Aggregation Of Bovine Serum Albumin Research Articles

Collection of serum albumin aggregate nanoparticles from human plasma by dielectrophoresis.

Dielectrophoresis (DEP) is a fast and reliable nanoparticle recovery method that utilizes nonuniform electric fields to manipulate particles based on their material composition and size, enabling recovery of biologically-derived nanoparticles from plasma for diagnostic applications. When applying DEP to undiluted human plasma, collection of endogenous albumin proteins was observed at electric field gradients much lower than predicted by theory to collect molecular proteins. To understand this collection, nanoparticle tracking analysis of bovine serum albumin (BSA) dissolved in 0.5× phosphate-buffered saline was performed and showed that albumin spontaneously formed aggregate nanoparticles with a mean diameter of 237nm. These aggregates experienceda dielectrophoretic force as a function of aggregate radius rather than the diameter of individual protein molecules which contributed to their collection. In high conductance buffer (6.8mS/cm), DEP was able to move these aggregates into regions of high electric field gradient, and in lower conductance buffer (0.68mS/cm), these aggregates could be moved into high or low gradient regions depending on the applied frequency. Disruption of BSA aggregates using a nonionic detergent significantly decreased the particle diameter, resulting in decreased dielectrophoretic collection of albumin which increased the collection consistency of particles of interest. These results provide techniques to manipulate albumin aggregates via DEP, which impacts collection of diagnostic biomarkers.

Affinity Constants of Bovine Serum Albumin for 5 nm Gold Nanoparticles (AuNPs) with ω-Functionalized Thiol Monolayers Determined by Fluorescence Spectroscopy.

A detailed understanding of the binding of serum proteins to small (dcore <10 nm) nanoparticles (NPs) is essential for the mediation of protein corona formation in next generation nanotherapeutics. While a number of studies have investigated the details of protein adsorption on large functionalized NPs, small NPs (with a particle surface area comparable in size to the protein) have not received extensive study. This study determined the affinity constant (Ka) of BSA when binding to three different functionalized 5 nm gold nanoparticles (AuNPs). AuNPs were synthesized using three ω-functionalized thiols (mercaptoethoxy-ethoxy-ethanol (MEEE), mercaptohexanoic acid (MHA), and mercaptopentyltrimethylammonium chloride (MPTMA)), giving rise to particles with three different surface charges. The binding affinity of bovine serum albumin (BSA) to the different AuNP surfaces was investigated using UV-visible absorbance spectroscopy, dynamic light scattering (DLS), and fluorescence quenching titrations. Fluorescence titrations indicated that the affinity of BSA was actually highest for small AuNPs with a negative surface charge (MHA-AuNPs). Interestingly, the positively charged MPTMA-AuNPs showed the lowest Ka for BSA, indicating that electrostatic interactions are likely not the primary driving force in binding of BSA to these small AuNPs. Ka values at 25 °C for MHA, MEEE, and MPTMA-AuNPs were 5.2 ± 0.2 × 107, 3.7 ± 0.2 × 107, and 3.3 ± 0.16 × 107 M-1 in water, respectively. Fluorescence quenching titrations performed in 100 mM NaCl resulted in lower Ka values for the charged AuNPs, while the Ka value for the MEEE-AuNPs remained unchanged. Measurement of the hydrodynamic diameter (Dh) by dynamic light scattering (DLS) suggests that adsorption of 1-2 BSA molecules is sufficient to saturate the AuNP surface. DLS and negative-stain TEM images indicate that, despite the lower observed Ka values, the binding of MPTMA-AuNPs to BSA likely induces significant protein misfolding and may lead to extensive BSA aggregation at specific BSA:AuNP molar ratios.

Glycyl-l-histidyl-l-lysine prevents copper- and zinc-induced protein aggregation and central nervous system cell death in vitro.

Common features of neurodegenerative diseases are oxidative and inflammatory imbalances as well as the misfolding of proteins. An excess of free metal ions can be pathological and contribute to cell death, but only copper and zinc strongly promote protein aggregation. Herein we demonstrate that the endogenous copper-binding tripeptide glycyl-l-histidyl-l-lysine (GHK) has the ability to bind to and reduce copper redox activity and to prevent copper- and zinc-induced cell death in vitro. In addition, GHK prevents copper- and zinc-induced bovine serum albumin aggregation and reverses aggregation through resolubilizing the protein. We further demonstrate the enhanced toxicity of copper during inflammation and the ability of GHK to attenuate this toxicity. Finally, we investigated the effects of copper on enhancing paraquat toxicity and report a protective effect of GHK. We therefore conclude that GHK has potential as a cytoprotective compound with regard to copper and zinc toxicity, with positive effects on protein solubility and aggregation that warrant further investigation in the treatment of neurodegenerative diseases.

