Mechanism of A beta(1-40) fibril-induced fluorescence of (trans,trans)-1-bromo-2,5-bis(4-hydroxystyryl)benzene (K114)

K114, (trans,trans)-1-bromo-2,5-bis(4-hydroxystyryl)benzene, is a fluorescent Congo Red analogue that binds tightly to amyloid fibrils, but not the monomeric proteins, with a concomitant enhancement in fluorescence. The mechanism for the low aqueous fluorescence and the subsequent enhancement by A beta(1-40) fibrils was investigated by fluorescence spectroscopy and binding analysis. K114's unusually low buffer fluorescence is due to self-quenching in sedimentable aggregates or micelles which upon interacting with amyloid fibrils undergo an enhancement in fluorescence intensity and shifts in the excitation and emission spectra. These spectral changes are suggestive of a stabilization of the phenolate anion, perhaps by hydrogen bonding, rather than an increase in the microenvironment dielectric constant or dye immobilization. 1,4-Bis(4-aminophenylethenyl)-2-methoxybenzene, which lacks the phenol moiety, and X-34, which contains a stabilized phenol (pK approximately 13.4), do not display the phenolate anion fluorescence in the presence of fibrils. The apparent affinity of K114 for fibril binding is 20-30 nM with a stoichiometry of 2.2 mol of K114/mol of A beta(1-40) monomer. Competition studies indicate that K114 and Congo Red share a site, but K114 does not bind to sites on A beta(1-40) fibrils for neutral benzothiazole (BTA-1), cationic thioflavin T, or the hydrophobic (S)-naproxen and (R)-ibuprofen molecules. Comparison of benzothiazole binding stoichiometry which has been suggested to reflect disease-relevant amyloid structures to that of Congo Red analogues which reflect total fibril content may be useful in defining biologically pertinent conformational forms of amyloid.     

Lability Landscape and Protease Resistance of Human Insulin Amyloid: A New Insight into Its Molecular Properties

Amyloid formation is a universal behavior of proteins central to many important human pathologies and industrial processes. The extreme stability of amyloids towards chemical and proteolytic degradation is an acquired property compared to the precursor proteins and is a major prerequisite for their accumulation. Here, we report a study on the lability of human insulin amyloid as a function of pH and amyloid ageing. Using a range of methods such as atomic force microscopy, thioflavin T fluorescence, circular dichroism, and gas-phase electrophoretic mobility macromolecule analysis, we probed the propensity of human insulin amyloid to propagate or dissociate in a wide span of pH values and ageing in a low concentration regime. We generated a three-dimensional amyloid lability landscape in coordinates of pH and amyloid ageing, which displays three distinctive features: (i) a maximum propensity to grow near pH 3.8 and an age corresponding to the inflection point of the growth phase, (ii) an abrupt cutoff between growth and disaggregation at pH 8-10, and (iii) isoclines shifted towards older age during the amyloid growth phase at pH 4-9, reflecting the greater stability of aged amyloid. Thus, lability of amyloid strongly depends on the ionization state of insulin and on the structure and maturity of amyloid fibrils. The stability of insulin amyloid towards protease K was assessed by using real-time atomic force microscopy and thioflavin T fluorescence. We estimated that amyloid fibrils can be digested both from the free ends and within the length of the fibril with a rate of ca 4 nm/min. Our results highlight that amyloid structures, depending on solution conditions, can be less stable than commonly perceived. These results have wide implications for understanding the propagation of amyloids via a seeding mechanism as well as for understanding their natural clearance and dissociation under solution conditions unfavorable for amyloid formation in biological systems and industrial applications.     

Visualization and classification of amyloid beta supramolecular assemblies

Deposition of amyloid beta (Abeta) fibrils has been suggested to play a central role in Alzheimer's disease. In clarifying the mechanism by which fibrils form and moreover in developing new treatments for amyloidosis, direct observation is important. Focusing on the interactions with surfaces at the early stages, we studied the spontaneous formation of Abeta(1-40) fibrils on quartz slides, monitored by total internal reflection fluorescence microscopy combined with thioflavin T, an amyloid-specific fluorescence dye. Self-assembly of Abeta(1-40), accelerated by a low concentration of sodium dodecyl sulfate, produced various remarkable amyloid assemblies. Densely packed spherulitic structures with radial fibril growth were typically observed. When the packing of fibrils was coarse, extremely long fibrils often protruded from the spherulitic cores. In other cases, a large number of wormlike fibrils were formed. Transmission electron microscopy and atomic force microscopy revealed relatively short and straight fibrillar blocks associated laterally without tight interaction, leading to random-walk-like fibril growth. These results suggest that, during spontaneous fibrillation, the nucleation occurring in contact with surfaces is easily affected by environmental factors, creating various types of nuclei, and hence variations in amyloid morphology. A taxonomy of amyloid supramolecular assemblies will be useful in clarifying the structure-function relationship of amyloid fibrils.     

