Characterization of the oligomeric states of insulin in self-assembly and amyloid fibril formation by mass spectrometry

The self-assembly and aggregation of insulin molecules has been investigated by means of nanoflow electrospray mass spectrometry. Hexamers of insulin containing predominantly two, but up to four, Zn(2+) ions were observed in the gas phase when solutions at pH 4.0 were examined. At pH 3.3, in the absence of Zn(2+), dimers and tetramers are observed. Spectra obtained from solutions of insulin at millimolar concentrations at pH 2.0, conditions under which insulin is known to aggregate in solution, showed signals from a range of higher oligomers. Clusters containing up to 12 molecules could be detected in the gas phase. Hydrogen exchange measurements show that in solution these higher oligomers are in rapid equilibrium with monomeric insulin. At elevated temperatures, under conditions where insulin rapidly forms amyloid fibrils, the concentration of soluble higher oligomers was found to decrease with time yielding insoluble high molecular weight aggregates and then fibrils. The fibrils formed were examined by electron microscopy and the results show that the amorphous aggregates formed initially are converted to twisted, unbranched fibrils containing several protofilaments. Fourier transform infrared spectroscopy shows that both the soluble form of insulin and the initial aggregates are predominantly helical, but that formation of beta-sheet structure occurs simultaneously with the appearance of well-defined fibrils.     

Independent heterologous fibrillation of insulin and its B-chain peptide

Insulin is very prone to form amyloid fibrils under slightly destabilizing conditions, and the B-chain region plays a critical role in the fibrillation. We show here that the isolated B-chain peptide of bovine insulin also forms fibrils at both acidic and neutral pH. When a mixture of insulin and the B-chain peptide was incubated at either acidic or neutral pH, the formation of fibrils was clearly separated into two phases, with the faster phase corresponding to the formation of homogeneous fibrils from the B-chain and the slower phase corresponding to homogeneous fibrillation of insulin. To further investigate the interaction (or lack thereof) between the two polypeptides, we examined the effects of cross-seeding. The results indicate that seeds of B-chain fibrils accelerate the fibrillation of insulin at pH 1.6 and inhibit the fibrillation at pH 7.5, but seeds of insulin fibrils have little effect on the fibrillation of the B-chain. We conclude that at pH 7.5 simultaneous independent homologous fibrillation occurs, but at low pH, heterologous fibrillation takes place, and with B-chain seeding of insulin, a unique conformation of fibrils is formed. Our results demonstrate that in the co-aggregation of closely related peptides each peptide species may undergo concurrent homogeneous or heterologous polymerization and that fibrils of one species may or may not seed fibrillation of the other. The results demonstrate the significant "species" barrier in amyloid fibril formation between fibrillation induced by different fibrils. A model for the fibrillation of the heterogeneous system of insulin and B-chain insulin is proposed.     

Role of aggregation conditions in structure, stability, and toxicity of intermediates in the Abeta fibril formation pathway

beta-amyloid peptide (Abeta) is one of the main protein components of senile plaques associated with Alzheimer's disease (AD). Abeta readily aggregates to forms fibrils and other aggregated species that have been shown to be toxic in a number of studies. In particular, soluble oligomeric forms are closely related to neurotoxicity. However, the relationship between neurotoxicity and the size of Abeta aggregates or oligomers is still under investigation. In this article, we show that different Abeta incubation conditions in vitro can affect the rate of Abeta fibril formation, the conformation and stability of intermediates in the aggregation pathway, and toxicity of aggregated species formed. When gently agitated, Abeta aggregates faster than Abeta prepared under quiescent conditions, forming fibrils. The morphology of fibrils formed at the end of aggregation with or without agitation, as observed in electron micrographs, is somewhat different. Interestingly, intermediates or oligomers formed during Abeta aggregation differ greatly under agitated and quiescent conditions. Unfolding studies in guanidine hydrochloride indicate that fibrils formed under quiescent conditions are more stable to unfolding in detergent than aggregation associated oligomers or Abeta fibrils formed with agitation. In addition, Abeta fibrils formed under quiescent conditions were less toxic to differentiated SH-SY5Y cells than the Abeta aggregation associated oligomers or fibrils formed with agitation. These results highlight differences between Abeta aggregation intermediates formed under different conditions and provide insight into the structure and stability of toxic Abeta oligomers.     

