Bovine Serum Albumin Monomer Research Articles

Reversible fast-dimerization of bovine serum albumin detected by fluorescence resonance energy transfer

Self-association of bovine serum albumin (BSA) was explored using fluorescence resonance energy transfer (FRET) between two populations of the protein labeled separately with either fluorescein-5′-isothiocyanate (FITC) or eosin-5′-isothiocyanate (EITC). The energy transfer reached the steady state after 5 s at 25 °C, indicating a fast exchange between oligomer subunits. The dependence of the energy transfer efficiency on the protein concentration and its reversion by unlabeled BSA demonstrate that association between BSA monomers occurs through a reversible path that involves specific interactions between the protein molecules. Because energy transfer took place even after blocking Cys 34 with iodoacetamide, this residue might not be involved in the reversible self-association process. The number of subunits forming the oligomer and its dissociation constant were determined from measurements of energy transfer as a function of the donor–acceptor ratio and of the total protein concentration. Analysis of these data indicated that BSA is in a monomer–dimer equilibrium with a dissociation constant of 10±2 μM at 25 °C in 10 mM MOPS-K (pH 5.8).

The cytochrome bc1 and cytochrome c oxidase complexes associate to form a single supracomplex in yeast mitochondria.

The mitochondrial electron transport chain complexes are large multisubunit complexes embedded in the inner membrane. We report here that in the yeast Saccharomyces cerevisiae, the cytochrome bc(1) and cytochrome c oxidase complexes co-exist as a larger complex of approximately 1000 kDa in the mitochondrial membrane. Following solubilization with a mild detergent, the cytochrome bc(1)-cytochrome c oxidase complex remains stable. It was analyzed using the techniques of gel filtration and blue native-polyacrylamide gel electrophoresis. Direct physical association of subunits of the cytochrome bc(1) complex with those of the cytochrome c oxidase complex was verified by co-immunoprecipitation analysis. Our data indicate that the cytochrome bc(1) complex is exclusively in association with the cytochrome c oxidase complex in yeast mitochondria. We term this complex the cytochrome bc(1)-cytochrome c oxidase supracomplex.

Effects of added yeast on protein transmission and flux in cross-flow membrane microfiltration

Microfiltration membranes may be used to separate valuable proteins from suspensions containing cells or cell debris. Although a clean microfiltration membrane allows for complete protein transmission and high flux, both of these quantities decline in time due to membrane fouling. Using bovine serum albumin (BSA) as a model protein, flux and protein transmission during cross-flow microfiltration were studied with and without added yeast cells. Cross-flow microfiltration of BSA-only solutions results in a BSA fouling layer with low permeability forming on the membrane surface. Due to this layer, the long-term BSA transmission is typically only 25-40%. In contrast, during microfiltration of yeast-BSA mixtures, the yeast forms a cake layer on the membrane surface. The yeast cake acts as a dynamic or secondary membrane, allowing BSA monomers to pass through but preventing protein aggregates from fouling the membrane. The result is slower flux decline and higher long-term BSA transmission of typically 60-90%. For filtration of yeast-BSA mixtures at low yeast concentrations (<1 g/L), 50-100% higher BSA recovery is obtained than for BSA-only solutions with the same BSA concentration. At high yeast concentrations (>5 g/L), the protein transmission remains high, but the recovery may be lower due to reduced flux.

Photophysical studies on the interaction of two water-soluble porphyrins with bovine serum albumin. Effects upon the porphyrin triplet state characteristics