Dissolution of beta2-microglobulin amyloid fibrils by dimethylsulfoxide

Increasing numbers of proteins have been found to aggregate into insoluble fibers, collectively referred to as amyloid fibrils. To address the conformational stability of amyloid fibrils, we studied the effects of dimethylsulfoxide (DMSO), 2,2,2-trifluoroethanol (TFE), and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) on beta(2)-microglobulin amyloid fibrils by circular dichroism, thioflavin T fluorescence, light scattering, and electron microscopy. When measured by circular dichroism and thioflavin T fluorescence, HFIP, and TFE dissolved the fibrils, producing predominantly helical conformations. However, these alcohols did not dissolve the amyloid fibrils completely as monitored by light scattering and electron microscopy. On the other hand, DMSO completely dissolved the amyloid fibrils although a high concentration [i.e., 80% (v/v)] was required. These results are consistent with the important role of hydrogen bonds in stabilizing amyloid fibrils.     

Modulating α-synuclein fibril formation using DNA tetrahedron nanostructures

The small presynaptic protein α-synuclein (α-syn) is involved in the etiology of Parkinson's disease owing to its abnormal misfolding. To date, little information is known on the role of DNA nanostructures in the formation of α-syn amyloid fibrils. Here, the effects of DNA tetrahedrons on the formation of α-syn amyloid fibrils were investigated using various biochemical and biophysical methods such as thioflavin T fluorescence assay, atomic force microscopy, light scattering, transmission electron microscopy, and cell-based cytotoxicity assay. It has been shown that DNA tetrahedrons decreased the level of oligomers and increased the level of amyloid fibrils, which corresponded to decreased cellular toxicity. The ability of DNA tetrahedron to facilitate the formation of α-syn amyloid fibrils demonstrated that structured nucleic acids such as DNA tetrahedrons could modulate the process of amyloid fibril formation. Our study suggests that DNA tetrahedrons could be used as an important facilitator toward amyloid fibril formation of α-synuclein, which may be of significance in finding therapeutic approaches to Parkinson's disease and related synucleinopathies.     

Hexafluoroisopropanol induces amyloid fibrils of islet amyloid polypeptide by enhancing both hydrophobic and electrostatic interactions

Although amyloid fibrils deposit with various proteins, the comprehensive mechanism by which they form remains unclear. We studied the formation of fibrils of human islet amyloid polypeptide associated with type II diabetes in the presence of various concentrations of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) under acidic and neutral pH conditions using CD, amyloid-specific thioflavin T fluorescence, fluorescence imaging with thioflavin T, and atomic force microscopy. At low pH, the formation of fibrils was promoted by HFIP with an optimum at 5% (v/v). At neutral pH in the absence of HFIP, significant amounts of amorphous aggregates formed in addition to the fibrils. The addition of HFIP suppressed the formation of amorphous aggregates, leading to a predominance of fibrils with an optimum effect at 25% (v/v). Under both conditions, higher concentrations of HFIP dissolved the fibrils and stabilized the α-helical structure. The results indicate that fibrils and amorphous aggregates are different types of precipitates formed by exclusion from water-HFIP mixtures. The exclusion occurs through the combined effects of hydrophobic interactions and electrostatic interactions, both of which are strengthened by low concentrations of HFIP, and a subtle balance between the two types of interactions determines whether the fibrils or amorphous aggregates dominate. We suggest a general view of how the structure of precipitates varies dramatically from single crystals to amyloid fibrils and amorphous aggregates.     