Investigating the binding interaction of quinoline yellow with bovine serum albumin and anti-amyloidogenic behavior of ferulic acid on QY-induced BSA fibrils

Protein aggregation induces profound changes in the structure along with the conformation of the protein, and is responsible for the pathogenesis of a number of neurodegenerative conditions such as Huntington’s, Creutzfeldt–Jacob, Type II diabetes mellitus, Alzheimer’s, etc. Numerous multi-spectroscopic approaches and in-silico experiments were utilized to investigate BSA’s biomolecular interaction and aggregation in the presence of quinoline yellow. The present research investigation evaluated the interaction of BSA with the food colorant (QY) at two different pH (7.4 and 2.0). The development of the BSA-QY complex was established with UV visible and fluorescence spectroscopy. The quenching of fluorescence upon the interaction of BSA with QY revealed the static nature of quenching mechanism. The Kb value obtained from our result is 4. 54 × 10−4 M−1. The results from the competitive site marker study infer that quinoline yellow is binding with the sub-domain IB of bovine serum albumin, specifically on site III. Three-dimensional fluorescence and synchronous fluorescence spectroscopy were applied for monitoring the alterations in the microenvironment of BSA upon the addition of quinoline yellow. The results from turbidity and RLS studies showed that higher concentrations of QY (80–400 µM) triggered bovine serum albumin (BSA) aggregation at pH 2.0. At pH 7.4, QY couldn’t manage to trigger bovine serum albumin aggregation, perhaps because of the repulsion between negatively charged dye (QY) and anionic bovine serum albumin. The results from far-UV CD, Congo Red, and scanning electron microscopy implicate that the QY-induced aggregates exhibit amyloid fibril-like structures. Molecular docking results revealed that hydrophobic interactions, hydrogen bonding, and Pi-Sulfur interactions contribute to QY-induced aggregation of BSA. Further, the amyloid inhibitory potential of ferulic acid (FA), a phenolic acid on QY-induced aggregation of BSA, has also been assessed. The QY-induced amyloid fibrils are FA-soluble, as confirmed by turbidity, RLS, and far-UV CD studies. Far-UV CD results showed that FA retains α helix and inhibits cross β sheet formation when the BSA samples were pre-incubated with increasing concentrations of FA (0–500 µM). Our findings conclude that QY dye successfully stimulates BSA aggregation, but ferulic acid inhibits QY-induced aggregation of BSA. Thus, FA can serve as a therapeutic agent and can help in the treatment of various amyloid-related conditions.

Effect and Mechanism of Soluble Starch on Bovine Serum Albumin Cold-Set Gel Induced by Microbial Transglutaminase: A Significantly Improved Carrier for Active Substances.

Soluble starch (SS) could significantly accelerate the process of bovine serum albumin (BSA) cold-set gelation by glucono-δ-lactone (GDL) and microbial transglutaminase (MTGase) coupling inducers, and enhance the mechanical properties. Hardness, WHC, loss modulus (G″) and storage modulus (G') of the gel increased significantly, along with the addition of SS, and gelation time was also shortened from 41 min (SS free) to 9 min (containing 4.0% SS); the microstructure also became more and more dense. The results from FTIR, fluorescence quenching and circular dichroism (CD) suggested that SS could bind to BSA to form their composites, and the hydrogen bond was probably the dominant force. Moreover, the ability of SS to bind the original free water in BSA gel was relatively strong, thereby indirectly increasing the concentration of BSA and improving the texture properties of the gel. The acceleration of gelling could also be attributed to the fact that SS reduced the negative charge of BSA aggregates and further promoted the rapid formation of the gel. The embedding efficiency (EE) of quercetin in BSA-SS cold-set gel increased from 68.3% (SS free) to 87.45% (containing 4.0% SS), and a controlled-released effect was detected by simulated gastrointestinal digestion tests. The work could put forward new insights into protein gelation accelerated by polysaccharide, and provide a candidate for the structural design of new products in the food and pharmaceutical fields.

