Unfolded Bovine Serum Albumin Research Articles

Drug Binding to Partially Unfolded Serum Albumin: Insights into Nonsteroidal Anti-Inflammatory Drug Naproxen-BSA Interactions from Spectroscopic and MD Simulation Studies.

Understanding the binding details of a small-molecule drug to a protein in its partially unfolded state is important for drug delivery as it provides insight into the overall drug-binding ability of the protein, even when the majority of binding pockets in its unfolded state are impaired. The interaction of partially unfolded proteins with drugs remains poorly understood due to a lack of structural information on proteins in their partially unfolded states. Here, we studied the interaction between serum albumin (bovine serum albumin (BSA) as a model system), an abundant protein in blood serum that is an effective carrier for numerous known drugs, and a nonsteroidal anti-inflammatory drug (NSAID) naproxen (NPS) using various spectroscopic and computational methods. Molecular dynamics simulations starting from the drug-unbound state and performed at physiological and higher temperatures revealed novel hydrophobic sites on the BSA surface. We analyzed the BSA-NPS interaction in the presence and absence of the cationic organized assembly CTAB and two oligosaccharides (β-CD and 2-HP-β-CD) at different excitation wavelengths. The solvation dynamics of BSA under NPS-bound conditions became ∼4.6% faster. Oligosaccharides were found to increase the solubility of NPS by providing a hydrophobic environment for the formation of inclusion complexes through host-guest interactions. These findings provide a comprehensive overview and uncover the binding model and mechanism of interaction of NPS with BSA, revealing hydrophobic and electrostatic interactions and hydrogen bonds required for BSA to bind NPS at these noncanonical sites. The molecular-level understanding of the binding mechanism of commonly used NSAIDs like NPS with partially unfolded BSA will be useful in designing pharmaceutically important molecules with efficient loading and delivery properties.

Ferguson plot analysis of multiple intermediate species of thermally unfolded bovine serum albumin

Ferguson plot was used to characterize the multiple intermediate species of bovine serum albumin (BSA) upon thermal unfolding. Differential scanning calorimetry showed an irreversible melting of BSA in Tris-HCl and phosphate buffers with a mid-transition temperature, Tm, of ∼68 °C. Thermally unfolded BSA was analyzed by agarose native gel electrophoresis stained by Coomassie blue and SYPRO Orange staining as a function of pH or protein concentration. SYPRO Orange was used to stain unfolded proteins. BSA heated at 70 and 80 °C, i.e., above the Tm, formed multiple intermediate species, which depended on the pH between 7.0 and 8.0, protein concentration and which buffer was used. These intermediate species were analyzed by Ferguson plot, which showed that BSA heated at 60 °C had a similar size to the native BSA, indicating that they are either native or native-like state consistent with no SYPRO Orange staining. The intermediate species observed at higher temperatures with the mobility less than that of the native BSA showed a steeper Ferguson plot and were stained by SYPRO Orange, indicating that these species had a larger hydrodynamic size than the native BSA and were unfolded.

α-Helix-Mediated Protein Adhesion.

Proteins have been adopted by natural living organisms to create robust bioadhesive materials, such as biofilms and amyloid plaques formed in microbes and barnacles. In these cases, β-sheet stacking is recognized as a key feature that is closely related to the interfacial adhesion of proteins. Herein, we challenge this well-known recognition by proposing an α-helix-mediated interfacial adhesion model for proteins. By using bovine serum albumin (BSA) as a model protein, it was discovered that the reduction of disulfide bonds in BSA results in random coils from unfolded BSA dragging α-helices to gather at the solid/liquid interface (SLI). The hydrophobic residues in the α-helix then expose and break through the hydration layer of the SLI, followed by the random deposition of hydrophilic and hydrophobic residues to achieve interfacial adhesion. As a result, the first assembled layer is enriched in the α-helix secondary structure, which is then strengthened by intermolecular disulfide bonds and further initiates stepwise layering protein assembly. In this process, β-sheet stacking is transformed from the α-helix in a gradually evolving manner. This finding thus indicates a valuable clue that β-sheet-featuring amyloid may form after the interfacial adhesion of proteins. Furthermore, the finding of the α-helix-mediated interfacial adhesion model of proteins affords a unique strategy to prepare protein nanofilms with a well-defined layer number, presenting robust and modulable adhesion on various substrates and exhibiting good resistance to acid, alkali, organic solvent, ultrasonic, and adhesive tape peeling.

