Neurochem Res (2014) 39:194–201 DOI 10.1007/s11064-013-1206-x

ORIGINAL PAPER

Nanomedicine: Action of Metal Nanoparticles on Neuronal Nitric Oxide Synthase—Fluorimetric Analysis on the Mechanism for Fibrillogenesis E. R. Padayachee • A. Arowolo • C. G. Whiteley

Received: 22 July 2013 / Revised: 16 September 2013 / Accepted: 20 November 2013 / Published online: 29 November 2013 Ó Springer Science+Business Media New York 2013

Abstract The incubation of neuronal nitric oxide synthase with the five amyloid peptide fragments [Ab17–21; Ab25–29; Ab29–33; Ab33–37; Ab25–37] catalyzed the formation of fibrils. The role of neuronal isomer (nNOS) involved the entrapment of free monomers and seed aggregates to initiate the events of nucleation and elongation, critical for the formation of fibrils. It was evident that the hydrophobic nature of Ab17–21, the three glycine zipper peptides [Ab25–29; Ab29–33; Ab33–37] and Ab25–37 was a trigger in the formation of fibrils and was a force critical in the association of the peptides with the enzyme. Gold and silver nanoparticles (average 4.0 nm) inhibited fibril formation when added to the induced fibrils from nNOS-Ab incubation. The addition of nNOS and/or Ab to co-incubated solutions of nanoparticle-Ab or nanoparticle-nNOS respectively did not prevent fibril formation but reversed it. Three mechanisms for this reversal were proposed: (1) depletion of free Ab monomer in solution and blocking potential aggregation sites on the nNOS molecule due to large surface area of the nanoparticle (2) hydrophobic interaction between the Ab peptide and nanoparticle (3) disruption of binary adducts between Ab-peptides and nNOS by nanoparticles. Keywords Fibrillogenesis  Neuronal nitric oxide synthase  Silver/gold nanoparticles  Amyloid peptides  Alzheimer’s disease

E. R. Padayachee  A. Arowolo  C. G. Whiteley (&) Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, P.O. Box 94, Grahamstown 6140, South Africa e-mail: [email protected]

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Introduction Nanomedicine is a term within the scientific and medical fraternity that defines characterisation, synthesis and applications of functional units on a nanoscale (10-9 m) and there is little doubt that a plethora of new opportunities in health care, environmental biology, chemical and materials sciences and communication has appeared [1]. More specifically, nanomedicine and nanotechnology can be exploited in the preparation and properties of metal nanoparticles [2, 3], dendrimers [4], drug and/or gene delivery [5], imaging [6], molecular diagnostics [7], nanoproteomics [7] and cardiac therapy [8]. Metal nanoparticles have been synthesised by both biological and chemical means [9–11] with the latter reported to be environmentally hazardous with limited appeal. Biological syntheses through the bioreduction of metal salts by both prokaryotic and eukaryotic organisms [12–14] is cost effective, simple and eco-friendly but with limitation in an ability to control the particle shape and size [15]. The use of a protein cage, like apoferritin, to confine particle growth in a homogeneous distribution as well as to stabilise against particle aggregation has been reported [16]. Apoferrtin has ferroxidase activity and catalyses the oxidation of Fe2? to Fe3? in the presence of molecular oxygen as an electron acceptor. Several other zero-valent encapsulated metal nanoparticles have been prepared within this protein cage including platinum [2], copper [17], palladium [18], cadmium [19] and cobalt and nickel [20]. Recently we reported [21, 22] an enhanced activity (110-fold) of ferroxidase inside an apoferritin cage in the presence of metal nanoparticles. As the particle itself became smaller and smaller, its association with the biomacromolecule changes inevitably leading these molecules to behave differently to the native ones. The key