The effect of fatty acids, ionic strength, and electric fields on the microscopic dynamics of BSA aggregates

This paper presents the microscopic dynamics of the concentrated suspensions of bovine serum albumin (BSA) proteins and their aggregates by dynamic light scattering (DLS) experiments. The effects of fatty acids binding to BSA, as well as the ionic strength and weak electric field, are discussed for affecting the stability of BSA suspensions against calcium-induced aggregation. By variation of the ionic strength, in the absence of an external electric field, DLS experiments show that monomer–BSA interactions (in the essentially fatty acid-free case) are overall repulsive but that, nevertheless, aggregation occurs to some extent. Also, the diffusive properties of different types of BSA are explored under an applied low-AC electric field by means of in situ electric small-angle depolarized DLS experiments, which reveal a significant decrease of the translational BSA–monomer diffusion coefficient with increasing frequency, while the aggregates indicate orientational motion via rotation on applying an electric field. These observations are interpreted in terms of (localized) orientation interactions obtained as oscillations in the intermediate scattering correlation function, as well as the anomalous slower relaxations as resulting in effective (collective) dynamics between monomeric BSA and their protein aggregates.

Surface Properties of Aqueous Dispersions of Bovine Serum Albumin Fibrils

The surface properties of aqueous dispersions of worm-like fibril aggregates of bovine serum albumin (BSA) differ from those of the adsorption layers of the native protein. The dispersions of BSA fibrils are characterized by slower changes of the surface tension and dynamic surface elasticity and also have different steady-state values of the surface properties. The fourfold compression of the adsorption layer of BSA fibrils leads to noticeably higher surface pressures than those of a compressed layer of the native protein, indicating the formation of a more rigid layer structure in the former case. The spreading of BSA fibrils onto a liquid surface from a concentrated dispersion reduces the effect of surface-active admixtures on the layer properties. The dependencies of the dynamic surface elasticity on surface pressure almost coincide for the spread layers of fibrils and the native protein in the range of low surface pressures, but only the spreading of the native protein can lead to surface pressures higher than 4 mN/m. This distinction is presumably caused by the formation of stable clusters of BSA fibrils at the interface and their slow propagation along the liquid surface.

Effect of 4-mercaptophenylboronic acid functionalized MoS2 quantum dots on amyloid aggregation of bovine serum albumin

In recent years, seeking and screening of compounds that can inhibit and/or depolymerize protein aggregation has been a hot issue in the field of pharmaceutical research. As a new material, quantum dots have been widely concerned by medical researchers. In present study, a novel 4-mercaptophenylboronic acid functionalized MoS2 quantum dots (4-MPBA-MoS2 QDs) was successfully synthesized through a one-pot hydrothermal approach by using molybdate dehydrate and 4-mercaptophenylboronic acid as Mo and S source, respectively. Transmission electron microscopy observation showed that the morphology and microstructure of the 4-MPBA-MoS2 QDs displayed uniform spherical shape with a diameter of approximately 1.5 ∼ 3.0 nm. The UV and fluorescence spectra experiments indicated that the prepared QDs had good water solubility and two weak absorption peaks were appeared at 280 nm and 310 nm. When the excitation wavelength was set to 310 nm, the 4-MPBA-MoS2 QDs had the strongest fluorescence intensity, and the maximum emission wavelength appeared at 405 nm. In vitro experiments showed that the 4-MPBA-MoS2 QDs could significantly reduce the aggregation of bovine serum albumin (BSA). Especially when the mass ratio of BSA to 4-MPBA-MoS2 QDs was 1:5, the inhibition rate could reach 76.4%. Cell experiment showed that the presence of 4-MPBA-MoS2 QDs could obviously decrease the cytotoxicity induced by BSA amyloid fibrils. Moreover, the depolymerization of BSA amyloid fibrils by 4-MPBA-MoS2 QDs and its excellent cell permeability were also observed. Molecular docking studies have shown that 4-MPBA-MoS2 QDs may stabilize the BSA structure through van der Waals forces, hydrophobic force, electrostatic interactions and hydrogen bonds formed between the outer layer of 4-MPBA and BSA to prevent fibrosis aggregation. The results of this study suggested that 4-MPBA-MoS2 QDs showed low cytotoxicity, good biocompatibility and solubility, and had a great potential in the design of new drugs for the treatment of amyloid-related diseases.

Serum Albumin Aggregation Facilitated by Cobalt and Chromium Metal Ions.