Amyloid-like coatings decorated electrodes boost the uranium electro-adsorption from seawater

Electro-adsorption has been regarded as one of the most effective methods for uranium extraction from seawater since this method could gather ultralow-concentration uranyl ions at the adsorbent surfaces for higher adsorption capacity and adsorption rates. However, the marine microorganisms and pollutants would also migrate to the adsorbent surfaces, causing serious inhibiting effects. Herein, the pristine electrodes were modified by the convenient amyloid-like assembly to solve the above problem. The unfolded bovine serum albumin (BSA) coatings not only offer adsorption sites toward uranium but also enable the electrodes with excellent anti-fouling and anti-adhesion properties. The modified electrodes achieved superior reusability and long-term stability even with the interferences of various pollutants and microorganisms. To the best of our knowledge, the overlooked but pernicious influences of the fouling phenomenon are firstly investigated in electro-adsorption. This finding not only removes the barriers in the practical applications of electro-adsorption, but also guides the design strategy of adsorbents for uranium extraction.

Patterns in Dried Droplets to Detect Unfolded BSA.

The morphological analysis of patterns in dried droplets has allowed the generation of efficient techniques for the detection of molecules of medical interest. However, the effectiveness of this method to reveal the coexistence of macromolecules of the same species, but different conformational states, is still unknown. To address this problem, we present an experimental study on pattern formation in dried droplets of bovine serum albumin (BSA), in folded and unfolded conformational states, in saline solution (NaCl). Folded proteins produce a well-defined coffee ring and crystal patterns all over the dry droplet. Depending on the NaCl concentration, the crystals can be small, large, elongated, entangled, or dense. Optical microscopy reveals that the relative concentration of unfolded proteins determines the morphological characteristics of deposits. At a low relative concentration of unfolded proteins (above 2%), small amorphous aggregates emerge in the deposits, while at high concentrations (above 16%), the “eye-like pattern”, a large aggregate surrounded by a uniform coating, is produced. The radial intensity profile, the mean pixel intensity, and the entropy make it possible to characterize the patterns in dried droplets. We prove that it is possible to achieve 100% accuracy in identifying 4% of unfolded BSA contained in a protein solution.

Binding interactions of cationic gemini surfactants with gold nanoparticles-conjugated bovine serum albumin: A FRET/NSET, spectroscopic, and docking study

This work demonstrates binding interactions of two cationic gemini surfactants, 12-4-12,2Br- and 12-8-12,2Br- with gold nanoparticles (AuNPs)-conjugated bovine serum albumin (BSA) presenting binding isotherms from specific binding to saturation binding regions of surfactants. The binding isotherm has been successfully constructed using Förster's resonance energy transfer (FRET) and nanometal surface energy transfer (NSET) parameters calculated based on fluorescence quenching of donor, tryptophan (Trp) residue by acceptor, AuNP. Energy transfer efficiency (ET) changes due to alteration in the donor-acceptor distance when surfactants interact with bioconjugates. A solid reverse relationship between α-helix and β-turn contents of BSA-AuNPs-conjugates is noted while interacting with surfactants. 12-8-12,2Br- shows stronger binding interactions with BSA-bioconjugates than 12-4-12,2Br-. The effect of bioconjugation on secondary/tertiary structures of BSA in the absence and presence of a surfactant is studied through circular dichroism, fluorescence, and Fourier transform infrared spectroscopic measurements. Motional restrictions imposed by AuNPs on Trp residues of folded and unfolded BSA have been investigated using red edge emission shift (REES) measurements. Finally, the molecular docking results present the modes of interactions of 12-4-12,2Br- and 12-8-12,2Br-, and Au-nanoclusters (Au92) with BSA. An approach to describe the binding isotherms of surfactants using AuNPs-bioconjugates as optical-based molecular ruler and possible effects of AuNPs on microenvironment and conformations of the protein is presented.