Inhibition of insulin fibrillogenesis with targeted peptides

Under conditions of acidic pH and elevated temperature, insulin partially unfolds and aggregates into highly structured amyloid fibrils. Aggregation of insulin leads to loss of activity and can trigger an unwanted immune response. Compounds that prevent protein aggregation have been used to stabilize insulin; these compounds generally suppress aggregation only at relatively high inhibitor concentrations. For example, effective inhibition of aggregation of 0.5 mM insulin required arginine concentrations of > or =100 mM. Here, we investigate a targeted approach toward inhibiting insulin aggregation. VEALYL, corresponding to residues B12-17 of full-length insulin, was identified as a short peptide that interacts with full-length insulin. A hybrid peptide was synthesized that contained this binding domain and hexameric arginine; this peptide significantly reduced the rate of insulin aggregation at near-equimolar concentrations. An effective binding domain and N-terminal placement of the arginine hexamer were necessary for inhibitory activity. The data were analyzed using a simple two-step model of aggregation kinetics. These results are useful not only in identifying an insulin aggregation inhibitor but also in extending a targeted protein strategy for modifying aggregation of amyloidogenic proteins.     

Strong inhibition of peptide amyloid formation by a fatty acid

The aggregation of peptides into amyloid fibrils is associated with several diseases, including Alzheimer’s and Parkinson’s disease. Because hydrophobic interactions often play an important role in amyloid formation, the presence of various hydrophobic or amphiphilic molecules, such as lipids, may influence the aggregation process. We have studied the effect of a fatty acid, linoleic acid, on the fibrillation process of the amyloid-forming model peptide NACore (GAVVTGVTAVA). NACore is a peptide fragment spanning residue 68–78 of the protein α-synuclein involved in Parkinson’s disease. Based primarily on circular dichroism measurements, we found that even a very small amount of linoleic acid can substantially inhibit the fibrillation of NACore. This inhibitory effect manifests itself through a prolongation of the lag phase of the peptide fibrillation. The effect is greatest when the fatty acid is present from the beginning of the process together with the monomeric peptide. Cryogenic transmission electron microscopy revealed the presence of nonfibrillar clusters among NACore fibrils formed in the presence of linoleic acid. We argue that the observed inhibitory effect on fibrillation is due to co-association of peptide oligomers and fatty acid aggregates at the early stage of the process. An important aspect of this mechanism is that it is nonmonomeric peptide structures that associate with the fatty acid aggregates. Similar mechanisms of action could be relevant in amyloid formation occurring in vivo, where the aggregation takes place in a lipid-rich environment.     

Interaction of the molecular chaperone alphaB-crystallin with alpha-synuclein: effects on amyloid fibril formation and chaperone activity

alpha-Synuclein is a pre-synaptic protein, the function of which is not completely understood, but its pathological form is involved in neurodegenerative diseases. In vitro, alpha-synuclein spontaneously forms amyloid fibrils. Here, we report that alphaB-crystallin, a molecular chaperone found in Lewy bodies that are characteristic of Parkinson's disease (PD), is a potent in vitro inhibitor of alpha-synuclein fibrillization, both of wild-type and the two mutant forms (A30P and A53T) that cause familial, early onset PD. In doing so, large irregular aggregates of alpha-synuclein and alphaB-crystallin are formed implying that alphaB-crystallin redirects alpha-synuclein from a fibril-formation pathway towards an amorphous aggregation pathway, thus reducing the amount of physiologically stable amyloid deposits in favor of easily degradable amorphous aggregates. alpha-Synuclein acts as a molecular chaperone to prevent the stress-induced, amorphous aggregation of target proteins. Compared to wild-type alpha-synuclein, both mutant forms have decreased chaperone activity in vitro against the aggregation of reduced insulin at 37 degrees C and the thermally induced aggregation of betaL-crystallin at 60 degrees C. Wild-type alpha-synuclein abrogates the chaperone activity of alphaB-crystallin to prevent the precipitation of reduced insulin. Interaction between these two chaperones and formation of a complex are also indicated by NMR spectroscopy, size-exclusion chromatography and mass spectrometry. In summary, alpha-synuclein and alphaB-crystallin interact readily with each other and affect each other's properties, in particular alpha-synuclein fibril formation and alphaB-crystallin chaperone action.     