The interaction of serum proteins with cobalt (Co) and chromium (Cr) ions is poorly understood, but it is suspected to result in protein aggregation, which may alter the corrosion process of biomedical CoCr alloys or result in adverse health effects. Here, we study the aggregation ability and mechanism of bovine serum albumin (BSA) induced or accelerated by aqueous Co(II) and Cr(III) ions. The metal salts were selected by chemical speciation modeling, and they did not affect the pH or precipitate under simulated physiological conditions (150 mM NaCl and pH 7.3). The counterion of Cr(III) influenced the binding to BSA only at physiologically irrelevant low ionic strength. This study used a variety of spectroscopic and light scattering methods. It was determined that both metal ions and an equimolar mixture of metal ions have the potential to induce protein aggregation. Melting curves collected by circular dichroism spectroscopy indicate that Co(II) significantly reduced BSA's melting temperature when compared with Cr(III) or an equimolar mixture of Co(II) and Cr(III), both of which increased the melting temperature of BSA. The metal ions in solution preferentially interacted with BSA, resulting in the depletion of metal ions from the surrounding protein-free solution. Finally, this study suggests that the likely mechanism for Co(II)- and Cr(III)-induced BSA aggregation is salt bridging between protein molecules.

A good and bad aggregation: Effect of imidazolium- and cholinium-based ionic liquids on the thermal stability of bovine serum albumin

The present work describes the effect of imidazolium- and cholinium-based ionic liquids (ILs) on the thermal stability, binding and aggregation of bovine serum albumin (BSA). The studied ILs include choline chloride [Ch][Cl], choline dihydrogenphosphate [Ch][DHP], choline acetate [Ch][OAc], 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF4], 1-octyl-3-methylimidazolium tetrafluoroborate [OMIM][BF4] 1-butyl-3-methylimidazolium trifluoromethanesulfonate [BMIM][TfO], 1-butyl-3-methylimidazolium acetate [BMIM][OAc], and 1-butyl-3-methylimidazolium methanesulfonate [BMIM][CH3SO3]. The state of BSA in presence of ILs was assessed by circular dichroism (CD), temperature dependent circular dichroism, fluorescence spectroscopy, and dynamic light scattering (DLS). The thermal stability of BSA in presence of ILs increased (above 85 °C) in all studied ILs except [OMIM][BF4]. Differences of binding ILs with BSA depending on alkyl chain length were estimated, the probable binding modes were identified using molecular docking. The influence of ILs on the aggregation of BSA at different temperatures was established. It was found that the ILs with short alkyl chain length (up to butyl) promote aggregation of the native form of BSA increasing its thermal stability.

Design and synthesis of piano-stool ruthenium(II) complexes and their studies on the inhibition of amyloid β (1–42) peptide aggregation

Misfolding and protein aggregation have been linked to numerous human neurodegenerative disorders such as Alzheimer's, prion, and Parkinson's diseases. Ruthenium (Ru) complexes have received considerable attention in studying protein aggregation due to their interesting photophysical and photo properties. In this study, we have synthesized the novel Ru complexes ([Ru(p-cymene)Cl(L-1)][PF6](Ru-1), and [Ru(p-cymene)Cl(L-2)][PF6](Ru-2)) and investigated their inhibitory activity against the bovine serum albumin (BSA) aggregation and the Aβ1–42 peptides amyloid formation. Several spectroscopic methods were used to characterize these complexes, and the molecular structure of the complex was determined by X-ray crystallography. Amyloid aggregation and inhibition activities were examined using the Thioflavin-T (ThT) assay, and the secondary structures of the protein were analyzed by circular dichroism (CD) spectroscopy and transmission electron microscopy (TEM). The cell viability assay was carried out on the neuroblastoma cell line, revealing that the complex Ru-2 showed better protective effects against Aβ1–42 peptide toxicity on neuro-2a cells than the complex Ru-1. Molecular docking studies elucidate the binding sites and interactions between the Ru-complexes and Aβ1–42 peptides. The experimental studies revealed that these complexes significantly inhibited the BSA aggregation and Aβ1–42 amyloid fibril formation at 1:3 and 1:1 molar concentrations, respectively. Antioxidant assays demonstrated that these complexes act as antioxidants, protecting from amyloid-induced oxidative stress. Molecular docking studies with the monomeric Aβ1–42 (PDB: 1IYT) show hydrophobic interaction, and both complexes bind preferably in the central region of the peptide and coordinate with two binding sites of the peptide. Hence, we suggest that the Ru-based complexes could be applied as a potential agent in metallopharmaceutical research against Alzheimer's disease.

