Insulin Amyloid Aggregation Research Articles

Study of Insulin Aggregation and Fibril Structure under Different Environmental Conditions.

Protein amyloid aggregation is linked with widespread and fatal neurodegenerative disorders as well as several amyloidoses. Insulin, a small polypeptide hormone, is associated with injection-site amyloidosis and is a popular model protein for in vitro studies of amyloid aggregation processes as well as in the search for potential anti-amyloid compounds. Despite hundreds of studies conducted with this specific protein, the procedures used have employed a vast array of different means of achieving fibril formation. These conditions include the use of different solution components, pH values, ionic strengths, and other additives. In turn, this variety of conditions results in the generation of fibrils with different structures, morphologies and stabilities, which severely limits the possibility of cross-study comparisons as well as result interpretations. In this work, we examine the condition-structure relationship of insulin amyloid aggregation under a range of commonly used pH and ionic strength conditions as well as solution components. We demonstrate the correlation between the reaction solution properties and the resulting aggregation kinetic parameters, aggregate secondary structures, morphologies, stabilities and dye-binding modes.

Mesalazine Inhibits Amyloid Formation and Destabilizes Pre-formed Amyloid Fibrils in the Human Insulin.

Amyloid formation due to protein aggregation is associated with several amyloid diseases (amyloidosis). The use of small organic ligands as inhibitors of protein aggregation is an attractive strategy for the treatment of these diseases. In the present study, we evaluated the in vitro inhibitory and destabilizing effects of Mesalazine on human insulin fibrillation. To induce fibrillation, human insulin was incubated in 50mM glycine buffer (pH 2.0) at 50°C. The effect of Mesalazine on insulin amyloid aggregation was studied using spectroscopic, imaging, and computational approaches.Based on the results, the Mesalazine in a concentration-dependent manner (different ratios (1:0.1, 1:0.5, 1:1, and 1:5) of the insulin to Mesalazine) prevented the formation of amyloid fibrils and destabilized pre-formed fibrils. In addition, our molecular docking study confirmed the binding of Mesalazine to insulin through hydrogen bonds and hydrophobic interactions. Our findings suggest that Mesalazine may have therapeutic potential in the prevention of insulin amyloidosis and localized amyloidosis.

Dissolution of amyloid aggregates by direct addition of alkali halides

The development of standard dissolution agents for various amyloid aggregates in vitro is essential to acquire basic information on pharmaceutical treatment strategies and improve the recovery rate for target expressed proteins. Nevertheless, no standard dissolution agents are currently applied to many amyloid aggregates. Thus, to proceed one step further, the dissolution ability of α-synuclein amyloid aggregates (α-synuclein amyloid) formed under mild conditions (pD 7.5 and 310 K) was investigated using the direct addition of alkali halides. Fourier-transform infrared spectroscopy and Congo red assay were used to evaluate the efficacy of the dissolution agents of alkali halides for amyloid aggregates. The effects of alkali halides on α- synuclein amyloid were classified into three types: (i) dissolution effect, (ii) no effect, and (iii) promotion effect. These effects correlate with the disruption ability of cations (the direct effect) and the solvation free energy difference (ΔΔGSolv) between the cations and anions (the indirect effect). Remarkably, lithium iodide (LiI) has the strongest dissolution ability for α-synuclein amyloid among the examined alkali halides. Furthermore, dialysis can recover the monomer state from α-synuclein amyloid dissolved in LiI. In addition, LiI also exhibits a strong dissolution ability for bovine insulin amyloid aggregates formed under extreme conditions (pD 1.6 and 363 K). These findings show that LiI has the potential as a standard dissolution agent applied to amyloid aggregates among the alkali halides.

The intriguing dose-dependent effect of selected amphiphilic compounds on insulin amyloid aggregation: Focus on a cholesterol-based detergent, Chobimalt.

