Bovine Serum Albumin Dimer Research Articles

Bovine Serum Albumin Effect on Collapsing PNIPAM Chains in Aqueous Solutions: Spin Label and Spin Probe Study.

The influence of bovine serum albumin (BSA) on collapsing poly(N-isopropylacrylamide) (PNIPAM) chains was studied with turbidimetry and spin probe and spin label electron paramagnetic resonance spectroscopy. An increased ratio of collapsed chains in aqueous solutions in the narrow temperature region near the LCST appeared in the presence of 2.5-10 wt% BSA. The spin probe EPR data indicate that the inner cavities of the BSA dimers are probably responsive to the capture of small hydrophobic or amphiphilic molecules, such as TEMPO nitroxyl radical. The observed features of the structure and dynamics of inhomogeneities of aqueous PNIPAM-BSA solutions, including their mutual influence on the behavior of the polymer and protein below the LCST, should be considered when developing and investigating PNIPAM-based drug delivery systems.

Probing protein aggregation at buried interfaces: distinguishing between adsorbed protein monomers, dimers, and a monomer-dimer mixture in situ.

Protein adsorption on surfaces greatly impacts many applications such as biomedical materials, anti-biofouling coatings, bio-separation membranes, biosensors, antibody protein drugs etc. For example, protein drug adsorption on the widely used lubricant silicone oil surface may induce protein aggregation and thus affect the protein drug efficacy. It is therefore important to investigate the molecular behavior of proteins at the silicone oil/solution interface. Such an interfacial study is challenging because the targeted interface is buried. By using sum frequency generation vibrational spectroscopy (SFG) with Hamiltonian local mode approximation method analysis, we studied protein adsorption at the silicone oil/protein solution interface in situ in real time, using bovine serum albumin (BSA) as a model. The results showed that the interface was mainly covered by BSA dimers. The deduced BSA dimer orientation on the silicone oil surface from the SFG study can be explained by the surface distribution of certain amino acids. To confirm the BSA dimer adsorption, we treated adsorbed BSA dimer molecules with dithiothreitol (DTT) to dissociate these dimers. SFG studies on adsorbed BSA after the DTT treatment indicated that the silicone oil surface is covered by BSA dimers and BSA monomers in an approximate 6 : 4 ratio. That is to say, about 25% of the adsorbed BSA dimers were converted to monomers after the DTT treatment. Extensive research has been reported in the literature to determine adsorbed protein dimer formation using ex situ experiments, e.g., by washing off the adsorbed proteins from the surface then analyzing the washed-off proteins, which may induce substantial errors in the washing process. Dimerization is a crucial initial step for protein aggregation. This research developed a new methodology to investigate protein aggregation at a solid/liquid (or liquid/liquid) interface in situ in real time using BSA dimer as an example, which will greatly impact many research fields and applications involving interfacial biological molecules.

Effects of molecule size and resin structure on protein adsorption on multimodal anion exchange chromatography media.

The effect of bead and ligand structure on protein adsorption was investigated for multimodal anion exchangers combining a quaternary ammonium ion group with hydrophobic moieties: Nuvia aPrime 1 and aPrime 2, based on a 54 μm diameter polymeric bead, and Capto Adhere ImpRes and Capto Adhere, based on agarose beads 51 and 78 μm diameter, respectively. Bovine serum albumin (BSA) monomer, BSA dimer, and thyroglobulin (Tg) were used as model proteins. Based on TEM imaging and iSEC, the Nuvia resins have a microgranular structure and large pores (110 nm radius), while the Capto resins have a fibrous structure and smaller pores (32-36 nm radius). Comparable binding capacities (80-110 mg/mL), decreasing as salt is added, are observed for all three proteins on the Nuvia resins. Higher capacities (110-130 mg/mL), also decreasing as salt is added, are observed for BSA monomer and dimer on the Capto resins. However, the Tg binding capacity is very low in this case and increases as salt is added. Confocal laser scanning microscopy show that the kinetics are controlled by pore diffusion for all four resins, but with diffusivities that decrease as the protein size increases especially for the Capto resins. For Tg at low salt, binding is restricted to a thin shell close to the bead surface for both Capto resins. The ratio of effective and free diffusivity is about 0.30, 0.18, and 0.08 for BSA monomer, BSA dimer, and Tg, respectively, on the Nuvia resin. These values decrease to about 0.11, 0.04, and 0.01, respectively, for the Capto resins as a result of diffusional hindrance. Dynamic binding capacities are consistent with the equilibrium and rate behaviors.