The interaction of two metal-free water soluble porphyrins (PPh): meso-tetrakis ( p-sulfo-natophenyl)porphyrin (TPPS 4) and meso-tetrakis(4- N-methyl-pyridiniumyl)porphyrin (TMPyP), with bovine serum albumin (BSA) was investigated in the pH range from 4.0 to 8.5 using the flash-photolysis technique in comparison with results obtained by optical absorption, fluorescence and resonance light scattering. It was found that in the presence of BSA, TPPS 4 can exist in aqueous solutions as free monomers, aggregates and/or monomers bound to a single BSA molecule and monomers inside the BSA aggregates, while TMPyP does not form aggregates at binding. Binding to BSA transforms the profile of the triplet decay curve from monoexponential to biexponential form with the component lifetimes and relative amplitudes depending on binding characteristics. The triplet lifetime of a bound porphyrin monomer is longer than that of a free one. The aggregation of TPPS 4 observed at [TPPS 4]/[BSA] > I reduces the T-T absorption since the lifetimes of the aggregate excited states are very short and/or quantum yield of the aggregate triplet state is very low. The porphyrin binding to BSA reduces the quenching constants of the porphyrin triplet states by molecular oxygen due to obstacles produced by binding. This effect is especially pronounced for the porphyrin molecules located inside BSA aggregates formed around the porphyrin molecules in excess BSA.

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.

Sequential adsorption of F(ab′)2 and BSA on negatively and positively charged polystyrene latexes

The aim of the present work is to study the sequential adsorption of F(ab')(2) and bovine serum albumin (BSA) molecules adsorbed onto positively and negatively charged polystyrene latexes. Cationic and anionic latexes were prepared by emulsifier-free emulsion polymerization. Adsorptions of F(ab')(2) on both latexes at a low ionic strength and different pHs were performed. The cationic latex showed a higher adsorption of F (ab')(2) molecules over a range of pH, which could be due to the formation of multilayers. Sequential adsorption of anti-CRP F(ab')(2) and monomeric BSA were performed at two different pre-adsorbed F(ab')(2) amounts on both types of latex. Displacement of F(ab')(2) occurred only when the preadsorbed amounts were larger than a certain critical value, which depends on the adsorption pH. A greater displacement of larger preadsorbed amounts might be the result of a weaker contact between the protein molecules and the polystyrene surface. The displacement of F(ab')(2) previously adsorbed onto both latexes occurred due to pH changes, an increase of ionic strength and the presence of BSA molecules. The effect caused by these three factors was studied independently. The main factors in the desorption of F(ab')(2) on the anionic latex are the changes in pH and ionic strength, whereas on the cationic latex the desorption is mainly caused by the increase of the ionic strength and the presence of BSA. The colloidal stability of the immunotatex was improved by BSA adsorption, especially on cationic latex. (c) 1995 John Wiley & Sons, Inc.

Adsorption of monomeric bovine serum albumin on sulfonated polystyrene model colloids 3. Colloidal stability of latex—protein complexes

The present work deals with the study of the colloidal stability of sulfonated polystyrene latex particles covered by a model protein such as bovine serum albumin (BSA). Two BSA forms, monomeric (m-BSA) and oligomeric (o-BSA), have been previously adsorbed onto two sulfonated latexes with the same particle size but having different surface charge densities. The latex particles, fully or partially covered by the protein (termed latex—protein complexes), were resuspended under several sets of conditions (different pH values and ionic strengths) and their colloidal stability against the addition of electrolyte was studied by turbidity measurements. The latex—protein complexes (with m-BSA and o-BSA) were stable at pH 7, when the protein is negatively charged, owing to the electrostatic repulsion between the particles. The estimation of the zeta potential of the complexes versus the degree of coverage by the proteins permits the confirmation of the electrostatic contribution to the colloidal stability. However, at pH 5, when the protein is uncharged, the complexes with m-BSA on their surface were unstable, while those with o-BSA were stable even at high ionic strength. The stability of the o-BSA—sulfonated latex complexes at pH 5 was confirmed by measuring the particle size of the complexes with a light scattering technique: the size of the complexes was similar to that of the bare particles. Again by zeta potential estimation it is possible to detect that there is no electrostatic stabilization for the m-BSA— or o-BSA—sulfonated polystyrene latex complexes at pH 5, and therefore there would be an additional steric stabilization due to the size of the oligomeric molecules (probably tetrameric) which are adsorbed on the particle surface.

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.