The amyloidogenic self-assembly of many peptides and proteins largely depends on external conditions. Among amyloid-prone proteins, insulin attracts attention because of its physiological and therapeutic importance. In the present work, the amyloid aggregation of insulin is studied in the presence of cholesterol-based detergent, Chobimalt. The strategy to elucidate the Chobimalt-induced effect on insulin fibrillogenesis is based on performing the concentration- and time-dependent analysis using a combination of different experimental techniques, such as ThT fluorescence assay, CD, AFM, SANS, and SAXS. While at the lowest Chobimalt concentration (0.1 µM; insulin to Chobimalt molar ratio of 1:0.004) the formation of insulin fibrils was not affected, the gradual increase of Chobimalt concentration (up to 100 µM; molar ratio of 1:4) led to a significant increase in ThT fluorescence, and the maximal ThT fluorescence was 3-4-fold higher than the control insulin fibril’s ThT fluorescence intensity. Kinetic studies confirm the dose-dependent experimental results. Depending on the concentration of Chobimalt, either (i) no effect is observed, or (ii) significantly, ∼10-times prolonged lag-phases accompanied by the substantial, ∼ 3-fold higher relative ThT fluorescence intensities at the steady-state phase are recorded. In addition, at certain concentrations of Chobimalt, changes in the elongation-phase are noticed. An increase in the Chobimalt concentrations also triggers the formation of insulin fibrils with sharply altered morphological appearance. The fibrils appear to be more flexible and wavy-like with a tendency to form circles. SANS and SAXS data also revealed the morphology changes of amyloid fibrils in the presence of Chobimalt. Amyloid aggregation requires the formation of unfolded intermediates, which subsequently generate amyloidogenic nuclei. We hypothesize that the different morphology of the formed insulin fibrils is the result of the gradual binding of Chobimalt to different binding sites on unfolded insulin. A similar explanation and the existence of such binding sites with different binding energies was shown previously for the nonionic detergent. Thus, the data also emphasize the importance of a protein partially-unfolded state which undergoes the process of fibrils formation; i.e., certain experimental conditions or the presence of additives may dramatically change not only kinetics but also the morphology of fibrillar aggregates.

Dual-Functional Antioxidant and Antiamyloid Cerium Oxide Nanoparticles Fabricated by Controlled Synthesis in Water-Alcohol Solutions.

Oxidative stress is known to be associated with a number of degenerative diseases. A better knowledge of the interplay between oxidative stress and amyloidogenesis is crucial for the understanding of both, aging and age-related neurodegenerative diseases. Cerium dioxide nanoparticles (CeO2 NPs, nanoceria) due to their remarkable properties are perspective nanomaterials in the study of the processes accompanying oxidative-stress-related diseases, including amyloid-related pathologies. In the present work, we analyze the effects of CeO2 NPs of different sizes and Ce4+/Ce3+ ratios on the fibrillogenesis of insulin, SOD-like enzymatic activity, oxidative stress, biocompatibility, and cell metabolic activity. CeO2 NPs (marked as Ce1–Ce5) with controlled physical–chemical parameters, such as different sizes and various Ce4+/Ce3+ ratios, are synthesized by precipitation in water–alcohol solutions. All synthesized NPs are monodispersed and exhibit good stability in aqueous suspensions. ThT and ANS fluorescence assays and AFM are applied to monitor the insulin amyloid aggregation and antiamyloid aggregation activity of CeO2 NPs. The analyzed Ce1–Ce5 nanoparticles strongly inhibit the formation of insulin amyloid aggregates in vitro. The bioactivity is analyzed using SOD and MTT assays, Western blot, fluorescence microscopy, and flow cytometry. The antioxidative effects and bioactivity of nanoparticles are size- or valence-dependent. CeO2 NPs show great potential benefits for studying the interplay between oxidative stress and amyloid-related diseases, and can be used for verification of the role of oxidative stress in amyloid-related diseases.

Advancements and future directions in research of the roles of insulin in amyloid diseases

Amyloid diseases, such as type 2 diabetes, Alzheimer's disease and Parkinson's disease are characterized by amyloid aggregates. Insulin is released from the pancreas, and it is known that insulin downstream signaling molecules are located majorly in the regions of cortex and hippocampus. Therefore, insulin plays crucial roles not only in the pancreas, but also in the brain. Recent studies have focused on the role of insulin in amyloid diseases. This review demonstrates the recent studies in which insulin affects amyloid aggregation. Specifically, molecular modeling studies provide insights into the molecular mechanisms of the effects of insulin in amyloid aggregates. Still, experimental studies are required to provide insights into the kinetics effects. This review opens new avenues for future studies on insulin molecules and amyloid aggregation.

