Aggregation Of Human Serum Albumin Research Articles

Tetrahydrobiopterin as a Trigger for Vitiligo: Phototransformation during UV Irradiation.

Vitiligo is a type of hypomelanosis. Tetrahydrobiopterin (H4Bip), the coenzyme of the initial stage of melanogenesis, appears to be a trigger for vitiligo. H4Bip is present in vitiligo in 3-5-fold excess and causes oxidative stress by triggering an autocatalytic cycle of excess hydrogen peroxide synthesis. Using quantum-chemical calculations, we have evaluated the possibility of H4Bip reactions occurring in the dark and under ultraviolet (UV) irradiation, including the formation of dihydropterin dimers. In order to simulate the oxidative stress, oxidative modification of human serum albumin (HSA) has been carried out in the presence of excessive H4Bip using the fluorescence method. The fraction of oxidized protein (FOP) has been calculated. It has been established that there is a strong oxidative modification of amino acids chromophores (tryptophan and tyrosine) in the protein (FOP 0.64). Under UV irradiation of the system (HSA + H4Bip), FOP is reduced to 0.39. Apparently, a part of H4Bip transforms into dihydropterin dimers and does not participate in the oxidative modification of the protein. The data on oxidative modification of HSA are consistent with dynamic light scattering: H4Bip promotes HSA aggregation with the formation of particles with a hydrodynamic radius Rh ≥ 2000 nm, which can become immunogenic.

Research on the secondary structure and hydration water around human serum albumin induced by ethanol with infrared and near-infrared spectroscopy

Given the role of ethanol played in the precipitation of various proteins, it is of great significance for the refined purification of proteins to understand the mechanism of protein secondary structure and hydration effect under the ethanol perturbation. Infrared (IR) spectroscopy and near-infrared (NIR) spectroscopy combined with chemometrics and aquaphotomics were utilized to get a generalized picture of the second structure of human serum albumin (HSA) and the surrounding hydrogen bond network of water after perturbation with different concentrations of ethanol ranging from 0% to 45% (v/v). The two-dimensional correlation (2DCOS) and moving window two-dimensional correlation analysis (MW-2DCOS) of IR spectroscopy provide evidence that the native structure of HSA was stabilized by the formation of α-helical structure at a low concentration of ethanol. However, the formation of intermolecular β-sheet structure by increasing ethanol is an important reason for the aggregation and precipitation of HSA. The analysis of NIR spectroscopy with the McCabe-Fisher method and aquaphotomics showed that free hydrogen bonded water was dynamic and fluctuating, 0%-20% ethanol could enhance the hydrogen bonded water cluster while the increasement of ethanol from 20% to 45% would lead to the formation of weak hydrogen bonded water cluster. These measurements provide a novel perspective on the relationship between ethanol and HSA, which is useful for maintaining activity and improving purity during the purification of proteins or other biological macromolecules.

4-Chloro-1,2-phenylenediamine induced structural perturbation and genotoxic aggregation in human serum albumin.

4-Chloro-1,2-phenylenediamine (4-Cl-OPD) is a halogenated aromatic diamine used as a precursor in permanent hair color production. Despite its well-documented mutagenic and carcinogenic effects in various in vitro and in vivo models, its role in fibrillar aggregate formation and their genotoxic effect in therapeutic proteins has received less attention. The significance of human serum albumin (HSA) arises from its involvement in bio-regulatory and transport processes. HSA misfolding and aggregation are responsible for some of the most frequent neurodegenerative disorders. We used various complementary approaches to track the formation of amyloid fibrils and their genotoxic effect. Molecular dynamics study demonstrated the complex stability. The impact of 4-Cl-OPD on the structural dynamics of HSA was confirmed by Raman spectroscopy, X-ray diffraction, HPLC and SDS-PAGE. Fibrilllar aggregates were investigated using Congo red assay, DLS, and SEM. The genotoxic nature of 4-Cl-OPD was confirmed using plasmid nicking assay and DAPI staining, which revealed DNA damage and cell apoptosis. 4-Cl-OPD provides a model system for studying fibrillar aggregation and their genotoxic potential in the current investigation. Future studies should investigate the inhibition of the aggregation/fibrillation process, which may yield valuable clinical insights.

Testing the link between isoaspartate and Alzheimer's disease etiology.