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Fig. 1 The amino acid sequence of Ab1–42 showing the N-terminal region Ab1–9, the hydrophobic pentapeptide, Ab17–21 and three glycine zipper motifs [G-X-X-X-G] (Ab25–29, Ab29–33 and Ab33–37) within the glycine zipper stretch (Ab25–37)

understanding for nanoparticle-biomacromolecule interactions has emerged from the role played by the dynamic layer (corona) [23–27]. The binding of the nanoparticles to the enzyme/protein would affect the protein structure to varying degrees and a study on the impact of these particles on biological systems, in particular fibrillogenesis, associated with Alzheimers disease (AD) may have far-reaching clinical implications. The role of nanoparticles on fibril formation has already been reported [28, 29] but with inconclusive findings. Fibrillogenesis is central to the pathogenesis of AD [30] and is initiated when amyloid peptides (Ab) undergo a conformational change from a random structure to a predominant b-sheet intermediate of aggregated protein oligomers. Mechanisms on the formation of these fibrils is an essential step into gaining insight into the disease and to assist in designing compounds that may inhibit, prevent or suppress fibril formation. Protein aggregation is initiated by the formation of an aggregated nucleus either from ‘seeds’ or from Ab-peptide concentration dependent ‘micelles’ [31] that are in rapidequilibrium with free peptide monomers. Three different kinetic phases are evident: (a) A lag phase in which an initial association occurs but no aggregation of the peptides; (b) An elongation phase/nucleation step in which aggregation proceeds rapidly by hydrophobic interaction after the formation of critical nucleii [32]; (c) a monomerfibril equilibrium phase in which, as the name implies, there is an equilibrium between monomers associating into fibril aggregates and fibril aggregates dissociating into monomers. Furthermore the ability of a protein/peptide to undergo an a to b conformational transition is facilitated by amino acid regions that adopt an a-helical conformation within its native structure while at the same time have a high propensity for the b-sheet structure [33]. There is strong evidence to suggest that Ab-peptides possess several criteria to initiate fibrillogenesis [34]. The N-terminus (Fig. 1) is thought to be important for initiating a–b conformational switching. Residues 1–9 exposed on the surface of the fibers may be involved in interaction between fibrils but not necessary in fibril formation [34]. The

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hydrophobic residues 17–21 and 29–37 are the major bsheet regions and function as key determinants in aggregation [35]. Furthermore strong evidence supports the role of aromatic residues [Phe19, Phe20] in the formation of fibrils by contributing p-p stacking interactions in the bsheet [36]. Intermediate helical states, hydrophobic interactions and hydrogen bonding are also important properties to promote b-sheet structure [37, 38]. Nitric oxide synthase (NOS) [EC. 1.14.13.39] is an enzyme that oxidises L-arginine to L-citrulline and nitric oxide (NO) (Fig. 2) and since there are elevated levels of arginine in the brain and cerebrospinal fluid of AD patients [39] it pointed towards a metabolic interference of this enzyme during the pathophysiology of the disease. Excess brain arginine is stored in neuroglial cells (astrocytes) which, in the diseased AD brain, are also surrounded by insoluble amyloid plaques [40] and so it would appear reasonable, in the etiology and pathogenesis of AD, to study NOS, especially its neuronal isomer (nNOS), and its intimate association with amyloid peptides deposits. Despite the knowledge that nNOS is found in the cytosolic fluid classical experiments were designed to examine Ab toxicities within the central nervous system (CNS) from the extracellular space. Evidence [41] suggested that intraneuronal accumulation of Ab is neurotoxic and may play an important role in the disease progression of AD. One of the means by which neurons accumulate intracellular Ab is through uptake of extracellular Ab peptides, and this process is a potential link between Ab generation, synaptic dysfunction, and AD pathology. In view of this, nNOS has the potential to interact with intracellular Ab peptides. A number of amyloidogenic proteins and short peptide fragments are capable of producing amyloid fibrils [42] and neurotoxic oligomers [43]. In a preliminary study from our laboratories it was shown nNOS, initially inhibited by Ab-peptides, eventually acted as a catalyst leading to the formation of fibrils [44– 46]. Though specific amino acids directly involved in inhibition and/or fibrillogenesis were not identified it did suggest that the hydrophobic pentapeptide (Ab17–21) and several glycine zipper motifs [G-X-X-X-G] (Ab25–29, Ab29–33, Ab33–37 and Ab25–37) (Fig. 1) were critical players in the mechanism. We were especially interested in the effect of nanoparticles, prepared within the apoferritin cage, on the interaction of Ab-peptides (Ab17–21, Ab25–29, Ab29–33, Ab33–37, Ab25–37) with nNOS. This approach would open up insights into the mechanisms of fibrillogenesis and agents that prevent the initial stages of Ab formation (Ab nucleation) could be more effective than those that merely block the final stages (Ab deposition). This would have future clinical implications in a de novo synthesis of small