Following Structural Changes by Thermal Denaturation Using Trapped Ion Mobility Spectrometry-Mass Spectrometry.

The behavior of biomolecules as a function of the solution temperature is often crucial to assessing their biological activity and function. While heat-induced changes of biomolecules are traditionally monitored using optical spectroscopy methods, their conformational changes and unfolding transitions remain challenging to interpret. In the present work, the structural transitions of bovine serum albumin (BSA) in native conditions (100 mM aqueous ammonium acetate) were investigated as a function of the starting solution temperature (T ∼ 23-70 °C) using a temperature-controlled nanoelectrospray ionization source (nESI) coupled to a trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) instrument. The charge state distribution of the monomeric BSA changed from a native-like, narrow charge state ([M + 12H]12+ to [M + 16H]16+ at ∼23 °C) and narrow mobility distribution toward an unfolded-like, broad charge state (up to [M + 46H]46+ at ∼70 °C) and broad mobility distribution. Inspection of the average charge state and collision cross section (CCS) distribution suggested a two-state unfolding transition with a melting temperature Tm ∼ 56 ± 1 °C; however, the inspection of the CCS profiles at the charge state level as a function of the solution temperature showcases at least six structural transitions (T1-T7). If the starting solution concentration is slightly increased (from 2 to 25 μM), this method can detect nonspecific BSA dimers and trimers which dissociate early (Td ∼ 34 ± 1 °C) and may disturb the melting curve of the BSA monomer. In a single experiment, this technology provides a detailed view of the solution, protein structural landscape (mobility vs solution temperature vs relative intensity for each charge state).

Protein exclusion is preserved by temperature sensitive PEG brushes

Poly(ethylene glycol) (PEG) is widely used in biotechnology-related applications yet its temperature-dependent functionality is not well understood. Here, we use bovine serum albumin (BSA) monomers and cross-linked dimers to directly probe the height of strongly stretched PEG brushes using surface plasmon resonance (SPR) in aqueous solution. Our results show that PEG brush height follows a smooth decrease as a function of increasing temperature commencing near room temperature. Measurements obtained by BSA monomers and dimers are comparable and suggest that BSA effectively probes the leading edge of the brush with minimal penetration into its interior being supported by SPR reflectivity calculations. Further, the BSA-PEG interaction remains largely inert over the entire temperature range. Overall, PEG brushes undergo a smooth conformational transition while fully preserving its protein excluding properties far from the lower critical solution temperature.

A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation.

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

Adsorption Behavior of Bovine Serum Albumin on Lowly Activated Anionic Exchangers Suggests a New Strategy for Solid-Phase Proteomics

Diluted solutions of bovine serum albumin (BSA) (e.g., 0.1 mg /mL) do not form detectable protein large aggregates. Using gel-filtration experiments, we determined that a diluted solution of BSA is 97% monomeric BSA and 3% dimeric. The adsorption of this diluted BSA on highly activated anionic exchangers (e,g., having 40 micromol/wet g) keeps this mainly monomeric form. When supports activated with 2 micromol/wet g are used, only dimers become adsorbed to the support, accounting for 100% of the offered BSA. When the diluted BSA solution is offered to very mildly activated anionic exchangers (even only 0.125 micromol/wet g), an unexpected adsorption of most of the BSA on the support was also observed. These very slightly activated supports are only able to adsorb very large proteins or very large protein-protein complexes, larger than BSA dimers. In fact, a rapid cross-linking of the adsorbed BSA with dextran-aldehyde reveals the formation of very large BSA-BSA complexes with molecular mass higher than 500 000 Da, complexes that may be observed for soluble BSA with very high concentrations but are not detectable at 0.1 mg/mL. Moreover, the size of the aggregates strongly depends on the concentration of the ionized groups on the support: the less activated the supports are, the higher the sizes of the complexes. It seems that the interaction of the BSA molecules on the margins of the BSA aggregate with the groups on the support may stabilize the whole protein aggregate, although some components are not interacting with the support. Aggregates could account for more than 40% of the BSA in the solution after 50 h of incubation. However, only these large BSA aggregates were adsorbed in the support.