Isoaspartate (isoAsp) is a damaging amino acid residue formed in proteins as a result of spontaneous deamidation. IsoAsp disrupts protein structures, making them prone to aggregation. Here we strengthened the link between isoAsp and Alzheimer's disease (AD) by novel approaches to isoAsp analysis in human serum albumin (HSA), the most abundant blood protein and a major carrier of amyloid beta (Aβ) and phosphorylated tau (p-tau) in blood. We discovered a reduced amount of anti-isoAsp antibodies (P < 0.0001), an elevated isoAsp level in HSA (P < 0.001), more HSA aggregates (P < 0.0001), and increased levels of free Aβ (P < 0.01) in AD blood compared to controls. We also found that deamidation significantly reduces HSA capacity to bind with Aβ and p-tau (P < 0.05). These suggest the presence in AD of a bottleneck in clearance of Aβ and p-tau, leading to their increased concentrations in the brain and facilitating their aggregations there.

Spontaneous and Ionizing Radiation-Induced Aggregation of Human Serum Albumin: Dityrosine as a Fluorescent Probe.

The use of spectroscopic techniques has shown that human serum albumin (HSA) undergoes reversible self-aggregation through protein–protein interactions. It ensures the subsequent overlapping of electron clouds along with the stiffening of the conformation of the interpenetrating network of amino acids of adjacent HSA molecules. The HSA oxidation process related to the transfer of one electron was investigated by pulse radiolysis and photochemical methods. It has been shown that the irradiation of HSA solutions under oxidative stress conditions results in the formation of stable protein aggregates. The HSA aggregates induced by ionizing radiation are characterized by specific fluorescence compared to the emission of non-irradiated solutions. We assume that HSA dimers are mainly responsible for the new emission. Dityrosine produced by the intermolecular recombination of protein tyrosine radicals as a result of radiolysis of an aqueous solution of the protein is the main cause of HSA aggregation by cross-linking. Analysis of the oxidation process of HSA confirmed that the reaction of mild oxidants (, , ) with albumin leads to the formation of covalent bonds between tyrosine residues. In the case of •OH radicals and partly, , species other than DT are formed. The light emission of this species is similar to the emission of self-associated HSA.

Crystallographic analysis of interaction between cisplatin and human serum albumin: Effect of fatty acid

Metallodrugs are important for anticancer treatments. They bind mainly to human serum albumin (HSA) in blood circulation, greatly modulating their pharmacokinetics and anticancer efficacy. Fatty acid (FA) is one of the most important endogenous ligands of HSA with tight binding to HSA and affecting its conformation. However, the effect of fatty acids on metallodrugs interaction with HSA is unknown. Here we identify the binding sites of a widely used metallodrug, cisplatin, in HSA in the presence or absence of a representative fatty acid, myristate, by X-ray crystallography. Our crystal structures indicate that the sidechain of residue Met548 becomes more exposed to solvent in the presence of fatty acid, and is the main Pt binding site together with Met329 in HSA:Myr:cisplatin ternary structure. An undoubted new Pt binding site is detected at His338 in the presence of fatty acid, and additional two sites are also identified at His146 and His440 + K436. In addition, we revealed the mechanism of cisplatin-induced HSA aggregation, which is due to the crosslinking between Met298 and His510 of two HSA molecules.

Mitigation of ribose and glyoxal induced glycation, AGEs formation and aggregation of human serum albumin by citrus fruit phytochemicals naringin and naringenin: An insight into their mechanism of action

Human serum albumin (HSA) being the most prevalent protein in the plasma is extremely vulnerable to glycation. Two flavonoids naringin and naringenin were tested for their effects on the glyoxal and ribose-induced glycation, advanced glycation end products (AGEs) and fibril formation of HSA. The inhibition of the formation of AGEs in the presence of both flavonoids demonstrated their antiglycating properties. The presence of fibrillar aggregates in the glyoxal and ribose modified HSA were also decreased by naringin and naringenin. The explanation for naringenin's stronger antiglycating potential than naringin was further investigated by examining their interactions with HSA. H-bonding and other non-covalent interactions with flavonoids stabilize HSA. Interactions of lysine and arginine residues with flavonoids may prevent the residues from getting modified during glycation process. Naringenin bind to both subdomains IIA and IIIA of HSA, protecting more residues than naringin, which only binds to subdomain IIA, may describe the higher inhibitory activity of naringenin.

Human Serum Albumin Aggregation and its Modulation Using Nanoparticles: A Review.