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Fig. 2 Enzymatic reaction for neuronal nitric oxide synthase

molecule inhibitors for the treatment of amyloid-forming diseases such as AD.

and kinetic parameters (Km and Vmax) established as previously described [44]. Assay for nNOS

Materials and Methods This assay was conducted as previuously described [44]. Materials Protein Determination Bovine brain was donated by Rosedale abattoir (Grahamstown, South Africa). All Ab peptides were synthesized by Biopep Peptide Research Group, Stellenbosch, South Africa, thioflavin-T (ThT) was purchased from Sigma Aldrich (South Africa). All reagents were of analytical grade and all solutions were prepared with deionized water obtained from a Milli-Q system. Fluorimetric analyses were carried out on PowerWave microplate spectrofluorimeter (Bio-Tek Instruments) with 96 well plates, operated at 1 nm bandwidth using the KC Junior software program. Purification of nNOS Neuronal NOS was purified as previously described [44]. In brief, bovine brain (374 g) was homogenized by sonication (10 W, 30 s intervals, 4 min) in HEPES buffer (50 mM, pH 7.6, 600 ml) that contained ethylenediaminetetraacetic acid (EDTA) (1.0 mM), NADPH (1.0 mM), dithiothreitol (0.5 mM) and phenylmethylsulphonylfluoride (0.43 mM). The cell debris was removed by centrifugation (10,0009g, 4 °C, 30 min) and the crude cellfree extract (20 ml), dialyzed and then applied to a DEAESepharose anion-exchanger resin, previously equilibrated with Tris–HCl buffer (50 mM, pH 7.6). Active fractions of nNOS were eluted with 0.75 M NaCl in the same buffer at a flow rate of 2 ml.min-1. Characterization of nNOS The purity of the enzyme was confirmed by SDS-PAGE analysis and its optimum temperature, pH, thermal stability

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The protein concentration for all experiments was routinely determined according to the method of Bradford [47]. The assay was performed in triplicate in a 96-well microplate. Enzyme extract (5 ll) was incubated (22 °C, 10 min) with Bradford reagent (245 ll), the absorbance measured at 595 nm and the concentration determined from a bovine serum albumen (BSA) standard curve. Synthesis and Characterization of Gold (Au) and Silver (Ag) Nanoparticles Au and Ag nanoparticles were biologically synthesized in the presence of horse spleen apoferritin (HSAF), as described [21, 22]. To summarise, HSAF was incubated with varying concentrations of K2PtCl4 to give a theoretical loading of 250–4,000 metal atoms per HSAF. The pH was maintained at pH 8 with (NaOH, 0.1 M) and the mixture stirred (1 h, 4 °C) after which NaBH4 (20-fold of metal salt concentration) was added. The solution was stirred for a further 1 h until full reduction was achieved as determined by visual change in color. After dialysis, and size-exclusion chromatography the presence, purity and stability of the metal-nanoparticle-HSA samples were assessed by native polyacrylamide gel electrophoresis. The particles were then characterized and size distribution visualized using transmission electron microscopy (TEM) (Fig. 3). The molar ratio of Au and Ag to HSAF was 500:1 and it was this solution that was used in subsequent experiments. On average, the size of Au and Ag nanoparticles was &4 nm, based on the size distribution of particles [Fig. 3a(ii), b(ii)]. Since nanoparticles were encased in HSAF a control contained only HSAF (0.3–0.12 lM).

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Fig. 3 TEM micrographs and size distribution of Au nanoparticles (a) and Ag nanoparticles (b) made from varying molar ratios of metal salts to HSAF. Scale bar = 100 nm. 200 particles were analyzed in

each group. (i) 250:1; (ii) 500:1; (iii) 1,000:1; (iv) 2,000:1 and (v) 4,000:1 (Sennuga et al., 2012a)

Co-incubation of nNOS and Ab-peptides: Effect of Nanoparticles

[Ab17–21, Ab25–29, Ab29–33, Ab33–37, Ab25–37] [50 lM] were then added to the co-incubated mixture and the ThT fluorescence monitored (10 s intervals; 60 s) at kex = 440 nm and kem = 482 nm and values obtained represented by the mean (±SEM) of three trials. A solution of ThT alone was monitored under the same conditions as above to establish the basal fluorescence and a solution of Ab-Tris HCl buffer, without ThT, was used as a control. The fluorescence intensity above the control was indicative of concentration of fibrils. The positive controls were Abpeptide-Au nanoparticle, Ab-peptide-Ag nanoparticle, and Ab-peptide.