FTIR characterization of the secondary structure of proteins encapsulated within PLGA microspheres

A commonly used technique for protein encapsulation in microspheres is the double-emulsion method wherein an initial water-in-oil (w/o) emulsion of protein and polymer is formed via sonication, and then a second emulsion (w/o)/w is formed by dispersion in an aqueous phase via homogenization. This approach is often used to produce microspheres of biodegradable poly(lactic- co-glycolic acid) (PLGA). The harsh processing associated with this method can cause denaturation of the encapsulated protein. Herein, we have used Fourier transform infrared (FTIR) spectroscopy to determine the secondary structures of two model proteins, bovine serum albumin (BSA) and chicken egg-white lysozyme, within PLGA microspheres. The α-helix content of both proteins in the microspheres was about a third lower than in the lyophilized state, indicating conformational changes upon protein entrapment within the microspheres. BSA microspheres containing the stabilizing excipient trehalose have a higher α-helix content than those without excipient, suggesting that trehalose partially prevents the denaturing effects incurred during processing. In addition, BSA released from microspheres is improved by incorporation of trehalose: analysis of the protein released from the microspheres indicates that there is less BSA dimer formation in the trehalose-containing microspheres than in those without trehalose.

The RNA molecule CsrB binds to the global regulatory protein CsrA and antagonizes its activity in Escherichia coli.

The RNA-binding protein CsrA (carbon storage regulator) is a new kind of global regulator, which facilitates specific mRNA decay. A recombinant CsrA protein containing a metal-binding affinity tag (CsrA-H6) was purified to homogeneity and authenticated by N-terminal sequencing, matrix-assisted laser desorption/ionization time of flight mass spectrometry, and other studies. This protein was entirely contained within a globular complex of approximately 18 CsrA-H6 subunits and a single approximately 350-nucleotide RNA, CsrB. cDNA cloning and nucleotide sequencing revealed that the csrB gene is located downstream from syd in the 64-min region of the Escherichia coli K-12 genome and contains no open reading frames. The purified CsrA-CsrB ribonucleoprotein complex was active in regulating glg (glycogen biosynthesis) gene expression in vitro, as was the RNA-free form of the CsrA protein. Overexpression of csrB enhanced glycogen accumulation in E. coli, a stationary phase process that is repressed by CsrA. Thus, CsrB RNA is a second component of the Csr system, which binds to CsrA and antagonizes its effects on gene expression. A model for regulatory interactions in Csr is presented, which also explains previous observations on the homologous system in Erwinia carotovora. A highly repeated nucleotide sequence located within predicted stem-loops and other single-stranded regions of CsrB, CAGGA(U/A/C)G, is a plausible CsrA-binding element.

Adsorption of Bovine Serum Albumin onto TiO2Particles

The adsorption of bovine serum albumin (BSA) at the TiO2–aqueous NaCl interface was studied as a function of pH and electrolyte concentration using kinetic and steady-state measurements. The electrokinetic properties of BSA-covered TiO2particles and the acid–base character of the protein were monitored by electrophoretic mobilities and acid–base potentiometric titration, respectively. Some experiments were also outlined to analyze the different affinities of BSA monomers, dimers, and polymers for the surface. The adsorption process is fast; less than 10 min is enough to achieve steady-state conditions. All measured isotherms showed high initial slopes even under adverse electrostatic conditions, resulting in high-affinity isotherms. Adsorption plateau values (Γmax) had a great pH dependence; Γmax–pH curves reached a maximum at around the isoelectric point of BSA and showed a drastic decrease on the acid side. Both structural and electrostatic effects must be invoked to explain the diminution of adsorbed BSA on either side of the isoelectric point. Structural effects were related to the different conformational states that BSA molecules adopt with pH changes, whereas electrostatic effects were analyzed assuming that BSA molecules behave as soft particles. This reasoning also allows us to explain the independence of the Γmax–pH curves from electrolyte concentration.