Mechanism of pre-ozonation in control of protein fouling of ultrafiltration membranes: Synergistic effect between ozone oxidation and aeration

Pre-ozonation is often used before ultrafiltration (UF) to control membrane fouling, usually by supplying ozone into water or wastewater via aeration. However, the role of aeration in pre-ozonation for mitigation of membrane fouling has not been well elucidated. Hence, in this study, bovine serum albumin (BSA) was used as a protein-type foulant in experiments on the outcomes and underlying mechanisms of pre-ozonation. The change in the membrane fouling behavior, the transformation of BSA, and the fouling-layer properties were investigated after pre-ozonation with and without aeration. The results showed that the membrane fouling notably declined after pre-ozonation, which mainly attributed to the synergistic effect between ozone oxidation and aeration. Ozone oxidation unfolded the BSA molecules and exposed their hydrophobic groups, and these unfolded BSA aggregated and adhered to the surface of the microbubbles produced by aeration. This induced BSA foaming and aggregation in the ozonated suspension. With pre-ozonation for a short contact time of 5 min, small BSA aggregates in water were retained on the UF membrane surface to form a highly porous deposition layer with a low filtration resistance. With pre-ozonation for a contact time of 10 min or longer, BSA foaming led to the formation of large aggregates that were efficiently removed from water in the ozone reactor, which greatly reduced the foulant loading of the water and its subsequent membrane fouling rate. In contrast, when quantitative and quiescent ozonation was performed without aeration, ozone oxidation alone worsened the membrane fouling propensity of the BSA solution.

Study of refolding of a denatured protein and microenvironment probed through FRET to a twisted intramolecular charge transfer fluorescent biosensor molecule

In the present study, Fӧrster Resonance Energy Transfer (FRET) from Tryptophan residues (Trp) of bovine serum albumin (BSA) to a nongenotoxic and nontoxic molecule, trans-2-[4-(dimethylamino)styryl]benzothiazole (DMASBT) resulting in fluorescence from the twisted intramolecular charge transfer (TICT) state of the latter has been explored to probe the gemini surfactant induced unfolding and β-cyclodextrin (β-CD) induced refolding of the protein. DMASBT as a potential acceptor in FRET process with its polarity-sensitive TICT fluorescence properties is found to be an efficient biosensor molecule to mark the changes in microenvironment as well as conformations of the protein. λmax of TICT fluorescence band of DMASBT in native protein at 486 nm is red-shifted to 503 nm in unfolded protein which is blue-shifted to 487 nm upon refolding of the protein. Process of BSA fluorescence quenching through FRET to DMASBT has been utilized to compare between the hydrophobic area and fractional accessibility to quencher in native and refolded protein. Fluorescence quenching data have also been used to explain the binding affinity of DMASBT with native, refolded and unfolded BSA. Study reveals the roles of spacer group of gemini surfactants, 12-4-12,2Br− and 12-8-12,2Br− on refolding and unfolding of BSA. A gemini surfactant with a longer spacer is found to be easily stripped off by β-CD resulting in better refolding of protein gaining a larger hydrophobic area. Results obtained corroborate well with the findings from other experimental measurements on circular dichroism, dynamic light scattering and Hg2+ ion-induced fluorescence quenching. An easy and efficient method to study the microenvironment, and unfolding and refolding of a protein is presented here.

Aggregation features of partially unfolded bovine serum albumin modulated by hydrogenated and fluorinated surfactants: Molecular dynamics insights and experimental approaches

Protein aggregation plays important roles in life science as, for instance, those associated to neurodegenerative diseases. Although extensive efforts have been done to elucidate all the possible variables related to the aggregation process, much has yet to be done to unveil the main pathways governing protein assembling. In the current work, we induce bovine serum albumin (BSA) association, at pH 3.7, by adding sodium dodecyl sulfate (SDS) and sodium perfluorooctanoate (SPFO) surfactants to BSA solution as promoters of protein aggregation. Firstly, we combine molecular dynamic simulations (MD) to obtain a partially unfolded state of BSA’s monomer at the acid pH and small angle X-ray scattering (SAXS) to validate the model. Interestingly, we found by SAXS that at pH 3.7 BSA monomers coexist with dimers in surfactant-free solution. Upon SDS and SPFO addition, the partial unfolded BSA may evolve to large aggregates depending on surfactant concentration. The threshold occurs at 30:1 and 45:1 SDS:BSA and SPFO:BSA molar ratio, respectively, according to turbidity, Thioflavin (ThT) fluorescence, synchrotron radiation circular dichroism (SRCD), SAXS and scanning electron microscopy (SEM) experiments. BSA aggregates are larger in the presence of SDS and structurally more defined upon SPFO binding. Isothermal titration calorimetry (ITC) results give support to infer that both surfactants initially bind to the BSA macromolecule forming a complex. Then, these complexes self-associate towards supramolecular aggregates. Taking into account the physicochemical characteristics of both surfactants and also MD simulations we may suggest that the higher rigidity of the fluorinated chains in respect to hydrogenated ones is crucial to induce more ordered and smaller BSA’s aggregates. Our results thus evidence that the ligand structural flexibility might be of a key importance in the pathway of protein aggregation and may pave the way to better understand the early steps of neurodegenerative disorders.