Direct evidence for excitonically coupled chlorophylls a and b in LHC II of higher plants by nonlinear polarization spectroscopy in the frequency domain

Human stefin B (cystatin B) is an intracellular cysteine proteinase inhibitor broadly distributed in different tissues. Here, we show that recombinant human stefin B readily forms amyloid fibrils in vitro. It dimerises and further oligomerises, starting from the native-like acid intermediate, I(N), populated at pH 5. On standing at room temperature it produces regular (over 4 microm long) fibrils over a period of several months. These have been visualised by transmission electron microscopy and atomic force microscopy. Their cross-sectional diameter is about 14 nm and blocks of 27 nm repeat longitudinally. The fibrils are smooth, of unbranched surface, consistent with findings of other amyloid fibrils. Thioflavin T fluorescence spectra as a function of time were recorded and Congo red dye binding to the fibrils was demonstrated. Adding 10% (v/v) trifluoroethanol resulted in an increased rate of fibrillation with a typical lag phase. The finding that human stefin B, in contrast to the homologue stefin A, forms amyloid fibrils rather easily should promote further studies of the protein's behaviour in vivo, and/or as a model system for fibrillogenesis.     

Michler's hydrol blue: a sensitive probe for amyloid fibril detection

Michler's hydrol blue (MHB) is investigated with respect to photophysical properties in varied solvent environment and when bound to insulin and lysozyme fibrils. The MHB chromophore is shown to act like a molecular rotor and bind well to amyloid fibrils, where it exhibits a characteristic red-shift in its excitation spectrum and an increase in the emission quantum yield upon binding. MHB is more sensitive to environmental changes than Thioflavin T (ThT) and furthermore, in contrast to the latter amyloid probe, can differentiate between insulin and lysozyme fibrils by a more red-shifted excitation spectrum for insulin fibrils. To support the experimental observations, time-dependent density functional theory (TDDFT) calculations were performed on MHB at several levels of theory. The predicted changes of spectral properties as a function of the environment are in good agreement with the experimental results. Linear dichroism (LD) is used to determine the orientation of the MHB within the fibrils. It was shown through LD and molecular modeling that MHB aligns itself preferentially parallel with the amyloid fiber at an angle of 14°-22° to the fibril axis and along the grooves of the β-sheet.     

Direct observation of amyloid fibril growth monitored by thioflavin T fluorescence

Real-time monitoring of fibril growth is essential to clarify the mechanism of amyloid fibril formation. Thioflavin T (ThT) is a reagent known to become strongly fluorescent upon binding to amyloid fibrils. Here, we show that, by monitoring ThT fluorescence with total internal reflection fluorescence microscopy (TIRFM), amyloid fibrils of beta2-microgobulin (beta2-m) can be visualized without requiring covalent fluorescence labeling. One of the advantages of TIRFM would be that we selectively monitor fibrils lying along the slide glass, so that we can obtain the exact length of fibrils. This method was used to follow the kinetics of seed-dependent beta2-m fibril extension. The extension was unidirectional with various rates, suggesting the heterogeneity of the amyloid structures. Since ThT binding is common to all amyloid fibrils, the present method will have general applicability for the analysis of amyloid fibrils. We confirmed this with the octapeptide corresponding to the C terminus derived from human medin and the Alzheimer's amyloid beta-peptide.     

Thioflavin T fluorescence assay for β-lactoglobulin fibrils hindered by DAPH

The molecule 4,5-dianilinophthalimide was recently found to be an efficient compound in disaggregating amyloid fibrils involved in the Alzheimer’s disease. In this study we have investigated whether the compound 4,5-dianilinophthalimide was able to disaggregate fibrils derived from β-lactoglobulin. In addition to a Thioflavin T fluorescence assay, flow-induced birefringence was used as an independent technique to measure the total length concentration of the fibrils. An additional advantage of the latter technique is that not only the total length concentration, but also the length distribution of the fibrils can be measured. The results from flow-induced birefringence showed that the total amount of fibrils and also the length distribution of the fibrils was not influenced by the addition of 4,5-dianilinophthalimide, even though this was suggested by the results of the Thioflavin T assay. The results of flow-induced birefringence were confirmed by rheological measurements and transmission electron microscopy. Our findings show that the use of a Thioflavin T assay in order to probe the possible disaggregating effect of certain compounds can give misleading results.     