Competitive adsorption of α-lactalbumin and bovine serum albumin to a sulfopropyl ion-exchange membrane

Breakthrough curves were measured for pure solutions of α-lactalbumin (ALA) and bovine serum albumin (BSA) individually, and for a binary mixture of the proteins, using a sulfopropyl ion-exchange membrane. The breakthrough curves were qualitatively consistent with local-equilibrium theory predictions. Competitive adsorption caused displacement of bound BSA monomer by the more strongly binding BSA dimer, illustrating that even apparently single-protein systems may display multicomponent competitive behavior. In the two-protein experiment, ALA was competitively displaced by the more strongly binding BSA monomer and dimer, indicating that the binding strength was in the order: BSA dimer > BSA monomer > ALA.

The influence of protein aggregates on the fouling of microfiltration membranes during stirred cell filtration

Although macromolecular fouling of microfiltration membranes is one of the critical factors governing the performance of these filtration processes, there is still little fundamental understanding of the underlying phenomena that influence the initiation, rate, and extent of fouling. We have obtained experimental data for the flux decline during the stirred cell filtration of different commercial preparations of bovine serum albumin (BSA) through asymmetric polyethersulfone microfiltration membranes. The fouling characteristics of these commercial solutions varied substantially, with the flux decline directly related to the technique utilized to initially precipitate and prepare the BSA. Prefiltration of BSA solutions prior to microfiltration substantially reduced their fouling tendency, with the degree of improvement increasing as the prefiltration was performed through smaller molecular weight cut-off membranes. The protein solutions were also characterized using gel permeation chromatography (GPC), with the fouling tendency of the different BSA preparations highly correlated with the concentration of BSA dimers and other high molecular weight species present in these BSA solutions. These results suggest that BSA fouling of these microfiltration membranes is associated with the deposition of trace quantities of aggregated and/or denatured BSA, with these fouling species serving as initiation sites for the continued deposition of bulk protein.

Preferential adsorption of bovine serum albumin dimer onto polymer microspheres having a heterogeneous surface consisting of hydrophobic and hydrophilic parts

The adsorption behaviors of bovine serum albumin (BSA) containing both dimeric and monomeric species onto polymer microspheres were examined using various homopolymers and poly(2-hydroxyethyl methacrylate)/polystyrene composite microspheres which were produced by the emulsifier-free (seeded) emulsion polymerization technique. The preferential adsorption of the BSA dimer was clearly observed in an optimum region of the surface hydrophilicities of the polymer microspheres. The preferential adsorption of the BSA dimer onto the composite polymer microspheres having heterogeneous surfaces consisting of hydrophilic and hydrophobic parts was more marked than those onto the homopolymer and copolymer microspheres having homogeneous surfaces.

Lipid-vesicle-surface chromatography

Egg-yolk phospholipid vesicles (liposomes) containing stearylamine cations or phosphatidylserine anions, were formed and entrapped in agarose gel beads (Sepharose 6B) by a dialysis procedure. On a column of entrapped phospholipid—stearylamine (4:1) (cationic) vesicles, 0.36 mg of ferritin was bound per μmol lipids at 0.05 M ionic strength and pH 7. About 30% of the vesicle surface thus became covered with ferritin. Only 0.04 mg of citraconylated myoglobin was bound per μmol lipids, as myoglobin is much smaller than ferritin. Haeme groups were readily inserted into the lipid bilayers. An excess amount of bovine serum albumin (BSA) or ribonuclease A was applied to entrapped ionic vesicles and the bound proteins were eluted by increasing the ionic strength from 0.01 ot 0.2 or 0.5 M. After three to five runs, 82–88% of the vesicles (the phospholipids) remained entrapped. The capacity of the cationic vesicle-column for BSA decreased more than did the amount of entrapped vesicles, which indicates a preferential loss of stearylamine. Ion-exchange experiments were done with human plasma and with BSA monomers and dimers on entrapped cationic vesicles. Plasma proteins could be separated. BSA dimers were eluted later than BSA monomers in a sodium chloride gradient and the separation was better than on DEAE-Sepharose. The contact area between the protein and the vesicle surface is important for the binding strength. Protein—vesicle surface interactions can be studied by chromatography on entrapped vesicles.