Spectroscopic probing of the refolding of an unfolded protein through the formation of mixed-micelles.

We report the unfolding of the globular protein, Bovine Serum Albumin (BSA) induced by anionic surfactant sodium dodecyl sulfate (SDS) and subsequently monitored the refolding of this denatured BSA using triblock copolymers F127 and P123 through the formation of mixed micelles. Our study exclusively represents the reversibility of this unfolding-refolding process using pluronic triblock copolymers F127/P123 as refolding agents. We confirm the recovery of its native state from its denatured state estimating the α-helical structure of the denatured protein from the CD data which support our steady state fluorescence spectra monitoring the fluorescence of the intrinsic Trp molecules present in BSA. Time resolved study also corroborates the stepwise recovery of the denatured BSA as well as the reversibility of the processes. Isothermal Titration Calorimetry (ITC) data explain the negligible interactions between the triblock copolymers and the native state of BSA. The high binding constant of SDS and triblock copolymers probably play the crucial role in the stepwise recovery of the unfolded BSA followed by reversibility of the refolding processes through the formation of the mixed micelles. The mechanism of mixed-micelle formation has been substantiated by the fact that the Guanidine Hydrochloride denatured BSA does not react with F127/P123 whereby no recovery of the protein was observed.

Unfolding and Refolding of Protein by a Combination of Ionic and Nonionic Surfactants.

The interaction of protein and surfactant yields protein–surfactant complexes which have a wide range of applications in the cosmetics, foods, and pharmaceutical industries among others. Ionic and nonionic surfactants are known to interact differently with the protein. The interplay of electrostatic and hydrophobic interactions governs the resultant structure of protein–surfactant complexes. The present study enlightens the paramount role of the hydrophobic interaction, tuned by the hydrophobic tail length of ionic surfactants, in the unfolding of anionic bovine serum albumin (BSA) protein. The unfolding of BSA in the presence of four different tail-length cationic surfactants, that is, C10TAB, C12TAB, C14TAB, and C16TAB, has been investigated by small-angle neutron scattering and dynamic light scattering. All cationic surfactants unfold the protein at a certain concentration range. The propensity of protein unfolding increases with increasing the hydrophobic tail length. The denatured structure of BSA upon addition of cationic surfactants is characterized by the random flight model representing a beads-on-a-string chain-like complex. The unfolded protein binds the surfactant micelles in the protein–surfactant cluster. The micelles get elongated with the increasing concentration of cationic surfactants, whereas the number of micelles per cluster is decreased. In the final stage, the protein–surfactant cluster merges to one large micelle with unfolded protein wrapping the micelle surface. The pathway of protein unfolding is described in terms of the changes in the micellar size, the number of micelles formed per cluster, the separation between the micelles in the cluster, the aggregation number of micelles, and the number of proteins per cluster. The protein–surfactant interaction is further examined in the presence of a nonionic surfactant, that is, C12E10. The nonionic surfactant significantly suppresses the interaction of BSA protein with ionic surfactants by forming mixed micelles. As a result of the mixed micelles formation by ionic–nonionic surfactants, the ionic surfactant moves out from the unfolded BSA protein, and this enables the protein to refold back to its native structure. The propensity of mixed micelle-driven refolding of proteins is significantly changed with changing the tail length of the ionic surfactant.