In order to induce fibrils the original Ab peptides [Ab17–21, Ab25–29, Ab29–33, Ab33–37, Ab25–37] [25 lM; 50 lM] were incubated with nNOS (5 ll, 32 nM) in a reaction mixture containing ThT (3 ll, 3.14 mM) in Tris HCl buffer (pH 7.6, 100 mM) in a final volume of 200 ll for 15 min at 25 °C. Solutions of Au or Ag nanoparticles (average 4.0 nm, 0.15 lM; 0.30 lM), were then added, separately, to solutions of induced fibrils and the ThT fluorescence monitored (10 s intervals; 60 s) at kex = 440 nm and kem = 482 nm and values obtained represented by the mean (±SEM) of three trials. A solution of ThT alone was monitored under the same conditions as above to establish the basal fluorescence and a solution of Ab-Tris HCl buffer, without ThT, was used as a control. The fluorescence intensity above the control was indicative of concentration of fibrils. The positive controls were Ab-peptide-Au nanoparticle, Ab-peptide-Ag nanoparticle, nNOS-Au nanoparticle, nNOS-Ag nanoparticle and nNOS. Co-incubation of nNOS and Nanoparticles: Effect of Ab-peptides In order to establish whether nanoparticles could influence the initial formation of fibrils by nNOS induction with Abpeptides, solutions of Au and Ag nanoparticles (average 4.0 nm, 0.30 lM) were incubated with nNOS (5 ll, 32 nM) in a reaction mixture containing ThT (3 ll, 3.14 mM) in Tris HCl buffer (pH 7.6, 100 mM) in a final volume of 200 ll for 15 min. The original Ab peptides

Co-incubation of Ab-peptides and Nanoparticles: Effect of nNOS In order to establish whether nanoparticles, in the presence of Ab-peptides, could influence fibrillogenesis by nNOS, solutions of Au and Ag nanoparticles (average 4.0 nm, 0.30 lM) were incubated with original Ab peptides [Ab17–21, Ab25–29, Ab29–33, Ab33–37, Ab25–37] [50 lM] in a reaction mixture containing ThT (3 ll, 3.14 mM) in Tris HCl buffer (pH 7.6, 100 mM) in a final volume of 200 ll for 15 min. The nNOS (5 ll, 32 nM) was then added to the Ab-nanoparticle solution and the ThT fluorescence monitored (10 s intervals; 60 s) at kex = 440 nm and kem = 482 nm and values obtained represented by the mean (± SEM) of three trials. A solution of ThT alone was monitored under the same conditions as above to establish the basal fluorescence and a solution of Ab-Tris HCl buffer, without ThT, was used as a control. The fluorescence intensity above the control was indicative of concentration

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Fig. 4 Change in ThT fluorescence indicating concentration of amyloid fibrills with respect to time. nNOS (32 nM) was incubated with Ab-peptide (50 lM) in a final volume (200 ll) in Tris HCl buffer (pH 7.6, 100 mM) at 25 °C for 15 min prior to addition of a Ag; b Au nanoparticles (average 4.0 nm; 0.30 lM) at time = 0 s. Inset Change in ThT fluorescence indicating concentration of amyloid fibrills with respect to time. nNOS (32 nM) was incubated with Abpeptide (25 lM) in a final volume (200 ll) in Tris HCl buffer (pH 7.6, 100 mM) at 25 °C for 15 min prior to addition of a Ag; b Au nanoparticles (average 4.0 nm, 0.15 lM) at time = 0 s

of fibrils. The positive controls were nNOS-Au nanoparticle, nNOS-Ag nanoparticle and nNOS. Statistical Analysis All experiments were carried out in triplicate. Mean and standard deviation calculations and comparison of data using analysis of variance (ANOVA) was performed to 5 % level of significance (p \ 0.05) using Statistica for Windows, version 8 (Statsoft Inc.) and Microsoft Excel 2010.