Luminescent imaging of insulin amyloid aggregation using a sensitive ruthenium-based probe in the red region

A sensitive and selective strategy to identify insulin fibrils remains a challenge for researchers in amyloid protein research. Thus, it is critical to detect, in vitro, the species generated during amyloid aggregation, particularly the fibrillar species. Here we demonstrate that the luminescent complex cis-[Ru(phen)2(3,4Apy)2]2+ (RuApy; phen = 1,10-phenanthroline; 3,4Apy = 3,4-diaminopyridine) is a rapid, low-cost alternative to in vitro detection of fibrillar insulin, using conventional optical techniques. The RuApy complex displays emission intensity enhancement at 655 nm when associated with insulin, which enables imaging of the conformational changes of the protein's self-aggregation. The complex shows high sensitivity to fibrillar insulin with a limit of detection of 0.85 μM and binding affinity of 12.40 ± 1.84 μM which is comparable to those of Thioflavin T and Congo red, with the advantage of minimizing background fluorescence, absorption of light by biomolecules, and light scattering from physiologic salts in the medium.

Root cause determination of intraperitoneal catheter obstructions: Insulin amyloid aggregates vs foreign body reaction

Continuous intraperitoneal insulin infusion, from an implanted insulin pump connected to a catheter that delivers insulin directly to the peritoneal cavity has many clinical advantages for patients with Type 1 diabetes. However, the ongoing incidence of catheter obstructions remains a barrier to the widespread use of this therapy. To date, the root cause of these obstructions remains unknown. Here, a two-year clinical investigation was conducted, along with the development of an animal model to enable a mechanistic investigation into this issue. This novel animal model was able to mimic the catheter obstructions that occur in patients and, fortuitously, at an accelerated rate. This model allowed for independent assessment of each potential cause associated with catheter obstructions to help identify the root cause. Both macroscopic and microscopic analysis were conducted with regards to the onset and progression of catheter obstructions, along with monitoring of insulin delivery. Interestingly, although insulin aggregation occurs in insulin pumps and insulin aggregates were found in some catheter obstructions, insulin is unlikely to be the root cause, since obstructions also occurred in the control groups where only diluent (no insulin) was administered to the animals. Inflammatory cells, different phenotypes of fibroblasts, as well as collagen were observed in all obstructed catheters explanted from the patients and the animals. The presence of these cells and collagen is indicative of a typical foreign body reaction. In addition, the dynamic change in the fibroblasts with respect to morphology, phenotype, and spatial distribution suggests that tissue irritation-mediated epithelial to mesenchymal transition plays a role in catheter obstructions.

Development of multifunctional nanocomposites for controlled drug delivery and hyperthermia

Magnetic nano/micro-particles based on clinoptilolite-type of natural zeolite (CZ) were fabricated and were expected to act as carriers for controlled drug delivery/release, imaging and local heating in biological systems. Adsorption of rhodamine B, sulfonated aluminum phthalocyanine and hypericin by magnetic CZ nano/micro-particles was investigated, as was the release of hypericin. Using an alternating magnetic field, local temperature increase by 10 °C in animal tissue with injected magnetic CZ particles was demonstrated. In addition, the CZ-based particles have been found to exhibit an anti-amyloidogenic effect on the amyloid aggregation of insulin and lysozyme in a dose- and temperature-dependent manner. Therefore, the mesoporous structure of CZ particles provided a unique platform for preparation of multifunctional magnetic and optical probes suitable for optical imaging, MRI, thermo- and phototherapy and as effective containers for controlled drug delivery. We concluded that magnetic CZ nano/micro-particles could be evaluated for further application in cancer hyperthermia therapy and as anti-amyloidogenic agents.

Osmoprotectant Coated Thermostable Gold Nanoparticles Efficiently Restrict Temperature-Induced Amyloid Aggregation of Insulin.

Naturally occurring osmoprotectants are known to prevent aggregation of proteins under various stress factors including extreme pH and elevated temperature conditions. Here, we synthesized gold nanoparticles coated with selected osmolytes (proline, hydroxyproline, and glycine) and examined their effect on temperature-induced amyloid-formation of insulin hormone. These uniform, thermostable, and hemocompatible gold nanoparticles were capable of inhibiting both spontaneous and seed-induced amyloid aggregation of insulin. Both quenching and docking experiments suggest a direct interaction between the osmoprotectant-coated nanoparticles and aggregation-prone hydrophobic stretches of insulin. Circular-dichroism results confirmed the retention of insulin's native structure in the presence of these nanoparticles. Unlike the indirect solvent-mediated effect of free osmolytes, the inhibition effect of osmolyte-coated gold nanoparticles was observed to be mediated through their direct interaction with insulin. The results signify the protection of the exposed aggregation-prone domains of insulin from temperature-induced self-assembly through osmoprotectant-coated nanoparticles, and such effect may inspire the development of osmolyte-based antiamyloid nanoformulations.