Amyloid fibrils are highly stable protein fibrillar aggregates believed to be involved in various neurodegenerative diseases, which include Alzheimer's disease, Parkinson's disease, and prion diseases. Inhibiting the aggregation process is a potential strategy to prevent diseases caused by amyloid formation. In this regard, nanoparticles have emerged as promising candidates owing to their unique physical/chemical properties of small size, large surface area, biocompatibility, biodegradability, non-toxicity, and ease of functionalization. Human Serum Albumin (HSA) is a soluble multidomain monomeric protein that interacts with various ligands and hormones, aiding in their transport, distribution, metabolism in the circulatory system, and also plays a vital role in extracellular fluid volume stabilization. Under certain in vitro conditions, HSA has been reported to undergo conformational changes leading to fibril formation and hence acts as a suitable model for studying amyloidogenesis. In this review, we have explored the effects of various nanoparticles on HSA aggregation and their mechanism of action. The study will throw light on the mechanistic details of nanoparticle-mediated amyloid modulation, which will help in the development of effective therapeutics against amyloidosis.

The inhibitory effect of Sunset Yellow on thermally induced Human Serum Albumin aggregates: Possible role in naturopathy

Intensive research in the field of protein aggregation confirmed that the deposition of amyloid fibrils of proteins are the major cause for the development of various neurotoxic and neurodegenerative diseases, which could be controlled by ensuring the efficient inhibition of aggregation using anti aggregation strategies. Herein, we elaborated the anti amyloidogenic potential of Sunset Yellow (SY) dye against Human Serum Albumin (HSA) fibrillogenesis utilising different biophysical, computational and microscopic techniques. The inhibitory effect of sunset yellow was confirmed by Rayleigh Light Scattering (RLS) measurements along with different dye binding assays (ANS, ThT and CR) by showing concentration dependent reduction in scattering intensity and fluorescence intensity respectively. Further, destabilization and anti fibrillation activity of HSA aggregates were characterized through spectroscopic techniques like Circular Dichroism (CD) and other microscopic techniques like Transmission Electron Microscopy (TEM) for elucidating the structural properties. The SDS-PAGE was also carried out that render the disaggregation effect of the dye on the protein. Moreover, Molecular Docking studies revealed the binding parameters justifying the stable protein-dye complex. Simulation studies were also performed accordingly. Thus, this dye which is used as food additive can serve as a potential aggregation inhibiting agent that can aid in the prevention of amyloidogenic diseases.

TTAB and CTAB in human serum albumin aggregation

TTAB and CTAB in human serum albumin aggregation

Deep eutectic solvents as modulator on the micellization behaviour of cationic surfactants and potential application in human serum albumin aggregation

Deep eutectic solvents (DESs) have been utilized in many important applications in interdisciplinary research areas including the investigation of self-assembly of surfactants. In present study, the micellization behaviour of two conventional cationic surfactants i.e., tetradecyltrimethylammonium bromide (TTAB) and cetyltrimethylammonium bromide (CTAB) are investigated in aqueous DESs solutions with the help of surface tension, conductivity, UV–vis spectroscopy, fluorescence and dynamic light scattering (DLS) techniques. The synthesis of the DESs is done by simply mixing of choline chloride (ChCl) and urea/ethylene glycol (EG) in 1 : 2 mol ratio at 353 K temperatureand it is characterized by using FTIR technique. Various interfacial parameters and thermodynamic properties are evaluated by using tensiometric and conductometric methods. A noteworthy decrease in the critical micelle concentration (CMC) and increase in the aggregation number (Nagg) are observed in presence of DESs. Thus signifying that the micellization process of the surfactants is much favored in the DESs solutions and these are further supported by DLS results. DLS results show an increase in the micellar size for all the micellar systems in aqueous DESs solutions. More importantly, the micellar systems of the cationic surfactants within DESs solutions were also utilized for the study of their interaction with human serum albumin (HSA) to see how this albumin protein is behaving within the cationic micellar solutions in presence of DESs. UV–vis spectroscopic evidences show attractive results. It is noteworthy that the albumin protein HSA shows stronger binding affinity towards the DES-surfactants systems. The present work may further enhance the potential application of these neoteric green solvent systems composed of water-surfactant-DESs in the field of colloids, protein biophysics, nanoscience, which in turn will be highly applicable in the fields of synthesis, catalysis, and pharmaceutics etc.