Use of a Small Peptide Fragment as an Inhibitor of Insulin Fibrillation Process: A Study by High and Low Resolution Spectroscopy

A non-toxic, nine residue peptide, NIVNVSLVK is shown to interfere with insulin fibrillation by various biophysical methods. Insulin undergoes conformational changes under certain stress conditions leading to amyloid fibrils. Fibrillation of insulin poses a problem in its long-term storage, reducing its efficacy in treating type II diabetes. The dissociation of insulin oligomer to monomer is the key step for the onset of fibrillation. The time course of insulin fibrillation at 62°C using Thioflavin T fluorescence shows an increase in the lag time from 120 min without peptide to 236 min with peptide. Transmission electron micrographs show branched insulin fibrils in its absence and less inter-fibril association in its presence. Upon incubation at 62°C and pH 2.6, insulin lost some α-helical structure as seen by Fourier transformed infra-red spectroscopy (FT-IR), but if the peptide is added, secondary structure is almost fully maintained for 3 h, though lost partially at 4 h. FT-IR spectroscopy also shows that insulin forms the cross beta structure indicative of fibrils beyond 2 h, but in the presence of the peptide, α-helix retention is seen till 4 h. Both size exclusion chromatography and dynamic light scattering show that insulin primarily exists as trimer, whose conversion to a monomer is resisted by the peptide. Saturation transfer difference nuclear magnetic resonance confirms that the hydrophobic residues in the peptide are in close contact with an insulin hydrophobic groove. Molecular dynamics simulations in conjunction with principal component analyses reveal how the peptide interrupts insulin fibrillation. In vitro hemolytic activity of the peptide showed insignificant cytotoxicity against HT1080 cells. The insulin aggregation is probed due to the inter play of two key residues, Phe^B24 and Tyr^B26 monitored from molecular dynamics simulations studies. Further new peptide based leads may be developed from this nine residue peptide.     

Investigation of the kinetics of insulin amyloid fibrils formation

Today, the investigation of the structure of ordered protein aggregates-amyloid fibrils, the influence of the native structure of the protein and the external conditions on the process of fibrillation-is the subject of intense investigations. The aim of the present work is to study the kinetics of formation of insulin amyloid fibrils at low pH values (conditions that are used at many stages of the isolation and purification of the protein) using the fluorescent probe thioflavin T. It is shown that the increase of the fluorescence intensity of ThT during the formation of amyloid fibrils is described by a sigmoidal curve, in which three areas can be distinguished: the lag phase, growth, and a plateau, which characterize the various stages of fibril formation. Despite the variation in the length of the lag phase at the same experimental conditions (pH and temperature), it is seen to drop during solution stirring and seeding. Data obtained by electron microscopy showed that the formed fibrils are long, linear filaments ～20 nm in diameter. With increasing incubation time, the fibril diameter does not change, while the length increases to 2–3 μm, which is accompanied by a significant increase in the number of fibril aggregates. All the experimental data show that, irrespective of the kinetics of formation of amyloid fibrils, their properties after the completion of the fibrillation process are identical. The results of this work, together with the previous studies of insulin amyloid fibrils, may be important for clarification the mechanism of their formation, as well as for the treatment of amyloidosis associated with the aggregation of insulin.     

Effects of confinement on insulin amyloid fibrils formation

Insulin, a 51-residue protein universally used in diabetes treatment, is known to produce amyloid fibrils at high temperature and acidic conditions. As for other amyloidogenic proteins, the mechanisms leading to nucleation and growth of insulin fibrils are still poorly understood. We here report a study of the fibrillation process for insulin confined in a suitable polymeric hydrogel, with the aim of ascertain the effects of a reduced protein mobility on the various phases of the process. The results indicate that, with respect to standard aqueous solutions, the fibrillation process is considerably slowed down at moderately high concentrations and entirely suppressed at low concentration. Moreover, the analysis of the initial stages of the fibrillation process in aqueous solutions revealed a large spatial heterogeneity, which is completely absent when the fibrillation is carried out in the hydrogel. We attribute this heterogeneity to the diffusion in solution of large amyloidal aggregates, which must be formed very fast compared to the average times for the whole sample. These findings are interpreted in the framework of recently suggested heterogeneous nucleation mechanisms. Moreover, they may be useful for the development of new insulin pharmaceutical formulations, more stable against adverse conditions.     