Effects of the aspect ratio of plasmonic gold nanorods on the inhibition of lysozyme amyloid formation.

Amyloid formation due to altered protein folding and aggregation has gained significant attention due to its association with neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and systemic lysozyme amyloidosis. Amyloids are characterized by parallel and anti-parallel cross-β-strands arranged to form stacks of sheets that provide stability and rigidity to the amyloid core. The prototypic protein Hen Egg White Lysozyme (HEWL) has been extensively used to understand protein hydrolysis, fragmentation, folding, misfolding, and amyloid formation. In the present study, we have examined the efficacy of plasmonic gold nanorods (GNRs) as an anti-amyloid agent against HEWL amyloids. Our results reveal that (i) the amyloid inhibition by plasmonic GNRs is dependent on their aspect ratio, (ii) the large aspect ratio GNRs ameliorate amyloid assembly completely, and (iii) GNRs interfere at several stages along the lysozyme fibril-formation pathway and block the conversion of monomeric and oligomeric intermediates into mature fibrils. Using a multi-parametric approach, we demonstrate that GNRs drive HEWL into off-pathway and amyloid-incompetent forms. To establish GNRs as generic amyloid inhibitors, we extended our studies to another archetypal protein, Bovine Serum Albumin (BSA), and observed similar results of GNRs inhibiting BSA aggregation. We believe that our results will pave the way for the potential use of GNRs with current therapeutics to reduce the burden of amyloid-related diseases.

High-Throughput Counting and Sizing of Therapeutic Protein Aggregates in the Nanometer Size Range by Nano-Flow Cytometry.

Protein aggregation is one of the greatest challenges in biopharmaceuticals as it could decrease therapeutic efficacy, induce immunogenicity, and reduce shelf life of protein drugs. However, there lacks high-throughput methods than can count and size protein aggregates in the nanometer size range, especially for those smaller than 100 nm. Employing a laboratory-built nano-flow cytometer (nFCM) that enables light scattering detection of single silica nanoparticles as small as 24 nm with sizing resolution and accuracy comparable to those of electron microscopy, here, we report a new benchmark to analyze single protein aggregates as small as 40 nm. With an analysis rate of up to 10,000 particles/min, the size distribution and particle concentration of nanometer protein aggregates can be acquired in 2-3 min. Employing heat-induced aggregation of bovine serum albumin (BSA) at high concentrations as the model system, effects of different categories of excipients, including sugars, polyols, salts, and amino acids on the inhibition of protein aggregation were investigated. Strikingly enough, as high as 1010 to 1012 particles/mL of protein aggregates were observed in the size range of 40 to 200 nm for therapeutic proteins of human serum albumin injection, reconstituted recombinant human interieukin-2 solution, and human immunoglobulin injection. nFCM opens a new avenue to count and size nanometer protein aggregates, suggesting its future usability in the quality assessment and formulation promotion of therapeutic proteins.

Counteraction of the cetyltrimethylammonium bromide-induced protein aggregation by heparin: Potential impact on protein aggregation and neurodegenerative diseases using biophysical approaches

Heparin, a member of the glycosaminoglycan (GAG) family, is an extremely acidic sulfur-containing polysaccharide which has been suggested to be coupled with protein aggregation-related diseases including Alzheimer's, Parkinson's and Prion diseases. Here, we study the effect of heparin on the cetyltrimethylammonium bromide (CTAB)-induced aggregation of bovine serum albumin (BSA) exploiting biophysical approaches. Kinetic measurements indicated that heparin caused complete inhibition of CTAB-induced aggregation of protein with a significant decrease in the apparent rate constant. Far- and near-UV CD measurements depicted that CTAB behaves as a protein-aggregating agent. However, the perturbation of secondary and tertiary structure-induced by CTAB and the CTAB-induced amyloid formation of protein were prevented by heparin. ITC measurements revealed that enhancement of aggregation induced by CTAB was as a result of sequential binding in which CTAB binds with the protein and accompanied by exothermic heat of interaction. ITC measurements also showed that CTAB and heparin form complex while the complex does not bind with protein. Thus, we hypothesize that CTAB-Heparin complex is engaged in the inhibition of protein aggregation. Therefore, the mechanism of action of CTAB-heparin complex in inhibition of protein aggregation may be used as a basis of the development of therapeutic strategies of various neurodegenerative complications.