Pharmaceutical acetylation can modulate the amyloidogenicity of human serum albumin

Human serum albumin (HSA) is an abundant carrier protein present in the blood plasma manifesting affinity for drugs and ligands. The bindings of several drugs can cause changes in the structural conformation of HSA that may affect its function. HSA is also known to in vitro form amyloid-like aggregates with fibrillar morphology as observed under TEM. Earlier, we showed that the HSA amyloid-like aggregates display self-seeding potential and detergent stability and the dimers of HSA, which are preferable for clinical applications due to their longer circulatory life, can also form amyloid-like aggregates. As aspirin, a commonly prescribed drug, was previously documented to acetylate HSA at one of its lysine residues, here, we examined if acetylation has any effect on the in vitro amyloid-like aggregation of HSA. We show that HSA acetylated in vitro using acetylsalicylic acid manifests relatively reduced levels of amyloid-specific properties such as turbidity, thioflavin-T-positive aggregation, -sheet content and stability against an ionic detergent. Also, TEM imaging shows that the acetylated HSA forms relatively less aggregates and with smaller sizeswhereas the aggregates of HSA are more abundant and larger in sizes with fibrillar morphology which further support that acetylation can attenuate the amyloid-like aggregation of HSA.

Nanoclay based study on protein stability and aggregation and its implication in human health

Protein aggregation is the major cause of several acute amyloid diseases such as Parkinson's, Huntington's, Alzheimer's, Lysozyme Systemic amyloidosis, Diabetes-II etc. While these diseases have attracted much attention but the cure is still unavailable. In the present study, Human Serum Albumin (HSA) and Human Lysozyme (HL) were used as the model proteins to investigate their aggregations. Nanoclays are hydrous silicates found in clay fraction of soil and known as natural nanomaterials. They have long been used in several applications in health-related products. In the present paper, the different types of nanoclays (MMT K-10, MMT K-30, Halloysite, Bentonite) were used to inhibit the process of HSA and HL aggregation. Aggregation experiments were evaluated using several biophysical tools such as Turbidity measurements, Intrinsic fluorescence, 1-anilino-8-naphthalene sulfonate (ANS), Thioflavin T (Th T), congo red (CR) binding assays and Circular dichroism. Results demonstrated that all the nanoclays inhibit the DTT-induced aggregation. However, bentonite and MMT K-10 were progressively intense and potent as they slowed down nucleation stage which can be perceived using several biophysical techniques. Hence, nanoclays can be used as an artificial chaperone and might provide effective treatment against several protein aggregation related disorders.

Protective actions of bioactive flavonoids chrysin and luteolin on the glyoxal induced formation of advanced glycation end products and aggregation of human serum albumin: In vitro and molecular docking analysis

The post-translational modification of proteins by nonenzymatic glycation (NEG) and the accumulation of AGEs are the two underlying factors associated with the long-term pathogenesis in diabetes. Glyoxal (GO) is a reactive intermediate which has the ability to modify proteins and generate AGEs at a faster rate. Human serum albumin (HSA) being the most abundant serum protein has a higher chance to be modified by NEG. The key objective of the present study is to investigate the potency of chrysin and luteolin as antiglycating and antifibrillating agents in the GO-mediated glycation and fibril formation of HSA. AGEs formation were confirmed from the absorption and fluorescence spectral measurements. Both the flavonoids were able to quench the AGEs fluorescence intensity in vitro indicating the antiglycating nature of the molecules. The formation of fibrils in the GO-modified HSA was confirmed by the Thioflavin T (ThT) fluorescence assay and the flavonoids were found to exihibit the antifibrillation properties in vitro. Docking results suggested that both the flavonoids interact with various amino acid residues of subdomain IIA including glycation prone lysines and arginines via non-covalent forces and further stabilized the structure of HSA, which further explains their mechanisms of action as antiglycating and antifibrillating agents.

Deciphering the Nature of Caffeic Acid to Inhibit the HSA Aggregation Induced by Glyoxal.