Study on interactions of cationic gemini surfactants with folded and unfolded bovine serum albumin: Effect of spacer group of surfactants

Interactions of three cationic gemini surfactants, 12-4-12, 2Br− 12-8-12, 2Br− and 12-4(OH)-12, 2Br− with natured and denatured protein, bovine serum albumin (BSA) have been studied by means of UV–Visible absorption, steady-state and time-resolved fluorescence, and circular dichromism (CD) spectroscopy. CD spectroscopic study shows the change in the α-helix and β-strand content of protein with the concentration of gemini surfactants. Gemini surfactant with hydroxyl group in the spacer decreases the α-helix of the BSA more efficiently than that without hydroxyl group in the spacer. Efficiency to decrease the α-helix of the protein increases with decreasing the hydrophobicity of the spacer group of the surfactants at lower concentration range following the order, 12-8-12, 2Br−<12-4-12, 2Br−<12-4(OH)-12, 2Br−. However, at higher concentration range of surfactant, the increasing order of providing hydrophobic environment to tryptophan (Trp) and tyrosine (Tyr) residues of the protein is as follows: 12-4(OH)-12<12-4-12<12-8-12. Gemini surfactant with hydrophobic spacer group provides more hydrophobic environment around Trp and Typ residues of the protein forming micelles like structures along the protein chain. In this concentration range, 12-8-12, 2Br− interacts differently as compared to other two surfactants which are evidenced by the data on excited state lifetime of the protein. It is more efficient to form a particular conformer and/or puckerd ring of Trp as compared to other two surfactants. The microenvironment around Trp residues of BSA is perturbed to a greater extent than that around Tyr residues in presence of gemini surfactants. Fluorescence from Trp and Tyr are quenched by acrylamide to a greater extent in presence of 12-8-12, 2Br−. Interactions of denatured BSA with gemini surfactants also have been studied. 12-8-12, 2Br− even interacts with the BSA unfolded by guanidine hydrochloride (GdHCl) to a greater extent than that by 12-4-12, 2Br− and 12-4(OH)-12, 2Br−.

Naked-Eye Detection of Reversible Protein Folding and Unfolding in Aqueous Solution

Students learning general chemistry and biochemistry often have difficulties understanding that proteins routinely exist in both folded and unfolded states, and that protein unfolding is not equivalent to irreversible denaturation or aggregation. To overcome the above misconceptions, we developed a novel demonstration that provides a simple and visually engaging illustration of the reversible interconversion between folded and unfolded protein states. Bovine serum albumin (BSA), a commercially available and inexpensive protein, binds to 8-anilino-1-naphthalenesulfonate (ANS) when folded in solution, and the system fluoresces brightly. Conversely, unfolded BSA (in the presence of ANS) and ANS alone do not fluoresce. Variations in pH and temperature, and addition of destabilizing and stabilizing cosolutes, lead to clear and visually appealing changes in fluorescence, illustrating BSA folding and unfolding. Overall, this novel lecture demonstration aims at overcoming common conceptual challenges, and it demy...

Effects of hydrocarbon chain length and temperature on the self-assembly of cationic single-chain and Gemini surfactants and their interactions with bovine serum albumin