Lyophilization of Curcumin-Albumin Nanoplex with Sucrose as Cryoprotectant: Aqueous Reconstitution, Dissolution, Kinetic Solubility, and Physicochemical Stability.

An amorphous curcumin (CUR) and bovine serum albumin (BSA) nanoparticle complex (nanoplex) was previously developed as a promising anticancer nanotherapy. The CUR-BSA nanoplex had been characterized in its aqueous suspension form. The present work developed a dry-powder form of the CUR-BSA nanoplex by lyophilization using sucrose as a cryoprotectant. The cryoprotective activity of sucrose was examined at sucrose mass fractions of 33.33, 50.00, and 66.66% by evaluating the lyophilized nanoplex’s (1) aqueous reconstitution and (2) CUR dissolution and kinetic solubility. The physicochemical stabilizing effects of sucrose upon the nanoplex’s 30-day exposures to 40 °C and 75% relative humidity were examined from (i) aqueous reconstitution, (ii) CUR dissolution, (iii) CUR and BSA payloads, (iv) amorphous form stability, and (v) BSA’s structural integrity. The good cryoprotective activity of sucrose was evidenced by the preserved BSA’s integrity and good aqueous reconstitution, resulting in a fast CUR dissolution rate and a high kinetic solubility (≈5–9× thermodynamic solubility), similar to the nanoplex suspension. While the aqueous reconstitution, CUR dissolution, and amorphous form were minimally affected by the elevated heat and humidity exposures, the treated nanoplex exhibited a lower BSA payload (≈7–26% loss) and increased protein aggregation postexposure. The adverse effects on the BSA payload and aggregation were minimized at higher sucrose mass fractions.

Antiproliferative Activities of the Lipophilic Fraction of Eucalyptus camaldulensis against MCF-7 Breast Cancer Cells, UPLC-ESI-QTOF-MS Metabolite Profile, and Antioxidative Functions.

Although a number of pharmacological properties have been linked to Eucalyptus camaldulensis leaf essential oil and extracts, the biological attributes of the lipophilic fraction remain unknown. Moreover, only a limited number of active compounds have so far been identified. This work aimed to investigate the anti-oxidative, anti-aggregation, and cytotoxic properties as well as profile the secondary metabolites in the lipophilic fraction of E. camaldulensis leaf extract (Lipo-Eucam) using UHPLC-ESI-QTOF-MS and gas chromatography–mass spectrometry (GC–MS). It was found that Lipo-Eucam possessed potent antioxidant properties against DPPH, ABTS, and FRAP with IC50 values of 31.46, 32.78, and 10.12 μg/mL, respectively. The fraction was able to attenuate metal-catalyzed oxidation of bovine serum albumin (BSA) in a dose-dependent manner (p < 0.05) and abrogated the aggregation of amyloidogenic BSA as revealed by the Congo red assay and transmission electron microscopy. Furthermore, Lipo-Eucam demonstrated potent cytotoxic effects against MCF-7 (IC50 7.34 μg/mL) but was noncytotoxic at used concentrations against HEK-293 cells (IC50 > 80 μg/mL), suggestive of its selective anticancer properties. Spectrophotometric, UHPLC–MS, and GC–MS analysis revealed that Lipo-Eucam is rich in phenolics, flavonoids, terpenoids, volatile constituents, and a plethora of active metabolites, probably responsible for the observed activities. These findings indicate that Lipo-Eucam is endowed with pharmacologically relevant active principles with strong potential for use in the amelioration of disease conditions related to oxidative stress, protein aggregation, and breast cancer and therefore worthy of further investigations.

Double-Site Binding and Anti-/Pro-oxidation of Luteolin on Bovine Serum Albumin Mediated by Copper(II) Coordination.