Ultrasonication-induced amyloid fibril formation of beta2-microglobulin

To obtain insight into the mechanism of fibril formation, we examined the effects of ultrasonication, a strong agitator, on beta2-microglobulin (beta2-m), a protein responsible for dialysis-related amyloidosis. Upon sonication of an acid-unfolded beta2-m solution at pH 2.5, thioflavin T fluorescence increased markedly after a lag time of 1-2 h with a simultaneous increase of light scattering. Atomic force microscopy images showed the formation of a large number of short fibrils 3 nm in diameter. When the sonication-induced fibrils were used as seeds in the next seeding experiment at pH 2.5, a rapid and intense formation of long fibrils 3 nm in diameter was observed demonstrating seed-dependent fibril growth. We then examined the effects of sonication on the native beta2-m at neutral pH, conditions under which amyloid deposits occur in patients. In the presence of 0.5 mm sodium dodecyl sulfate, a model compound of potential trigger and stabilizer of amyloid fibrils in patients, a marked increase of thioflavin T fluorescence was observed after 1 day of sonication at pH 7.0. The products of sonication caused the accelerated fibril formation at pH 7.0. Atomic force microscopy images showed that the fibrils formed at pH 7.0 have a diameter of more than 7 nm, thicker than those prepared at pH 2.5. These results indicate that ultrasonication is one form of agitation triggering the formation of amyloid fibrils of beta2-m, producing fibrils adapted to the respective pH.     

Oleic acid inhibits amyloid formation of the intermediate of alpha-lactalbumin at moderately acidic pH

The effects of oleic acid on amyloid formation of Ca2+-depleted bovine alpha-lactalbumin (apo-BLA) at low pH and the biological impact of the effects were investigated by using thioflavin T, Congo red, far-UV circular dichroism, atomic force microscopy, transmission electron microscopy, and other biophysical methods. The results from the phase diagram method of fluorescence show that two intermediates exist in the conformational transition of apo-BLA induced by low pH. One intermediate populated at pH 3.0 is characterized as a molten globule state and the other accumulates with stable secondary structure and exposed hydrophobic surface at pH 4.0-4.5. Amyloid formation of apo-BLA takes place upon decreasing the pH to 4.5 and is accelerated remarkably as the pH is decreased further. However, amyloid fibrils of apo-BLA are not observed in the pH range of 5.0-7.0 on a time-scale of 30 days. The lag time of fibrillation at pH 4.0 is greatly elongated by the presence of oleic acid, accompanied by a remarkable decline of the maximum thioflavin T intensity. Furthermore, amyloid formation of apo-BLA at pH 4.5 is inhibited completely by oleic acid, and insoluble aggregates are observed. In contrast, the effects of oleic acid on amyloid formation are not remarkable at pH 3.0 or at pH 2.0. Our data demonstrate that oleic acid specifically induces the intermediate of apo-BLA at pH 4.0-4.5 to form insoluble amorphous aggregates, which is responsible for the inhibition of amyloid formation of the protein by oleic acid in this range of pH values.     

Template-directed self-assembly and growth of insulin amyloid fibrils

The formation of amyloid aggregates in tissue is a pathological feature of many neurodegenerative diseases and type II diabetes. Amyloid deposition, the process of amyloid growth by the association of individual soluble amyloid molecules with a pre-existing amyloid template (i.e., plaque), is known to be critical for amyloid formation in vivo. The requirement for a natural amyloid template, however, has made amyloid deposition study difficult and cumbersome. In the present work, we developed a novel, synthetic amyloid template by attaching amyloid seeds covalently onto an N-hydroxysuccinimide-activated surface, where insulin was chosen as a model amyloidogenic protein. According to ex situ atomic force microscopy observations, insulin monomers in solution were deposited onto the synthetic amyloid template to form fibrils, like hair growth. The fibril formation on the template occurred without lag time, and its rate was highly accelerated than in the solution. The fibrils were long, over 2 mum, and much thinner than those in the solution, which was caused by limited nucleation sites on the template surface and lack of lateral twisting between fibrils. According to our investigations using thioflavin T-induced fluorescence, birefringent Congo red binding, and circular dichroism, fibrils grown on the template were identified to be amyloids that formed through a conformational rearrangement of insulin monomers upon interaction with the template. The amyloid deposition rate followed saturation kinetics with respect to insulin concentration in the solution. The characteristics of amyloid deposition on the synthetic template were in agreement with previous studies performed with human amyloid plaques. It is demonstrated that the synthetic amyloid template can be used for the screening of inhibitors on amyloid deposition in vitro.     