Stability of Aβ (1-42) peptide fibrils as consequence of environmental modifications

β-Amyloid peptide (Aβ) plays a key role in the pathogenesis of Alzheimer disease (AD). Monomeric Aβ undergoes aggregation, forming oligomers and fibrils, resulting in the deposition of plaques in the brain of AD patients. A widely used protocol for fibril formation in vitro is based on incubation of the peptide at low pH and ionic strength, which generates Aβ fibrils several microns long. What happens to such fibrils once they are brought to physiological pH and ionic strength for biological studies is not fully understood. In this investigation, we show that these changes strongly affect the morphology of fibrils, causing their fragmentation into smaller ones followed by their aggregation into disordered structures. We show that an increase in pH is responsible for fibril fragmentation, while increased ionic strength is responsible for the aggregation of fibril fragments. This behavior was confirmed on different batches of peptide either produced by the same company or of different origin. Similar aggregates of short fibrils are obtained when monomeric peptide is incubated under physiological conditions of pH and ionic strength, suggesting that fibril morphology is independent of the fibrillation protocol but depends on the final chemical environment. This was also confirmed by experiments with cell cultures showing that the toxicity of fibrils with different initial morphology is the same after addition to the medium. This information is of fundamental importance when Aβ fibrils are prepared in vitro at acidic pH and then diluted into physiological buffer for biological investigations.     

N- and C-terminal hydrophobic patches are involved in fibrillation of glucagon

Recent work suggests that the molecular structure of amyloid-like fibrils is determined by environmental conditions as well as amino acid sequence. To probe the involvement of side chains in fibrillation of the 29-residue hormone glucagon, we have measured fibrillation kinetics of 15 alanine mutants. At acidic pH, all of the mutants are able to form fibrils. However, substitution of hydrophobic residues in the N- and C-termini (in particular Phe6, Tyr10, Val23, and Met27) decelerates fibrillation dramatically. This indicates that the hydrophobicity and/or high beta-sheet propensity of these residues may be important for fibrillation. In contrast, substitution of Leu14 increases fibrillation propensity compared to that of the wild type. Nevertheless, despite identical fibrillation conditions, the thioflavin T and tryptophan fluorescence spectra of fibrils formed by mutants Tyr13, Leu14, and Asp15 are significantly different from those of other mutants, indicating that substitution of these residues may influence not only the fibrillation kinetics and fibril stability but also the preferred final structure of the fibrils that is formed, in line with the general structural polymorphism of glucagon fibrils. In contrast, under alkaline conditions, only a handful of the alanine mutants are capable of forming fibrils, suggesting that more side chains are involved in stabilizing interactions here. In addition, fibrils formed by wild-type glucagon at alkaline pH appear very stable, compared to fibrils formed at acidic pH. This suggests that the distribution of charges determines the number of different fibrillated states available to a peptide, since these can block formation of metastable fibrillated states.     

Fibrillation of human insulin B-chain by pulsed hydrogen-deuterium exchange mass spectrometry

Insulin forms amyloid fibrils under slightly destabilizing conditions, and B-chain residues are thought to play an important role in insulin fibrillation. Here, pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS), far-UV circular dichroism spectroscopy, thioflavin T (ThioT) fluorescence, turbidity, and soluble fraction measurements were used to monitor the kinetics and mechanisms of fibrillation of human insulin B-chain (INSB) in acidic solution (1 mg/mL, pH 4.5) under stressed conditions (40°C, continuous shaking). Initially, INSB rapidly formed β-sheet-rich oligomers that were protected from HD exchange and showed weak ThioT binding. Subsequent fibril growth and maturation was accompanied by even greater protection from HD exchange and stronger ThioT binding. With peptic digestion of deuterated INSB, HDX-MS suggested early involvement of the N-terminal (1–11, 1–15) and central (12–15, 16–25) fragments in fibril-forming interactions, whereas the C-terminal fragment (25–30) showed limited involvement. The results provide mechanistic understanding of the intermolecular interactions and structural changes during INSB fibrillation under stressed conditions and demonstrate the application of pulsed HDX-MS to probe peptide fibrillation.     

Supersaturation-limited and Unlimited Phase Transitions Compete to Produce the Pathway Complexity in Amyloid Fibrillation

Although amyloid fibrils and amorphous aggregates are two types of aggregates formed by denatured proteins, their relationship currently remains unclear. We used β₂-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, to clarify the mechanism by which proteins form either amyloid fibrils or amorphous aggregates. When ultrasonication was used to accelerate the spontaneous fibrillation of β2m at pH 2.0, the effects observed depended on ultrasonic power; although stronger ultrasonic power effectively accelerated fibrillation, excessively strong ultrasonic power decreased the amount of fibrils formed, as monitored by thioflavin T fluorescence. An analysis of the products formed indicated that excessively strong ultrasonic power generated fibrillar aggregates that retained β-structures but without high efficiency as seeds. On the other hand, when the spontaneous fibrillation of β2m was induced at higher concentrations of NaCl at pH 2.0 with stirring, amorphous aggregates became more dominant than amyloid fibrils. These apparent complexities in fibrillation were explained comprehensively by a competitive mechanism in which supersaturation-limited reactions competed with supersaturation-unlimited reactions. We link the kinetics of protein aggregation and a conformational phase diagram, in which supersaturation played important roles.     