Results and Discussion According to results nNOS is shown to be an amyloidogenic enzyme catalyzing the formation of fibrils after a

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15 min incubation with each of the Ab-peptides, at starting concentrations of both 50 and 25 lM as indicated (Fig. 4; Fig. 4 inset) [time = 0 s]. The concentration of fibrils produced by Ab17–21 and Ab29–33 were greater (ThT fluorescence = 35, 40 respectively after 15 min incubation) than with Ab25–29, Ab33–37 and Ab25–37 [ThT fluorescence = 25, 20, 20 respectively after 15 min incubation. This supports discussion earlier that not only do the hydrophobic residues 17–21 and 29–33 function towards aggregation [35] but aromatic residues [Phe19, Phe20] contribute favourably towards the formation of fibrils by p-p stacking interactions within the b-sheet [36]. Surprisingly, for reasons unknown, the non-polar glycine zipper (Ab33–37) supported only mild aggregation. The formation of more fibrils from Ab17–21 and Ab29–33 by nNOS, can also be attributed to their tighter binding affinity to the enzyme compared to the affinity of the other Ab peptides [Ab17–21 (Ki = 5 lM; Kd = 6 lM) and Ab29–33 (Ki = 9 lM; Kd = 2.7 lM)] [46]. These findings indicated that for Ab17–21 and Ab29–33, it was not the tight binding affinity that determined stronger inhibition, but it was the nature of the amino acid sequence and the identity of the amino acids which influenced binding [46]. Ab17–21 and Ab29–33 are the only two sequences, compared to Ab25–29, Ab33–37 and Ab25–37 which contain double repeat phenylalanine and isoleucine amino acids. The interactions of these amino acids with nNOS accelerated the catalysis of fibrils from these peptide sequences. With the addition of either Ag- or Au-nanoparticles (average 4.0 nm) there was a dramatic decrease ([90 %) in fibril concentration over the first 10 s of incubation (Fig. 4) gradually decreasing to nearly zero after 1 min. Crucial to reactivity in nanotechnology is the surface-to-volume ratio of the nanoparticle: the greater this ratio the more the reactivity. It is, therefore, this explanation that offers a reason why silver particles, with a larger surface area to volume, indicated a more pronounced effect than gold nanoparticles. Since the particles themselves had not interfered with the fluorescence of ThT alone (data not shown) we were confident that the nanoparticles had, in fact, reversed the aggregation of the fibrils into individual oligomers or, indeed, monomers. Though it may appear (Fig. 4) that Ab29–33 was more influential in this reversal process when compared to other amyloid peptides there was little significance between the findings even at the different concentrations of nanoparticles (Fig. 4, inset). What was clearly significant, however, was a fibril concentration of zero after one minute of incubation with silver nanoparticles (0.3 lM) no matter what amyloid peptide had been used. At a lower concentration of nanoparticles 3–5 % fibrils remained after 1 min incubation. It also made silver nanoparticles superior to gold nanoparticles in the fibril to oligomer/monomer transition since 5–7 % fibrils

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Fig. 5 Change in ThT fluorescence indicating concentration of amyloid fibrills with respect to time. a nNOS (32 nM) was incubated with Ag- and/or Au-nanoparticles (average 4.0 nm, 0.3 lM) in a final volume (200 ll) in Tris HCl buffer (pH 7.6, 100 mM) at 25 °C for 15 min prior to addition of Ab-peptides (50 lM) at time = 0 s. b Abpeptides (50 lM) was incubated with Ag- and/or Au-nanoparticles (average 4.0 nm, 0.3 lM) in a final volume (200 ll) in Tris HCl buffer (pH 7.6, 100 mM) at 25 °C for 15 min prior to addition of nNOS (32 nM) at time = 0 s

remained after 1 min incubation with gold particles no matter at what concentration. We turned our attention to whether nanoparticles could influence the initial formation of fibrils from the Ab-peptides by nNOS induction. In this case Ag- and Au-nanoparticles were incubated, separately, with either nNOS or each of the Ab-peptides for 15 min prior to the addition of respective amyloid peptides or nNOS. Obviously, without any amyloid peptide or nNOS present, the ThT fluorescence remained at base-line (time = 0 s) (Fig. 5a, b). When the Ab-peptides were added to the incubation mixture, however, there was a rapid formation of fibrils as indicated by the initial increase in ThT fluorescence (time = 10 s; Fig. 5a). These values were substantially more than the corresponding scenario (time = 10 s; Fig. 4) reflecting that the nanoparticles, under the present experimental conditions, were reversing the formation of fibrils and not preventing their formation. The differentiation in