Trehalose inhibits fibrillation of A53T mutant alpha-synuclein and disaggregates existing fibrils

The aggregation of alpha-synuclein (AS) is pivotally implicated in the development of Parkinson's disease (PD), inhibiting this process might be effective in treating PD. Here, by using circular dichroism spectroscopy, thioflavin T fluorescence, and atomic force microscopy, we found that trehalose at low concentration disaggregates preformed A53T AS protofibrils and fibrils into small aggregates or even random coil structure, while trehalose at high concentration slows down the structural transition into β-sheet structure and completely prevents the formation of mature A53T AS fibrils. Further work in vivo will be needed to evaluate its potential as a novel strategy for treating PD.     

In vitro characterization of lactoferrin aggregation and amyloid formation

Lactoferrin has previously been identified in amyloid deposits in the cornea, seminal vesicles, and brain. We report in this paper a highly amyloidogenic region of lactoferrin (sequence of NAGDVAFV). This region was initially identified by sequence comparison with medin, a 5.5 kDa amyloidogenic fragment derived from lactadherin. Subsequent characterization revealed that this peptide forms amyloid fibrils at pH 7.4 when incubated at 37 degrees C. Furthermore, although full-length lactoferrin does not by itself form amyloid fibrils, the protein does bind to the peptide fibrils as revealed by an increase in thioflavin T fluorescence and the presence of enlarged fibrils by transmission electron microscopy and polarized light microscopy. The binding of lactoferrin is a selective interaction with the NAGDVAFV fibrils. Lactoferrin does not bind to insulin or lysozyme fibrils, and the NAGDVAFV fibrils do not bind to soluble insulin or lysozyme. The lactoferrin appears to coat the peptide fibril surface to form mixed peptide/protein fibrils, but again there is no evidence for the formation of lactoferrin-only fibrils. This interaction, therefore, seems to involve selective binding rather than conventional seeding of fibril formation. We suggest that such a process could be generally important in the formation of amyloid fibrils in vivo since the identification of both full-length protein and protein fragments is common in ex vivo amyloid deposits.     

Stimulation of α-synuclein amyloid formation by phosphatidylglycerol micellar tubules

α-Synuclein (α-Syn) is a presynaptic protein that is accumulated in its amyloid form in the brains of Parkinson's patients. Although its biological function remains unclear, α-syn has been suggested to bind to synaptic vesicles and facilitate neurotransmitter release. Recently, studies have found that α-syn induces membrane tubulation, highlighting a potential mechanism for α-syn to stabilize highly curved membrane structures which could have both functional and dysfunctional consequences. To understand how membrane remodeling by α-syn affects amyloid formation, we have studied the α-syn aggregation process in the presence of phosphatidylglycerol (PG) micellar tubules, which were the first reported example of membrane tubulation by α-syn. Aggregation kinetics, β-sheet content, and macroscopic protein-lipid structures were observed by Thioflavin T fluorescence, circular dichroism spectroscopy and transmission electron microscopy, respectively. Collectively, the presence of PG micellar tubules formed at a stochiometric (L/P = 1) ratio was found to stimulate α-syn fibril formation. Moreover, transmission electron microscopy and solid-state nuclear magnetic resonance spectroscopy revealed the co-assembly of PG and α-syn into fibril structures. However, isolated micellar tubules do not form fibrils by themselves, suggesting an important role of free α-syn monomers during amyloid formation. In contrast, fibrils did not form in the presence of excess PG lipids (≥L/P = 50), where most of the α-syn molecules are in a membrane-bound α-helical form. Our results provide new mechanistic insights into how membrane tubules modulate α-syn amyloid formation and support a pivotal role of protein–lipid interaction in the dysfunction of α-syn.     

Inhibitor discovery targeting the intermediate structure of beta-amyloid peptide on the conformational transition pathway: implications in the aggregation mechanism of beta-amyloid peptide

Abeta peptides cleaved from the amyloid precursor protein are the main components of senile plaques in Alzheimer's disease. Abeta peptides adopt a conformation mixture of random coil, beta-sheet, and alpha-helix in solution, which makes it difficult to design inhibitors based on the 3D structures of Abeta peptides. By targeting the C-terminal beta-sheet region of an Abeta intermediate structure extracted from molecular dynamics simulations of Abeta conformational transition, a new inhibitor that abolishes Abeta fibrillation was discovered using virtual screening in conjunction with thioflavin T fluorescence assay and atomic force microscopy determination. Circular dichroism spectroscopy demonstrated that the binding of the inhibitor increased the beta-sheet content of Abeta peptides either by stabilizing the C-terminal beta-sheet conformation or by inducing the intermolecular beta-sheet formation. It was proposed that the inhibitor prevented fibrillation by blocking interstrand hydrogen bond formation of the pleated beta-sheet structure commonly found in amyloid fibrils. The study not only provided a strategy for inhibitor design based on the flexible structures of amyloid peptides but also revealed some clues to understanding the molecular events involved in Abeta aggregation.     

