has been observed that cysteine coated AuNPs are capable of suppressing the heat induced aggregation of BSA and citrate synthase [45]. On
Scheme 1. Schematic representation of coupled mechanism of heat stressed -lg amyloid fibrillation in absence and presence of AuNPs of ∼20 nm diameter.
the other hand the bare AuNPs and the copolymeric nanoparticles have been used as rescuing molecules in the retardation of A-40 fibrilization [46,47]. Based on this study, it appears that AuNPs may process the capability of suppressing the aggregation of proteins and facilitate the formation of dimeric and native-like monomeric structure. Results of previous studies and our current observations show that this aggregation inhibitive property of AuNPs is not only dependent on their size and charge on their surface but also depends on their concentration. The Scheme 1 described a simple mechanism that can explain nicely our experimental observations and protein aggregation inhibitory activity of AuNPs. Heating at 75 ◦ C in absence of AuNPs, the Cys121 of -lg becomes exposed on thermal denaturation and forms covalently linked (s-s bond) ‘intermediates’ like dimer, trimer and tetramer, etc. The presence of monomer along with other oligomeric forms can be explained in the view of intra and intermolecular disulfide bond exchange mechanism. These disulfide linked intermediates undergo further aggregation via non-covalent interactions like hydrophobic interactions to yield larger aggregate. Aggregation would not be limited to the formation of linear aggregates but branched aggregates appear after a longer period of time. But incubation of -lg at 75 ◦ C in presence of AuNP helps (leads to the formation of dimer and smaller aggregates of -lg). At high temperature and concentration, AuNPs may tend to form nano-cages (appears in the TEM image, Fig. 9). Gold nano-cages likely to have higher heat capacities, knowing as nano-fluids [48]. The smaller nanoparticle having greater surface area, enhance the formation of nano-cages. The cage-trapped protein may be thermally shielded, as some excess heat is likely to be adsorbed by nano surface leading to the refolding of -lg into its native-like structure. Moreover the interaction of the protein molecules, on the surface of nano-cluster reduces the hydrophobic interaction between the protein molecules and thereby inhibiting aggregation. 5. Conclusion In this study we have examined the effects of citrate stabilized AuNP on heat induced aggregation of an important globular protein beta lactoglobulin (-lg). It was observed that AuNPs are able to inhibit the formation of amyloid fibrillar aggregates of lg under thermal condition in a concentration dependent manner. Such inhibition may be due to the stabilization of -lg on the nanoparticle surface. In the earlier reports, the bare nanoparticles showed similar activities against amyloidosis [46]. Previous studies
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also showed that these smaller gold nanoparticles (