Dependence of preferential bovine serum albumin oligomer adsorption on the surface properties of monodisperse polystyrene latices

Protein adsorption of several bovine serum albumin (BSA) samples, containing different amounts of dimeric and/or polymeric species, onto polystyrene (PS) latices, each having a different surface charge density, was studied under various conditions by high-performance liquid chromatography (HPLC). Depletion of each individual component in the solution demonstrated that in nearly all experiments the BSA dimer and/or polymer preferentially adsorb to the BSA monomer. The extent of preferential adsorption depends on BSA sample, surface charge of PS latex, BSA-to-surface area ratio, and solution conditions. The same variables also influence the total amount of adsorbed BSA.

Conformational studies of BSA using laser light scattering

AbstractThe usefulness of laser light scattering as a technique for determining protein conformation has been investigated by studying the self‐association and drug binding of bovine serum albumin (BSA). The diffusion coefficients of BSA monomers and dimers have been measured and the ratio of these two quantities indicates that in the dimer, the subunit separation is 2.2 times the monomeric hydrodynamic radius. The binding of salicylate to BSA causes an increase in its diffusion coefficient corresponding to a reduction in the frictional drag of the solvent on the protein molecules. It has been found that data obtained using laser light scattering may be interpreted confidently only when proper care has been taken in sample preparation and the scattered intensity autocorrelation function has been appropriately analyzed.

The interaction of dodecyl and tetradecyl sulfate with proteins during polyacrylamide gel electrophoresis

1. 1. The affinity of tetradecyl sulfate for many unfolded proteins is greater than that of dodecyl sulfate. 2. 2. It is the presence of tetradecyl sulfate that results in the staining of proteins by pinacryptol yellow seen by Stoklosa and Latz (Stoklosa, J.T. and Latz, H.W. (1974) Biochem. Biophys. Res. Commun. 58, 74–79), as some tetradecyl sulfate remains associated with proteins during electrophoresis at room temperature (as opposed to dodecyl sulfate which, within the limit of detection, is completely removed). 3. 3. Tetradecyl sulfate has a greater capacity to dissociate protein aggregates which consist of identical peptide chains, such as Glycophorin dimers and bovine serum albumin dimers, than does dodecyl sulfate.

Electrophoresis of Cottage Cheese Whey Proteins and Their Polymers

Cottage cheese whey solutions containing sodium dodecyl sulfate were resolved electrophoretically on 5% sodium dodecyl sulfate polyacrylamide gels. Major bands were identified by comparing their electrophoretic mobilities to those of known whey components. Other components were identified principally from molecular weights determined from a calibration correlating mobility and molecular weight. Conditions which affect polymerization of proteins were studied in whey and solutions of purified β-lactoglobulin. Formation of a number of polymers was induced by concentrating the whey samples, lowering the temperature, adjusting pH, or adding salts. The dimer, trimer, tetramer, and octamer of β-lactoglobulin, the dimer and trimer of bovine serum albumin, and several unidentified components in the 100,000 to 300,000 molecular weight range were observed. The octamer state of β-lactoglobu-lin was observed in whey at pH values between 5.1 and 8.0, at temperatures below 10C, and with .2M addition of potassium thiocyanate, potassium iodide, calcium chloride, or sodium acetate. Similar polymer formation, and temperature and pH effects, were observed with solutions of purified β-lactoglobulin, which contained dimers, trimers, and tetramers. The β-lactoglobulin octamer in whey samples could be dissociated by the addition of acid. The bovine serum albumin dimer appeared in whey at pH above 6.2 and at –1C.

Light scattering in mixtures of BSA, BSA dimers, and fibrinogen under the influence of electric fields

We report the development of a new technique for analyzing a mixture of different charged macromolecules. This technique combines light scattering and electrophoresis to permit the simultaneous determination of the electrophoretic mobilities and diffusion coefficients of charged macromolecules in solution. High resolution experiments on bovine serum albumin (BSA) have provided both the electrophoretic mobility and diffusion coefficient within a standard deviation of 5%. We report also the first observation of analytical separation of multicomponent systems by this technique. Distinct resolution for the systems BSA-BSA dimers and BSA-BSA dimersfibrinogen has been achieved. Finally we discuss potential extensions and applications of this technique.