Guiding protein aggregation with macromolecular crowding

Macromolecular crowding is expected to have a significant effect on protein aggregation. In the present study we analyzed the effect of macromolecular crowding on fibrillation of four proteins, bovine S-carboxymethyl-alpha-lactalbumin (a disordered form of the protein with reduced three out of four disulfide bridges), human insulin, bovine core histones, and human alpha-synuclein. These proteins are structurally different, varying from natively unfolded (alpha-synuclein and core histones) to folded proteins with rigid tertiary and quaternary structures (monomeric and hexameric forms of insulin). All these proteins are known to fibrillate in diluted solutions, however their aggregation mechanisms are very divers and some of them are able to form different aggregates in addition to fibrils. We studied how macromolecular crowding guides protein between different aggregation pathways by analyzing the effect of crowding agents on the aggregation patterns under the variety of conditions favoring different aggregated end products in diluted solutions.     

The kinetic behavior of insulin fibrillation is determined by heterogeneous nucleation pathways

When subjected to acidic conditions and high temperature, insulin is known to produce fibrils that display the common properties of disease amyloids. Thus, clarifying the mechanisms of insulin fibrillation can help the general understanding of amyloidal aggregation. Insulin fibrillation exhibits a very sharp time dependence, with a pronounced lag phase and subsequent explosive growth of amyloidal aggregates. Here we show that the initial stages of this process can be well described by exponential growth of the fibrillated proteins. This indicates that the process is mainly controlled by a secondary nucleation pathway.     

Quaternary conformational stability: The effect of reversible self‐association on the fibrillation of two insulin analogs

Under conditions relevant to the manufacturing of insulin (e.g., pH 3, room temperature), biosynthetic human insulin (BHI), and Lispro insulin (Lispro) require a nucleation step to initiate aggregation. However, upon seeding with preformed aggregates, both insulins rapidly aggregate into nonnative fibrils. Far ultraviolet circular dichroism (far‐UV CD) and second derivative Fourier transform infrared (2D‐FTIR) spectroscopic analyses show that the fibrillation process involves a change in protein secondary structure from α‐helical in native insulin to predominantly β‐sheet in the nonnative fibrils. After seeding, Lispro aggregates faster than BHI, likely because of a reduced propensity to reversibly self‐associate. Composition gradient multi‐angle light scattering (CG‐MALS) analyses show that Lispro is more monomeric than BHI, whereas their conformational stabilities measured by denaturant‐induced unfolding are statistically indistinguishable. For both BHI and Lispro, as the protein concentration increases, the apparent first‐order rate constant for soluble protein loss decreases. To explain these phenomena, we propose an aggregation model that assumes fibril growth through monomer addition with competitive inhibition by insulin dimers. Biotechnol. Bioeng. 2011;108: 2359–2370. © 2011 Wiley Periodicals, Inc.     

Fibrillation of human insulin A and B chains

Human insulin, which consists of disulfide cross-linked A and B polypeptide chains, readily forms amyloid fibrils under slightly destabilizing conditions. We examined whether the isolated A and B chain peptides of human insulin would form fibrils at neutral and acidic pH. Although insulin exhibits a pH-dependent lag phase in fibrillation, the A chain formed fibrils without a lag at both pHs. In contrast, the B chain exhibited complex concentration-dependent fibrillation behavior at acidic pH. At higher concentrations, e.g., >0.2 mg/mL, the B chains preferentially and rapidly formed stable protofilaments rather than mature fibrils upon incubation at 37 degrees C. Surprisingly, these protofilaments did not convert into mature fibrils. At lower B chain concentrations, however, mature fibrils were formed. The explanation for the concentration dependence of B chain fibrillation is as follows. The B chains exist as soluble oligomers at acidic pH, have a beta-sheet rich conformation as determined by CD, and bind ANS strongly, and these oligomers rapidly form dead-end protofilaments. However, under conditions in which the B chain monomer is present, such as low B chain concentration (<0.2 mg/mL) or in the presence of low concentrations of GuHCl, which dissociates the soluble oligomers, mature fibrils were formed. Thus, both A and B chain peptides can form amyloid fibrils, and both are likely to be involved in the interactions leading to the fibrillation of intact insulin.     