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concentration of fibrils was again dependent on the identity of the amyloid peptide and influenced on the binding affinities of the peptides to the enzyme. Furthermore after incubation had continued for a full minute no further change seemed probable and about 20–25 % of the fibrils remained (based on original amount, Fig. 4, time = 0 s). A similar argument can be made when nNOS was added to the incubation mixture of Ab-peptide/nanoparticle with slightly higher ThT fluorescence and consequently higher fibril concentration (time = 10 s; Fig. 5b). Ironically slightly less fibrils remained after the incubation had proceeded for 1 min. Research has shown that soluble Ab (prefibrillar oligomers) are primary causative agents of AD compared to insoluble Ab (fibrils) that are inert or protective [48]. Furthermore it was realised that Ab fibrils act as nucleation centres, increasing the local concentration of monomers and speeding up their assembly into toxic oligomers [49]. Any molecule that targets the secondary pathway by binding amyloid fibrils or inhibiting their interaction with monomers will prevent the toxic formation of oligomers and their inherent toxicity [49]. Hence, since fibrils catalyse oligomer formation, the use of nanoparticles as therapeutic agents to reverse the formation of fibrils is indeed beneficial. Reversing fibril formation would in turn reverse oligomer formation leading to the significance of the present research. The existence of a rapid elongation/growth phase and an equilibrium phase in pointed towards a nucleated-polymerization model for fibrillogenesis. The results (Fig. 4) have suggested that nNOS, in the presence of amyloid peptides, catalyzed the formation of fibrils through an AbnNOS complex. This goes one step further with nNOS acting as a ‘chaperone’ on Ab-peptide assembly either by increasing the ‘seeds’ necessary for the nucleation step or by stimulating fibril elongation. First, free Ab monomers, in solution, bind to nNOS, formulating a nucleus and initiating their aggregation. Second, these seed aggregates become entrapped by nNOS and couple to the existing aggregated monomers, leading to an elongated fibril. According to our findings (Fig. 5), just as the formation of fibrils occurred so too did the reverse of formation. The amount of monomers or oligomers in solution decreased due to both the Ab-monomer/nanoparticle complex and the trapping of the monomers by nNOS. This, in turn, disturbed the monomer-oligomer equilibrium, affecting the nucleation step, eliminating the lag phase and reversing fibril formation (Fig. 6). According to the ‘corona effect’ [23–27] the nanoparticles would be surrounded by the amyloid peptide molecules in vivo creating an intense hydrophobic environment around the particle. This would, effectively, deplete the concentration of monomeric peptides and not allow any

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Fig. 6 Diagram illustrating a monomer peptides in solution; b monomer getting trapped by a nanoparticle; c less free monomer in solution; d fibril formation prevented

‘lag’ time, nor prevent the formation of critical nuclei for the initial association phase and inevitably prevent fibril initiation and elongation. Finally we consider a mechanism for the reversal of fibril formation by the nanoparticles. Accordingly a direct interaction between nNOS (A1) and Ab (B) leads to the formation of a binary adduct (A1B), preceding the formation of the fibril. The addition of a nanoparticle (A2) to this binary adduct disrupts the complex such that the peptide can no longer bind to the enzyme suppressing the probability of fibrillogenesis and consequently leading to a decrease in ThT fluorescence.

Conclusions The mechanism for the induction of fibrils by the interaction of nNOS with Ab-peptides was dependent on several factors: (1) the binding affinity that each peptide had for the enzyme especially Ab17–21 and Ab29–33; (2) the hydrophobic nature of the peptides and the p-p stacking within the b-sheet by the aromatic residues [Phe19, Phe20]; (3) the entrapment of free monomers and seed aggregates by the enzyme to initiate the events of nucleation and elongation; (4) the identity of a probable binding site that is the subject of present research and will be reported elsewhere. This study has shown more insight into the mechanism of fibrillogenesis with the fact that nanoparticles did not prevent the process but reversed it. Acknowledgments The authors wish to thank Medical Research Council (South Africa) and Rhodes University, South Africa for financial support. Conflict of interest

None.

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Nanomedicine: action of metal nanoparticles on neuronal nitric oxide synthase-fluorimetric analysis on the mechanism for fibrillogenesis.

The incubation of neuronal nitric oxide synthase with the five amyloid peptide fragments [Aβ17-21; Aβ25-29; Aβ29-33; Aβ33-37; Aβ25-37] catalyzed the f...
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