Protein-surfactant interaction has been a subject of extensive studies over the past few decades due to its importance in manifold applications, such as food industry, drug delivery, skin and body care products, and cosmetics. Mild surfactants are often used to extract membrane proteins while preserving native structure and functional properties of protein, whereas the strong ionic surfactants are known to bind to oppositely charged protein molecules, resulting in denaturation of native protein and complete loss of protein activity. In general, the ionic surfactants bind strongly to protein through electrostatic attraction between surfactant headgroups and oppositely charged amino acids residues, and through hydrophobic interaction between surfactant hydrophobic tails and non-polar amino acids residues. Extensive studies on the interactions between ionic surfactants and protein have been reported. However, most of them focus on single-chain surfactants such as sodium dodecyl sulfate (SDS) or alkyltrimethylammonium bromide (C n TAB). In recent years, cationic gemini surfactant, a kind of surfactant consisting of two identical hydrophobic chains and two polar headgroups covalently linked by a spacer group, has stimulated the considerable interests due to its lower critical micelle concentration (CMC), stronger surface activity, better solubility, stronger hydrophobic microdomain, and so on. Thus, gemini surfactant is expected to exhibit quite different interaction behavior with protein from single-chain surfactant. This work has investigated the effect of hydrophobic chain length and temperature on the self-assembly of two series of cationic surfactants, gemini hexamethylene-1,6-bis(alkyldimethylammonium bromide) (C n C6C n Br2, n =10, 12, and 14) and single-chain alkyl trimethylammonium bromide (C n TAB, n =10, 12, 14), and their interactions with BSA using well-established ITC technique. For the surfactant self-assembly process, the micellization enthalpy change ( Δ H mic) values are more exothermic with the hydrophobic chain increasing. The free energy changes of micellization ( Δ G mic) mainly come from the micellization entropy changes ( Δ S mic). Temperature has a slight effect on the critical micelle concentration (CMC), but it has a significant effect on the Δ H mic. In the studied temperature range, the Δ H mic values are negative, whereas the Δ S mic values are positive, and the absolute values of T Δ S mic are larger than those of Δ H mic, proving the micellization process is mainly entropy-driven. With the temperature increasing, the contribution of enthalpy change to the micellization becomes larger, whereas the contribution of entropy change to the micellization becomes smaller. For the surfactant and bovine serum albumin (BSA) interaction process, at different surfactant concentration regions, the binding of the surfactants with BSA shows different interaction patterns. For most calorimetric curves of interactions, two endothermic and two exothermic processes are observed, and accompanied by two endothermic peaks. The first endothermic process is the dehydration of BSA upon the binding of the surfactant monomers. The first exothermic process involves the interaction of the surfactant monomers with surfactant molecules already bound on BSA molecule and the resultant formation of the micelle-like hydrophobic aggregates. The protein conformation change and the further dehydration of BSA molecules account for the second endothermic process. The following second exothermic process is the binding of surfactant monomers or micelles to BSA again leads to the formation of more micelle-like aggregates along the unfolded BSA molecules. The hydrophobic chain length remarkably affects the second endothermic process and the second exothermic process. This is because these two processes relate to the formation of the micelle-like aggregates along the BSA chain, whereas the micelle- like aggregates are easily formed at relatively high surfactant concentration range. The temperature has a marked effect on the protein/surfactant interaction in the first exothermic process and the second endothermic process. In particular, temperature strongly affects the appearance and amplitude of the second maximum endothermic peak, which can be explained by the effect of temperature on the protein conformation and the enthalpy change of micellization. In addition, gemini surfactants show much stronger binding ability with BSA as compared with single-chain surfactants due to their double hydrophobic chains and double charges.

Role of pH-induced structural change in protein aggregation in foam fractionation of bovine serum albumin

For reducing protein aggregation in foam fractionation, the role of pH-induced structural change in the interface-induced protein aggregation was analyzed using bovine serum albumin (BSA) as a model protein. The results show that the decrease in pH from 7.0 to 3.0 gradually unfolded the BSA structure to increase the molecular size and the relative content of β-sheet and thus reduced the stability of BSA in the aqueous solution. At the isoelectric point (pH 4.7), BSA suffered the lowest level in protein aggregation induced by the gas–liquid interface. In the pH range from 7.0 to 4.7, most BSA aggregates were formed in the defoaming process while in the pH range from 4.7 to 3.0, the BSA aggregates were formed at the gas–liquid interface due to the unfolded BSA structure and they further aggregated to form insoluble ones in the desorption process.

Protein aggregation in foam fractionation of bovine serum albumin: Effect of protein concentration

In foam fractionation of proteins, a clear understanding of their aggregation induced by the gas-liquid interface has great importance to reducing the loss of their bioactivity. In this work, the effects of the concentration of bovine serum albumin (BSA) on its aggregation induced by the gas-liquid interface were investigated at the molecular level to clearly explain how this protein aggregated in foam fractionation. The results show that reducing the BSA concentration made the protein structure more unfolded so that it enhanced the protein aggregation induced by the gas-liquid interface. In foam fractionation, the BSA aggregates were mainly formed in the desorption of the BSA molecules from the gas-liquid interface. Their formation was closely related to the BSA enrichment ratio. In the concentration from 0.05g/L to 0.20g/L, the highly unfolded BSA structure and the high enrichment jointly resulted in the formation of insoluble aggregates.