Hydrophobic tail length plays a pivotal role in amyloid beta (25–35) fibril–surfactant interactions

The amyloid β‐peptide fragment comprising residues 25–35 (Aβ_25‐35) is known to be the most toxic fragment of the full length Aβ peptide which undergoes fibrillation very rapidly. In the present work, we have investigated the effects of the micellar environment (cationic, anionic, and nonionic) on preformed Aβ_25‐35 fibrils. The amyloid fibrils have been prepared and characterized by several biophysical and microscopic techniques. Effects of cationic dodecyl trimethyl ammonium bromide (DTAB), cetyl trimethylammonium bromide (CTAB), anionic sodium dodecyl sulfate (SDS), and nonionic polyoxyethyleneoctyl phenyl ether (Triton X‐100 or TX) on fibrils have been studied by Thioflavin T fluorescence, UV–vis spectroscopy based turbidity assay and microscopic analyses. Interestingly, DTAB and SDS micelles were observed to disintegrate prepared fibrils to some extent irrespective of their charges. CTAB micelles were found to break down the fibrillar assembly to a greater extent. On the other hand, the nonionic surfactant TX was found to trigger the fibrillation process. The presence of a longer hydrophobic tail in case of CTAB is assumed to be a reason for its higher fibril disaggregating efficacy, the premise of their formation being largely attributed to hydrophobic interactions. Proteins 2016; 84:1213–1223. © 2016 Wiley Periodicals, Inc.     

Inhibition of Alzheimer amyloid aggregation with sulfated glycopolymers

Glycopolymers carrying sulfated saccharides with modest sugar contents (11% and 28%) were found to suppress the formation of amyloid fibrils by amyloid beta peptides (Abeta(1-42), Abeta(1-40), and Abeta(25-35)), as evaluated by thioflavin T assays and atomic force microscopy observation. Circular dichroism spectra showed that the conformation of amyloid beta peptides depended on the glycopolymer additives, and that the glycopolymer additives reduced the beta-sheet contents. Neutralization activity was confirmed by in vitro assay with HeLa cells. The sulfate group and the appropriate sugar contents were essential for the inhibitory effect.     

Anti-amyloidogenic effects of soybean isoflavones in vitro: Fluorescence spectroscopy demonstrating direct binding to Aβ monomers, oligomers and fibrils

Alzheimer's disease is characterized by the presence of extracellular deposits of amyloid, primarily composed of the amyloid β-protein (Aβ). A growing body of evidence indicates that oligomeric forms of Aβ play a critical role in disease causation. Soybean isoflavones are flavonoids with an isoflavone backbone. Isoflavones have been reported to protect against Aβ-induced neurotoxicity in cultured cell systems, the molecular mechanisms remain unclear. Our previous studies demonstrated that red wine-related flavonoids with a flavone backbone are able to inhibit Aβ assembly and destabilize preformed Aβ aggregates. Here, we show that isoflavones, especially glycitein and genistein, have anti-fibrillization, anti-oligomerization and fibril-destabilizing effects on Aβ(1-40) and Aβ(1-42)in vitro at physiological pH and temperature, by using nucleation-dependent polymerization monitored by thioflavin T fluorescence, atomic force microscopy, electron microscopy, and photo-induced cross-linking of unmodified proteins followed by SDS-PAGE. Our three-dimensional fluorescence spectroscopic analyses demonstrated that glycitein interacted with Aβ monomers, oligomers and fibrils, indicating specific binding of glycitein to these Aβ species. Glycitein also interacted with different Aβ fragments (Aβ(1-42), Aβ(1-40), Aβ(1-16) and Aβ(25-35)), exhibiting the highest fluorescence enhancement with Aβ(25-35). We speculated that glycitein's anti-amyloidogenic properties are specifically mediated by its binding to Aβ monomers, oligomers and fibrils. Isoflavones may hold promise as a treatment option for preventative strategies targeting amyloid formation in Alzheimer's disease.