Under certain circumstances, the path for protein folding deviates and attains an alternative path forming misfolded states, which are the key precursors for protein aggregation. Protein aggregation is associated with variety of diseases and leads to the cytotoxicity. These protein aggregate related diseases have been untreated so far. However, extensive attempts have been applied to develop anti-aggregating agents as possible approaches to overcome protein aggregation. Different types of substances have been reported to halt or decrease the formation of ordered protein aggregates both in vitro and in vivo, such as polyphenols and metal ions. In the present study the in vitro aggregation of human serum albumin (HSA) by using a reactive dicarbonyl glyoxal has been investigated, simultaneously an attempt has been done to inhibit the glyoxal (GO) induced aggregation of (HSA) by caffeic acid (CA). Different methods have been employed to investigate the process, fluorescence spectroscopy, circular dichroism, cango red binding assay, thioflavin T dye binding, turbidimetric analysis, docking study and transmission electron microscopy. Results have shown that elevated concentration of GO forms aggregates of HSA, and the activity of CA suggested the possibility of inhibiting the HSA aggregation at higher concentrations, and this compound was found to have an anti-aggregation property. The present study explained that micro molar concentrations of CA inhibits the aggregation of HSA and showed pronounced anti-aggregation effect at increasing concentrations in the presence of GO which is elevated in diabetic and hyperglycaemia conditions.

Aggregate Removal Nanofiltration of Human Serum Albumin Solution Using Nanocellulose-Based Filter Paper.

This study is dedicated to the rapid removal of protein aggregates and viruses from plasma-derived human serum albumin (HSA) product to reduce the risk of viral contamination and increase biosafety. A two-step filtration approach was implemented to first remove HSA aggregates and then achieve high model virus clearance using a nanocellulose-based filter paper of different thicknesses, i.e., 11 μm (prefilter) and 22 μm (virus filter) at pH 7.4 and room temperature. The pore size distribution of these filters was characterized by nitrogen gas sorption analysis. Dynamic light scattering (DLS) and size-exclusion high performance liquid chromatography (SE-HPLC) were performed to analyze the presence of HSA aggregates in process intermediates. The virus filter showed high clearance of a small-size model virus, i.e., log10 reduction value (LRV) > 5, when operated at 3 and 5 bar, but a distinct decrease in LRV was detected at 1 bar, i.e., LRV 2.65–3.75. The throughput of HSA was also dependent on applied transmembrane pressure as was seen by Vmax values of 110 ± 2.5 L m−2 and 63.6 ± 5.8 L m−2 at 3 bar and 5 bar, respectively. Protein loss was low, i.e., recovery > 90%. A distribution of pore sizes between 40 nm and 60 nm, which was present in the prefilter and absent in the virus filter, played a crucial part in removing the HSA aggregates and minimizing the risk of virus filter fouling. The presented results enable the application of virus removal nanofiltration of HSA in bioprocessing as an alternative to virus inactivation methods based, e.g., on heat treatment.

T-Butanol Enables Dual Functionality of Mannitol: A Cryoprotectant in Frozen Systems and Bulking Agent in Freeze-Dried Formulations.

The effect of tertiary butyl alcohol (TBA) as a cosolvent on the phase behavior of mannitol in frozen and freeze-dried systems was characterized using differential scanning calorimetry (DSC) and X-ray diffractometry (XRD; laboratory and synchrotron sources). Solutions of mannitol (2 and 5% w/w) in TBA-water systems of different compositions (5 to 30% w/w TBA) were characterized, both during cooling and warming using DSC and XRD. At and below the TBA-water eutectic composition (22.5% w/w TBA), mannitol crystallization was completely inhibited in the frozen state, while it crystallized as anhydrous δ-mannitol in the final lyophile. The presence of mannitol did not affect the phase behavior of TBA. The ability of mannitol to serve as a cryoprotectant in frozen solutions, and as a bulking agent in final lyophile was evaluated using human serum albumin (HSA) as a model protein. When HSA in a TBA (5% w/w)-water solution containing mannitol (2% w/w) was freeze-thawed or freeze-dried, there was no evidence of HSA aggregation. Thus, when TBA was used as a cosolvent, mannitol exhibited dual functionality, serving as a cryoprotectant in frozen solutions and as a bulking agent in the final lyophile.

Conformation and Aggregation of Human Serum Albumin in the Presence of Green Tea Polyphenol (EGCg) and/or Palmitic Acid.