Lysozyme Fibrils Alter the Mechanism of Insulin Amyloid Aggregation

Protein aggregation into amyloid fibrils is linked to multiple disorders. The understanding of how natively non-harmful proteins convert to these highly cytotoxic amyloid aggregates is still not sufficient, with new ideas and hypotheses being presented each year. Recently it has been shown that more than one type of protein aggregates may co-exist in the affected tissue of patients suffering from amyloid-related disorders, sparking the idea that amyloid aggregates formed by one protein may induce another protein’s fibrillization. In this work, we examine the effect that lysozyme fibrils have on insulin amyloid aggregation. We show that not only do lysozyme fibrils affect insulin nucleation, but they also alter the mechanism of its aggregation.

Exploring the occurrence of thioflavin-T-positive insulin amyloid aggregation intermediates.

The aggregation of proteins is considered to be the main cause of several neurodegenerative diseases. Despite much progress in amyloid research, the process of fibrillization is still not fully understood, which is one of the main reasons why there are still very few effective treatments available. When the aggregation of insulin, a model amyloidogenic protein, is tracked using thioflavin-T (ThT), an amyloid specific dye, there is an anomalous occurrence of double-sigmoidal aggregation kinetics. Such an event is likely related to the formation of ThT-positive intermediates, which may affect the outcome of both aggregation kinetic data, as well as final fibril structure. In this work we explore insulin fibrillization under conditions, where both normal and double-sigmoidal kinetics are observed and show that, despite their dye-binding properties and random occurrence, the ThT-positive intermediates do not significantly alter the overall aggregation process.

Modification of insulin amyloid aggregation by Zr phthalocyanines functionalized with dehydroacetic acid derivatives.

Amyloid fibrils are widely studied both as target in conformational disorders and as basis for the development of protein-based functional materials. The three Zr phthalocyanines bearing dehydroacetic acid residue (PcZr(L1)2) and its condensed derivatives (PcZr(L2)2 and PcZr(L3)2) as out-of-plane ligands were synthesized and their influence on insulin fibril formation was studied by amyloid-sensitive fluorescent dye based assay, scanning electron microscopy, fluorescent and absorption spectroscopies. The presence of Zr phthalocyanines was shown to modify the fibril formation. The morphology of fibrils formed in the presence of the Zr phthalocyanines differs from that of free insulin and depends on the structure of out-of-plane ligands. It is shown that free insulin mostly forms fibril clusters with the length of about 0.3–2.1 μm. The presence of Zr phthalocyanines leads to the formation of individual 0.4–2.8 μm-long fibrils with a reduced tendency to lateral aggregation and cluster formation (PcZr(L1)2), shorter 0.2–1.5 μm-long fibrils with the tendency to lateral aggregation without clusters (PcZr(L2)2), and fibril-like 0.2–1.0 μm-long structures (PcZr(L3)2). The strongest influence on fibrils morphology made by PcZr(L3)2 could be explained by the additional stacking of phenyl moiety of the ligand with aromatic amino acids in protein. The evidences of binding of studied Zr phthalocyanines to mature fibrils were shown by absorption spectroscopy (for PcZr(L1)2 and PcZr(L2)2) and fluorescent spectroscopy (for PcZr(L3)2). These complexes could be potentially used as external tools allowing the development of functional materials on protein fibrils basis.

Unfolding the Role of a Flavone-Based Fluorescent Antioxidant towards the Misfolding of Amyloid Proteins: An Endeavour to Probe Amyloid Aggregation.

4'-N,N-Dimethylamino-3-hydroxyflavone (DMAHF), a synthetic fluorescent flavone analogue with potent antioxidant activity, was explored as a molecular rotor-like fluoroprobe for amyloid aggregations, a causative factor in Alzheimer's disease, Parkinson's disease, type-2 diabetes, etc. During its interactions with (human) insulin amyloid aggregation (IAA), its microenvironment was changed. This instigated a drastic change in its excited-state intramolecular proton transfer-based dual emission behavior, which was tracked to monitor its amyloid probing activity. Thus, the amyloid probing potential of DMAHF was originated from its interactions with IAA, which were studied by various spectroscopic techniques and molecular docking and quantum-mechanical calculations. Morphological changes of the IAA in the presence of DMAHF were studied by scanning electron microscopy. DMAHF also probed efficiently the islet amyloid polypeptide deposition in the pancreatic β-cells of diabetic mice. DMAHF showed significant sensitivity and specificity towards amyloid aggregation without having any complexity in its photophysical behavior. This indicates its potential as an ideal bio-friendly and cost-effective fluoroprobe for amyloid proteins.