Adsorption of monomeric bovine serum albumin on sulfonated polystyrene model colloids 1. Adsorption isotherms and effect of the surface charge density

The adsorption of monomeric bovine serum albumin (m-BSA) onto sulfonated polystyrene model colloids was studied by determining the adsorption isotherms under various pH and ionic strength conditions. The amount of m-BSA adsorbed was higher for the latex with a higher surface charge density for all the pH values pH (3–8) and ionic strengths (2 and 50 mM) studied. The maximum amount adsorbed was obtained at pH 5 (near the isoclectric point (IEP) of the protein), when the ionic strength was 2 mM, and shifted to pH 4 when the ionic strength was 50 mM. This amount (3.4mg m −2) was higher compared with that obtained with a conventional sulfate latex, but lower in comparison with that obtained with oligomeric BSA (o-BSA) (5.5 mg m −2). No significant desorption of m-BSA and o-BSA was detected after the redispersion of the latex-protein complexes under several, including extreme, experimental conditions. These results together with those obtained from the adsorption isotherms (with only one plateau) seem to indicate no multilayer formation or aggregation of BSA molecules. The flexibility of the BSA molecules and the asymmetry of the charge distribution seem to be the reasons for the high amount of protein adsorbed on sulfonated latexes.

Ultrastructural localization of albumin transport across the cerebral microvasculature during experimental meningitis in the rat.

Injury to the blood brain barrier (BBB) is a fundamental sequela of bacterial meningitis, yet the precise mechanism facilitating exudation of albumin across the endothelium of the cerebral microvasculature remains conjectural. After intracisternal inoculation of Escherichia coli (0111:B4) lipopolysaccharide in rats to elicit a reversible meningitis and BBB injury, we utilized in situ tracer perfusion and immunolabeling procedures to identify by transmission electron microscopy the precise topography and microvascular exit pathway(s) of bovine serum albumin (BSA). Results revealed that during meningitis there was: (a) an inducible increase in immunodetectable monomeric BSA binding to the luminal membrane of all microvascular segments in the pia-arachnoid and superficial brain cortex; (b) similar uptake of both colloidal Au-BSA (as well as monomeric BSA) by plasmalemmal vesicles but no detectable transcytosis to the abluminal side; and (c) predominant exit of both perfused Au-BSA and immunodetectable monomeric BSA through open intercellular junctions of venules in the pia-arachnoid. This was corroborated in separate experiments documenting focal pial venular leaks of in situ perfused 0.01% colloidal carbon black during experimental meningitis. These results provide precise localization of BBB injury in meningitis to meningeal venules, confirm a paracellular exit pathway of albumin via open intercellular junctions, and suggest an injury mechanism amenable to specific therapeutic intervention.

The adsorption of bovine serum albumin on positively and negatively charged polystyrene latices

The present study deals with the electrostatic aspects of the interaction between monomeric bovine serum albumin (BSA) and polystyrene latex particles. Adsorption and electrophoresis experiments have been performed as a function of pH, using both negatively and positively charged latices with different surface charge densities. The adsorption isotherms develop well-defined plateaus. These plateaus vary with pH, showing a maximum at the isoelectric point of the protein-covered polystyrene particles, rather than that of the isoelectric point of the protein itself. Co-adsorption of low-molecular-weight ions is shown to be an important parallel process. Studying it helps to discriminate between electrical and chemical contributions to the binding affinity.