Polyphenols such as epigallocatechin gallate (EGCg) may have roles in preventing some chronic diseases when they are ingested as components of plant-based foods and beverages. Human serum albumin (HSA) is a multi-domain protein that binds various ligands and aids in their transport, distribution, and metabolism in the circulatory system. In the present study, the HSA-EGCg interaction in the absence or presence of fatty acid has been investigated. Förster resonance energy transfer (FRET) was used to determine inter- and intra-domain distances in the protein with and without EGCg and palmitic acid (PA). By labeling Cys-34 with 7-(diethyl amino)-4-methylcoumarin 3-maleimide (CPM), the distance between Trp-214 at domain IIA and CPM-Cys-34 at domain IA could be established. A small amount of PA decreased the distance, while a large amount increased the distance up to 5.4 Å. EGCg increased the inter-domain distance in HSA and HSA-PA up to 2.8 and 7.6 Å, respectively. We concluded that PA affects protein conformation more significantly compared to EGCg. Circular dichroism (CD) established that EGCg affects protein secondary structure more significantly than PA. PA had little effect on the α-helix content of HSA, while EGCg decreased the α-helix content in a dose-dependent fashion. Moreover, EGCg decreased α-helix content in HSA and HSA-PA to the same level. Dynamic light scattering (DLS) data revealed that both PA and EGCg increased HSA aggregation. EGCg increased HSA aggregation more significantly and promoted formation of aggregates that were more heterogenous. Any of these effects could impact the ability of serum albumin to transport and stabilize ligands including EGCg and other polyphenols.

Inhibition of Glycation-Induced Aggregation of Human Serum Albumin by Organic-Inorganic Hybrid Nanocomposites of Iron Oxide-Functionalized Nanocellulose.

Protein aggregation leads to the transformation of proteins from their soluble form to the insoluble amyloid fibrils and these aggregates get deposited in the specific body tissues, accounting for various diseases. To prevent such an aggregation, organic–inorganic hybrid nanocomposites of iron oxide nanoparticle (NP, ∼6.5–7.0 nm)-conjugated cellulose nanocrystals (CNCs) isolated from Syzygium cumini (SC) and Pinus roxburghii (PR) were chemically synthesized. Transmission electron microscopy (TEM) images of the nanocomposites suggested that the in situ-synthesized iron oxide NPs were bound to the CNC surface in a uniform and regular fashion. The ThT fluorescence assay together with 8-anilino-1-naphthalenesulfonic acid, Congo Red, and CD studies suggested that short fiber-based SC nanocomposites showed better inhibition as well as dissociation of human serum albumin aggregates. The TEM and fluorescence microscopy studies supported similar observations. Native polyacrylamide gel electrophoresis results documented dissociation of higher protein aggregates in the presence of the developed nanocomposite. Interestingly, the dissociated proteins retained their biological function by maintaining a high amount of α-helix content. The in vitro studies with HEK-293 cells suggested that the developed nanocomposite reduces aggregation-induced cytotoxicity by intracellular reactive oxygen species scavenging and maintaining the Ca2+ ion-channel. These results indicated that the hybrid organic–inorganic nanocomposite, with simultaneous sites for hydrophobic and hydrophilic interactions, tends to provide a larger surface area for nanocomposite–protein interactions, which ultimately disfavors the nucleation step for fibrillation for protein aggregates.

Synergistic effects of metal-induced aggregation of human serum albumin.

Exposure to cobalt (Co), chromium (Cr), and nickel (Ni) occurs often via skin contact and from different dental and orthopedic implants. The metal ions bind to proteins, which may induce structural changes and aggregation, with different medical consequences. We investigated human serum albumin (HSA) aggregation in the presence of CoII, CrIII, and/or NiII ions and/or their nanoparticle precipitates by using scattering, spectroscopic, and imaging techniques, at simulated physiological conditions (phosphate buffered saline - PBS, pH 7.3) using metal salts that did not affect the pH, and at HSA:metal molar ratios of up to 1:8. Co ions formed some solid nanoparticles in PBS at the investigated conditions, as determined by nanoparticle tracking analysis, but the CrIII anions and NiII ions remained fully soluble. It was found that all metal ions induced HSA aggregation, and this effect was significantly enhanced when a mixture of all three metal ions was present instead of any single type of ion. Thus, the metal ions induce aggregation synergistically. HSA aggregates formed linear structures on a mica surface in the presence of CrIII ions. A clear tendency of aggregation and linearly aligned aggregates was seen in the presence of all three metal ions. Spectroscopic investigations indicated that the majority of the HSA molecules maintained their alpha helical secondary structure and conformation. This study highlights the importance of synergistic effects of metal ions and/or their precipitates on protein aggregation, which are highly relevant for implant materials and common exposures to metals.