Modified polymer nanospheres – Characterization and their anti-amyloid activity to insulin amyloid aggregation

Polylactic acid-based nanoparticles are promising materials due to their tunable properties that are connected to surface modification. We have prepared polylactic acid nanospheres (PLA) with a non-magnetic phase (PLA loaded with various bovine serum albumin (BSA) concentrations) and magnetic phase (PLA loaded with magnetite nanoparticles coated with different amount of BSA (MFBSA)). The morphology, physico-chemical, magnetic and anti-amyloid properties were characterized by several biophysical methods. The amount of BSA used for PLA nanospheres or MFBSA modification played an important role in controlling the hydrodynamic size and zeta potential. In addition, magnetite loaded PLA nanospheres and MFBSA were all superparamagnetic. The potential of modified PLA nanospheres compared to MFBSA and 'naked' PLA to inhibit insulin amyloid fibrillation and destroy mature insulin fibrils was studied in vitro for samples with BSA/Fe3O4 weight ratio of 0.5. Destroying potential of samples was size–dependent, with the smallest MFBSA0.5 having the most significant effect. The strongest inhibitory effect on insulin fibrillation showed PLA-MFBSA0.5, slightly smaller than BSA modified PLA nanospheres.

Complementary experimental and computational analysis of the effects of non-ionic detergents and phospholipids on insulin amyloid aggregation

Amphiphilic compounds, both detergents and lipids, are important tools for in vitro analysis of water-soluble and integral membrane proteins. A key question is whether these two groups of amphiphilic molecules use the same pathway to affect structural and functional integrity of proteins. In the present study, we tested the effect of non-ionic detergent dodecyl maltoside (DDM), two phospholipids, 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), and the detergent-phospholipid mixtures on insulin amyloidogenesis in vitro. Amyloidogenesis of insulin is significantly affected by DDM in a time-and dose-dependent manner, but only slightly affected by either of phospholipids. Addition of DHPC or DMPC to detergent does not alter the inhibiting pattern, suggesting that DDM preferably binds to insulin. The molecular modeling revealed that DDM and the phospholipids occupy equivalent binding sites. DDM, due to the presence of maltose with several oxygen atoms (hydroxylic, glycosidic and ring) is involved in more hydrogen bonds than DHPC or DMPC. Hydrophobic interactions are important factors to stabilize both, DDM and phospholipids in their binding sites. Our results indicate that certain detergents (applying DDM as an example) and selected phospholipids are not always interchangeable in their use to investigate the effect of amphiphilic compounds on the behavior of amyloid-prone proteins.

Hydroxytyrosol Inhibits Protein Oligomerization and Amyloid Aggregation in Human Insulin.

Hydroxytyrosol (HT), one of the main phenolic components of olive oil, has attracted considerable interest for its biological properties, including a remarkable antioxidant and anti-inflammatory power and, recently, for its ability to interfere with the amyloid aggregation underlying several human diseases. We report here a broad biophysical approach and cell biology techniques that allowed us to characterize the molecular mechanisms by which HT affects insulin amyloid aggregation and the related cytotoxicity. Our data show that HT is able to fully inhibit insulin amyloid aggregation and this property seems to be ascribed to the stabilization of the insulin monomeric state. Moreover, HT completely reverses the toxic effect produced by amyloid insulin aggregates in neuroblastoma cell lines by fully inhibiting the production of toxic amyloid species. These findings suggest that the beneficial effects of olive oil polyphenols, including HT, may arise from multifunctional activities and suggest possible a application of this natural compound in the prevention or treatment of amyloid-associated diseases.