The interactions of luteolin (Lut) with bovine serum albumin (BSA) mediated by Cu(II) were investigated by spectroscopic, calorimetric, and molecular dynamic (MD) methods. Fluorescence studies showed that the binding of Lut to BSA was significantly enhanced by Cu(II) coordination with the number of binding sites and binding constant increasing from n = 1 and Ka = 3.2 × 105 L·mol–1 for Lut to n = 2 and Ka = 7.1 × 105 L·mol–1 for a 1:1 Cu(II)–luteolin complex, in agreement with the results from isothermal titration calorimetry (ITC). Site-specific experiments with warfarin and ibuprofen and MD confirmed that two binding sites of BSA were sequentially occupied by two Cu(II)–luteolin complexes. Cu(II) coordination increased the antioxidant activity of luteolin by 60% in the inhibition of carbonyl formation from the oxidation of amino groups in the side chain of BSA induced by the peroxyl radical ROO•; however, it counteracted the antioxidant effects of luteolin and played pro-oxidative roles in BSA aggregation induced by •OH.

Amyloid-Like Protein Aggregation Toward Pesticide Reduction.

Pesticide overuse is a major global problem and the cause of this problem is noticeable pesticide loss from undesired bouncing of sprayed pesticide droplets and rain erosion. This further becomes a primary source of soil and groundwater pollution. Herein, the authors report a method that can enhance pesticide droplet deposition and adhesion on superhydrophobic plant leave surfaces by amyloid‐like aggregation of bovine serum albumin (BSA). Through the reduction of the disulfide bond of BSA by tris(2‐carboxyethyl) phosphine hydrochloride (TCEP), the amyloid‐like phase transition of BSA is triggered that rapidly affords abundant phase‐transitioned BSA (PTB) oligomers to facilitate the invasion of the PTB droplet into the nanostructures on a leaf surface. Such easy penetration is further followed by a robust amyloid‐mediated interfacial adhesion of PTB on leaf surface. As a result, after mixing with pesticides, the PTB system exhibits a remarkable pesticide adhesion capacity that is more than 10 times higher than conventional fixation of commercial pesticides. The practical farmland experiments show that the use of PTB aggregation could reduce the use of pesticides by 70–90% while ensuring yield. This work demonstrates that current pesticide dosage in actual agriculture production may be largely reduced by utilizing eco‐friendly amyloid‐like protein aggregation.

Ultrasonic-assisted foam fractionation for recovering bovine serum albumin from aqueous solution: Structure and function evaluation

In this work, a technology of ultrasonic-assisted foam fractionation had been used to recover bovine serum albumin (BSA) from its aqueous solution. First, the operation parameters (e.g., height ratio of liquid phase and foam phase, aperture size of gas distributor and gas flow rate) of foam fractionation were optimized. Subsequently, ultrasonic treatment was assembled into foam fractionation operation. Experimental results indicated that the surface excess of BSA molecules first increased and then decreased with increasing ultrasonic intensity from 0 W/cm2 to 75 W/cm2. Considering enrichment ratio and recovery percentage of BSA, the suitable intensity of ultrasonic treatment was 45 W/cm2. Fourier transform infrared (FTIR) spectroscopy and SDS-PAGE analysis suggested that ultrasonic-assisted foam fractionation could not only destroy the intramolecular covalent bonds within BSA, but also prevent the oxidation of free sulfhydryl groups. The circular dichroism spectroscopy showed that the secondary structure of BSA had become loose and well-ordered after suffering foam fractionation with assistance of ultrasonic treatment. Ultrasound wave contributed to inhibiting the formation of BSA aggregates in foam fractionation. Furthermore, the surface hydrophobicity, emulsibility, and foaming property of BSA could be effectively maintained by assembling ultrasonic treatment into foam fractionation.

Analysis of proteins by agarose native gel electrophoresis in the presence of solvent additives.

Solvent additives, including NaCl, arginine hydrochloride (ArgHCl), glycine and sucrose, are used to enhance protein stability or reduce protein aggregation. Here, we studied the effects of these additives on proteins using agarose native gel electrophoresis. Since these additives are used at relatively high concentration, we first confirmed that they do not interfere with the performance of the native gel electrophoresis. Agarose native gel electrophoresis showed that aggregation of bovine serum albumin (BSA) induced by heating was slightly reduced by NaCl and ArgHCl. On the contrary, glycine and sucrose had marginal effects. ArgHCl and NaCl promoted heat aggregation of monoclonal antibody (mAb), while glycine and sucrose stabilized the native mAb. Arginine methyl ester inhibited heat aggregation of lysozyme and, to a much lesser extent, BSA. These results show that agarose native gel electrophoresis can be used to analyze the effects of solvent additives on proteins subjected to heat stresses. SYPRO Orange that stains only unfolded proteins confirmed unfolded structures of soluble aggregates.