Identification of a mutant amyloid peptide that predominantly forms neurotoxic protofibrillar aggregates

The amyloid peptide (Abeta), derived from the proteolytic cleavage of the amyloid precursor protein (APP) by beta- and gamma-secretases, undergoes multistage assemblies to fibrillar depositions in the Alzheimer's brains. Abeta protofibrils were previously identified as an intermediate preceding insoluble fibrils. While characterizing a synthetic Abeta variant named EV40 that has mutations in the first two amino acids (D1E/A2V), we discerned unusual aggregation profiles of this variant. In comparison of the fibrillogenesis and cellular toxicity of EV40 to the wild-type Abeta peptide (Abeta40), we found that Abeta40 formed long fibrillar aggregates while EV40 formed only protofibrillar aggregates under the same in vitro incubation conditions. Cellular toxicity assays indicated that EV40 was slightly more toxic than Abeta40 to human neuroblastoma SHEP cells, rat primary cortical, and hippocampal neurons. Like Abeta40, the neurotoxicity of the protofibrillar EV40 could be partially attributed to apoptosis since multiple caspases such as caspase-9 were activated after SHEP cells were challenged with toxic concentrations of EV40. This suggested that apoptosis-induced neuronal loss might occur before extensive depositions of long amyloid fibrils in AD brains. This study has been the first to show that a mutated Abeta peptide formed only protofibrillar species and mutations of the amyloid peptide at the N-terminal side affect the dynamic amyloid fibrillogenesis. Thus, the identification of EV40 may lead to further understanding of the structural perturbation of Abeta to its fibrillation.     

Tyrosine Autofluorescence as a Measure of Bovine Insulin Fibrillation

The traditional approach to investigating the partial unfolding and fibrillation of insulin, and proteins at large, has involved use of the dyes 1-anilinonaphthalene-8-sulphonic acid (ANS) and Thioflavin T (ThT), respectively. We compare the kinetic profiles of ThT, ANS, light scattering, and intrinsic Tyr fluorescence during insulin fibrillation. The data reveal that the sequence of structural changes (dimers → monomers → partially unfolded monomers → oligomeric aggregates → fibrils) accompanying insulin fibrillation can be detected directly using intrinsic Tyr fluorescence. The results indicate that at least two distinguishable structural intermediates precede fibril development. There is no evidence of tyrosinate or dityrosine during insulin aggregation. Obtaining such critical information from the protein itself is complementary to existing aggregation probes and affords the advantage of directly examining structural changes that occur at the molecular level, providing concrete details of the early events preceding fibrillation.     

Heparin‐induced amyloid fibrillation of β2‐microglobulin explained by solubility and a supersaturation‐dependent conformational phase diagram

Amyloid fibrils are fibrillar deposits of denatured proteins associated with amyloidosis and are formed by a nucleation and growth mechanism. We revisited an alternative and classical view of amyloid fibrillation: amyloid fibrils are crystal‐like precipitates of denatured proteins formed above solubility upon breaking supersaturation. Various additives accelerate and then inhibit amyloid fibrillation in a concentration‐dependent manner, suggesting that the combined effects of stabilizing and destabilizing forces affect fibrillation. Heparin, a glycosaminoglycan and anticoagulant, is an accelerator of fibrillation for various amyloidogenic proteins. By using β₂‐microglobulin, a protein responsible for dialysis‐related amyloidosis, we herein examined the effects of various concentrations of heparin on fibrillation at pH 2. In contrast to previous studies that focused on accelerating effects, higher concentrations of heparin inhibited fibrillation, and this was accompanied by amorphous aggregation. The two‐step effects of acceleration and inhibition were similar to those observed for various salts. The results indicate that the anion effects caused by sulfate groups are one of the dominant factors influencing heparin‐dependent fibrillation, although the exact structures of fibrils and amorphous aggregates might differ between those formed by simple salts and matrix‐forming heparin. We propose that a conformational phase diagram, accommodating crystal‐like amyloid fibrils and glass‐like amorphous aggregates, is important for understanding the effects of various additives.