Amyloid Aggregation of Insulin: An Interaction Study of Green Tea Constituents

Exogenous insulin, used as a therapeutic agent for diabetes, forms insoluble deposits containing amyloid fibrillar structures near the administration site. We have analyzed the in vitro anti-amyloid activity of four green tea constituents: (-)-epigallocatechin gallate (EGCG), (-)-epicatechin (EC), gallic acid (GA), caffeine (CF), and their equimolar mixtures. Regarding individually tested compounds, only EGCG inhibited the fibrillization process. The individual EC, GA, and CF molecules were ineffective. The presence of EGCG in equimolar combinations with GA, EC, or CF was required for the inhibitory activity of most mixtures. Molecular docking revealed that EGCG interacts with an essential amyloidogenic region of insulin chain B. Individually inactive GA had a potentiating effect on the activity of EGCG. In contrast, EC and CF had a negative impact on the activity of the mixtures. We have observed diverse morphology and the amount of insulin amyloid aggregates formed in the presence of studied compounds. The distinct types of amyloid aggregates created in vitro in the presence of EGCG and other green tea constituents were characterized. Results indicate that the biological activity of individual molecules is not directly applicable to the pooled samples effects prediction.

Ferrocene-modified peptides as inhibitors against insulin amyloid aggregation based on molecular simulation.

Peptide-based inhibitors have gradually been implicated as drugs for treating protein folding diseases because of their favorable biocompatibility and low toxicity. To develop potential therapeutic strategies for amyloid-related disorders, short peptides modified by Fc, ferrocene-l-Phe-l-Phe (Fc-FF) and ferrocene-l-Phe-l-Tyr (Fc-FY), were used as inhibitors for the investigation of the aggregation behavior of insulin. Firstly, molecular docking predicted the interaction between both Fc-peptides and insulin. Then, the experimental data from ThT, DLS, CD and TEM confirmed that Fc-FF and Fc-FY effectively inhibited insulin fibrillation and disaggregated mature insulin fibrils. Based on a dose-dependent manner, both Fc-peptides can strongly inhibit insulin fibrillation, extend lag phase time, reduce final fibril formation (beyond 99% by Fc-peptides of 400 µM), decrease the formation of high-content β-sheet structures and reduce the size of insulin fibrils. Additionally, we found that compared with Fc-FY, the better inhibitory effect of Fc-FF at concentration below 400 µM was mainly resulted from the difference in π-π interaction and hydrogen bonds between Fc-peptides and insulin, according to molecular dynamics analysis. Our results demonstrated Fc-peptides, Fc-FF and Fc-FY, may play effective roles in the development of new therapeutic drugs or strategies for amyloid-related disorders, and the molecular dynamics simulation may be helpful for designing appropriate inhibitors of anti-amyloidosis diseases.

Study of tetraphenylporphyrins as modifiers of insulin amyloid aggregation.

Amyloid fibrils are rigid β-pleated protein aggregates that are connected with series of harmful diseases and at the same time are promising as base for novel nanomaterials. Thus, design of compounds able to inhibit or redirect those aggregates formation is important both for the biomedical aims and for nanotechnology applications. Here, we studied the effect of tetraphenylporphyrins (metal free, their Cu and Pd complexes, and those functionalized by carboxy and amino groups on periphery) on insulin amyloid self-assembling. The strongest impact on insulin aggregation was demonstrated by a metal-free porphyrin bearing four carboxy groups. This compound strongly suppresses insulin aggregation (about 88% reduction in amyloid-sensitive probe emission) inducing formation of fibrils with the length close to this of free insulin (1.7 ± 0.6 μm as compared with 1.4 ± 0.4 μm, respectively) with an essentially reduced tendency to lateral aggregation. Contrarily, the presence of tetraphenylporphyrin containing four amino groups only slightly affects fibrils' morphology and makes weaker impact on insulin aggregation yield (about 44% reduction). This is explained by the ability of aromatic carboxy groups of 5,10,15,20-(tetra-4-carboxyphenyl)porphyrin to interact with complementary protein-binding groups and thus stabilize the supramolecular complex. For 5,10,15,20-(tetra-4-aminophenyl)porphyrin, full protonation takes place in acidic medium of protein aggregation reaction; this results in the high positive charge of TPPN4 (equal or close to +6) and hence higher contribution of coulombic repulsion to interaction of TPPN4 with insulin. One more possible mechanism of the lower inhibition effect of TPPN4 as compared with TPPC4 could be the more restricted possibility of the former as compared with the latter to form H bonds with insulin groups. It was also shown that metal-free, Pd-containing, and Cu-containing tetraphenylporphyrins without peripheral substituents make almost the same impact on the protein self-assembling. We suppose this to be due to coordination saturation of these metal atoms.