Differential Roles of the Protein Corona in the Cellular Uptake of Nanoporous Polymer Particles by Monocyte and Macrophage Cell Lines

Many biomolecules, mainly proteins, adsorb onto polymer particles to form a dynamic protein corona in biological environments. The protein corona can significantly influence particle-cell interactions, including internalization and pathway activation. In this work, we demonstrate the differential roles of a given protein corona formed in cell culture media in particle uptake by monocytes and macrophages. By exposing disulfide-stabilized poly(methacrylic acid) nanoporous polymer particles (PMASH NPPs) to complete cell growth media containing 10% fetal bovine serum, a protein corona, with the most abundant component being bovine serum albumin, was characterized. Upon adsorption onto the PMASH NPPs, native bovine serum albumin (BSA) was found to undergo conformational changes. The denatured BSA led to a significant decrease in internalization efficiency in human monocytic cells, THP-1, compared with the bare particles, due to reduced cell membrane adhesion. In contrast, the unfolded BSA on the NPPs triggered class A scavenger receptor-mediated phagocytosis in differentiated macrophage-like cells (dTHP-1) without a significant impact on the overall internalization efficiency. Taken together, this work demonstrates the disparate effects of a given protein corona on particle-cell interactions, highlighting the correlation between protein corona conformation in situ and relevant biological characteristics for biological functionalities.

Suitability of commercial hydrophobic interaction sorbents for temperature-controlled protein liquid chromatography under low salt conditions

The effect of temperature in the range from 10°C to 40°C and comparatively low ammonium sulfate (AS) concentrations of up to 0.5M on the adsorption of bovine serum albumin (BSA) on four different commercially available sepharose-based stationary phases was investigated. The determined isotherms were fitted by the Langmuir equation, and thermodynamic values were calculated by van’t Hoff analysis. The adsorption of BSA onto the chromatographic resin Butyl Sepharose 4FF showed the strongest temperature influence; however, protein unfolding effects occurred when characterizing this system by dynamic column experiments, with an unfolded BSA fraction strongly attached to the sorbent. The percentage of the unfolding fraction was determined for different operating conditions and found to increase with the concentration of the cosmotropic salt, but even stronger with increasing temperature. Temperature-induced cyclic adsorption and desorption experiments were carried out to investigate the long-term performance of Butyl Sepharose 4FF by applying purely temperature-controlled regeneration. Over a period of five cycles, the working capacity remained stable, but BSA also started to accumulate on the column due to incomplete regeneration. Finally, the possibility to fractionate different proteins with a single temperature shift was shown by the complete separation of lysozyme and BSA. The results presented indicate that temperature-induced binding and elution may offer a possibility to shift the operation conditions of HIC resins toward reduced salt concentrations, thus saving chemicals and facilitating salt removal in further downstream processing stages.

Bovine Serum Albumin Bioconjugated Gold Nanoparticles: Synthesis, Hemolysis, and Cytotoxicity toward Cancer Cell Lines

Bovine serum albumin (BSA) conjugated gold (Au) nanoparticles (NPs) were synthesized to explore their applications as drug delivery vehicles in systemic circulation. They showed little hemolysis and cytotoxic responses essentially required for such applications. This study shows some of the important physiochemical aspects needed for an appropriate synthesis of BSA-conjugated NPs where unfolded BSA is an essential reaction component. Unfolding of BSA was carried out under different experimental conditions in the presence of different ionic/zwitterionic surfactants and monitored simultaneously by UV–visible studies. Cationic surfactants induced unfolding at relatively lower temperatures than anionic and zwitterionic surfactants due to stronger electrostatic interactions with BSA. TEM analysis revealed the presence of NPs with almost similar shapes and sizes for different samples, and all NPs were stabilized by a coating of unfolded BSA. Isoelectric point of unfolded BSA coating on NP surface was close to 4...

About the albumin structure in solution and related electro-spinnability issues

In this work, we study the relationship between the shape and form of bovine serum albumin (BSA) protein in different solutions and their ability to form electrospun nanofibers. Small-angle X-ray scattering (SAXS) of the BSA in a water environment, in a 2,2,2-trifluoroethanol (TFE) environment, and in a TFE/beta-mercaptoethanol (β-ME) environment demonstrated an unfolding pathway; folded, partially unfolded and unfolded states of the protein, respectively. The scattering plot of BSA in water is characterized by a strong peak, thereby describing a solution of densely packaged globules. The scattering of BSA in TFE is attributed to a chain with compact folded-domains along its length, where the scattering of BSA in a mixture of TFE and β-ME suggests that the protein molecule adopts a freely coiled conformation in this solution. The zeta potential for both solutions of BSA in TFE was found to have an almost zero net charge, while the BSA solution in the water was highly negatively charged. This unfolding between three conformational states was correlated with the changes in electro-spinnability. Results show that the unfolded BSA is the only spinnable solution, producing long and continuous fibers with good mechanical stability.