Predicting the performance of gel-filtration chromatography of proteins

A method for determining the column and operating variables for the preparative separation of proteins by gel-filtration chromatography (GFC) of proteins is presented. The revocery of bovine serum albumin monomer from its dimer and higher molecular weight aggregates on high-performance GFC columns of various dimensions (column diameter d c = 0.75–10.8 cm, column length Z = 10–80 cm, particle diameter d p = 10–17 μm) was chosen as a model separation system. In the calculation method, the maximum sample feed volume V F,M that satisfies a specified purity ratio Q P and revocery ratio Q R for a given column length Z and particle diameter d P at a certain linear mobile phase velocity u was sought. The productivity P, defined as the amount of the recovered protein per unit column volume per unit time, was then calculated. The effects of Z and d P on the P— u relationship were examined. It was found that in general P increases with increasing u and the slope of the P— u curve becomes steep with decreasing d P and/or Z. A maximum in the P— u relationship was observed when the separation was difficult. The results show that it is not advantageous to employ larger particle diameter packings and/or a longe column in scaling-up. It is rather recommended that a short column packed with small gels be operated at relatively low flow-rates in the preparative GFC of proteins. It is also suggested that the calculation method presented is useful for scaling up GFC columns.

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.

Increased immunogenicity of a non-amphipathic protein (BSA) after inclusion into iscoms

Bovine serum albumin (BSA) was used as a non-amphipathic model protein to be included into iscoms. Pretreatment at an acidic pH (2.5) was used to reveal hydrophobic regions, after which BSA could be integrated. In immunization experiments in mice the BSA iscoms induced long lasting and considerably higher serum antibody responses than non-treated monomeric BSA or BSA aggregated by acidic treatment.

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.

Interactions between Orange II and Components in Heat-Treated Bovine Serum Albumin

In the earlier date, interactions between dyes and heat-treated bovine serum albumin (BSA) were studied using the whole solutions of heat-treated BSA themselves. Since it is known that heat-treated BSA consists of several components, interactions between dye and some of the individual components isolated from heat-treated solution of BSA are to be studied.By use of the native BSA monomer, components (1') and (2), spectrophotometric measurements were made at pH 7.O and 25°C on systems of Orange II and these albumin species. Components (1') and (2) were isolated from BSA solution heat-treated at pH 9 and 65°C for 60 min. Spectrophotometric data were analyzed by Terada-Miyake's method [Kolloid Z. Z. Polym., 251, 139 (1973)] to determine binding parameters. For the native BSA monomer, n (number of binding sites on the protein for the dye) was 6.4 and K (equilibrium constant of the system) was O.92 × 105 M-1. For components (1') and (2), values of n and K were different from those of the native BSA monomer.

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 use of o-phthaldialdehyde in the detection of proteins and peptides

A highly sensitive method utilizing o-phthaldialdehyde for an automated fluorescent assay of proteins and peptides is described in this paper. As an example of the utility of o-phthaldialdehyde, the separation of bovine serum albumin monomer from bovine serum albumin oligomers is discussed.

Heat denaturation of bovine serum albumin in alkaline pH region

The heat denaturation of bovine serum albumin was studied at 65°C in 0.1 M Tris-EDTA-boric acid buffer at pH 9.0. The gel electrophoresis indicated that the heat-treated bovine serum albumin solution contained several components, 1, 1′, 2, 3 and others. It was concluded that the component 1′ is a monomeric bovine serum albumin modified by the intramolecular SH-S-S exchange reaction. When the SH-blocked bovine serum albumin was heated in the presence of cysteine, a component having the same mobility as the component 1′, was formed by the reaction between the SH group of cysteine and the SS bond in bovine serum albumin, or by the SH-catalyzed structural alteration. (Components 1, 2 and 3 are a monomer, dimer and probably trimer of bovine serum albumin, respectively, as has already been concluded.) The amount of 1′ depended on the concentration of Tris-EDTA-boric acid buffer. The higher concentration of the buffer favored the formation of 1′, i.e. the intramolecular reaction. Components 1, 1′, 2 and 3 were isolated by the preparative disc gel electrophoresis. Studies on each component revealed the following: (a) The extinction coefficient E 1 cm 1% was the same for components 1, 1′, 2 and 3, being 6.6–6.7 at 279 nm, (b) The sedimentation coefficient of 1 and 1′ differed slightly, and (c) The helical content decreased in the order 1 > 1′ > 2 > 3. An evidence to support the concept of microheterogeneity of bovine serum albumin was presented. Additionally, the alkali denaturation of bovine serum albumin was studied in the pH range 10.5–12.0.