Bovine Serum Albumin Surface Research Articles

Selective adsorption of small proteins on large-pore anion exchangers coated with medium size proteins

A new anion exchanger support has been designed for the selective adsorption of small proteins. This has been achieved activating an aminated support with glutaraldehyde and further coating the support surface with bovine serum albumin (BSA). In this support, "wells" are generated by two neighborhoods BSA molecules, on the bottom of those "wells" glutaraldehyde groups are exposed out ready to react with small molecules that have a size small enough to be accommodated between two BSA molecules on the pre-existing support. However, the BSA surface was not inert enough adsorbing many proteins, thereby reducing the selectivity of the system. A further solution was coating the immobilized BSA molecules with dextran, reducing the adsorption of protein on the BSA surface. This new matrix has been evaluated in the selective adsorption of the very small beta-lactoglobulins and alpha-lactalbumin from dairy whey, achieving the selective adsorption of both small proteins while other larger proteins from dairy whey remained in the supernatant. Moreover, a protein crude extract has been offered to the new matrix, and only small proteins could be adsorbed on the support (as probed by gel filtration). Thus this amino-glutaraldehyde-BSA-dextran-Sepharose is a matrix that may be used to selectively ionically adsorb proteins that were smaller than BSA (62 kDa). This strategy may be used for any other kind of adsorbing groups (chelating agents, boronic acid, etc.), or using proteins with different sizes to coat the support, designing tailor-made supports that may permit the fractioning of proteins following their sizes and by adsorption/desorption on different matrices.

Effect of pH on the Formation of a Bovine Serum Albumin Layer on a Poly(stryren-co-maleic Acid) Surface

The layer formation of bovine serum albumin (BSA) on a poly(styrene-co-maleic acid) (PSMA) surface was investigated by using quartz crystal microbalance (QCM) technique at various pH values. The formation of a BSA surface was examined by atomic force microscopy (AFM). To study the effect on the layer formation, the pH of solution was varied from 2 to 7.4 while the concentration of BSA was in the range of 0.01 to 5 mg/ml during the layer absorption. It was found that the BSA adsorption strongly depends on the pH of solution, and the concentration of BSA. The absorption layer occurred maximum at the pH value of 3.5 which resulted from the charge of PSMA and BSA molecules. The layer formation reached the saturate value at the concentration higher than 3 mg/ml. The molecular packing of the BSA layer at different pH values was determined by AFM and total mass change of QCM.

The utilization of BSA-modified chip on the investigation of ligand/protein interaction with surface plasma resonance

The kinetic behavior of glutathione (GSH)/ glutathione-S-transferase (GST) was investigated using surface plasmon resonance (SPR). Here, an alkanethiol-modified chip incorporated with bovine serum albumin (BSA) was employed. Subsequently, GSH was anchored on BSA surface only in the experimental channel and the without-active BSA surface was designed as the reference channel to improve the quality of the binding data and prevent a number of experimental artifacts to complicate the final biosensor analysis. Our results demonstrated that the BSA-modified chip was effective not only in binding the target proteins but also in suppressing the nonspecific binding (NSB) of proteins.

Cargo surface hydrophobicity is sufficient to overcome the nuclear pore complex selectivity barrier

To fulfil their function, nuclear pore complexes (NPCs) must discriminate between inert proteins and nuclear transport receptors (NTRs), admitting only the latter. This specific permeation is thought to depend on interactions between hydrophobic patches on NTRs and phenylalanine-glycine (FG) or related repeats that line the NPC. Here, we tested this premise directly by conjugating different hydrophobic amino-acid analogues to the surface of an inert protein and examining its ability to cross NPCs unassisted by NTRs. Conjugation of as few as four hydrophobic moieties was sufficient to enable passage of the protein through NPCs. Transport of the modified protein proceeded with rates comparable to those measured for the innate protein when bound to an NTR and was relatively insensitive both to the nature and density of the amino acids used to confer hydrophobicity. The latter observation suggests a non-specific, small, and plant interaction network between cargo and FG repeats.

Investigation of enzyme reaction by surface plasmon resonance (SPR) technique

An investigation into the surface plasmon resonance (SPR) sensor surface for the detection of enzyme reaction is reported. The thin polymeric film is prepared by spin casting poly (styrene-co-maleic acid) [PSMA] on a chromium/gold-coated SPR substrate. Bovine serum albumin (BSA) is adsorbed onto this surface with a mix of side-on and end-on orientation. The adsorbed BSA was tested against the protease where the cleavage reaction was followed by the change in the resonance angle of the SPR. The kinetic of the enzyme reaction follows the Michaelis–Menten equation where the reaction depends on the concentration of protease. The protease concentration is in the range of 2.5 μg/ml to 1.25 mg/ml. The different topology of protein surface before and after enzyme cleavage was observed by atomic force microscope (AFM). The AFM result shows uncleaved BSA on the surface even at highest protease concentrations owing to the steric hindrance of the adsorbed protease on the BSA surface. The sensor surface can be cleaned by cleaning solution and the surface can be reused, which avoids repetition of the complicated preparing of the sensor surface.

Interactions of Bovine Serum Albumin with Ethylene Oxide/Butylene Oxide Copolymers in Aqueous Solution

The interactions of bovine serum albumin (BSA) with three ethylene oxide/butylene oxide (E/B) copolymers having different block lengths and varying molecular architectures is examined in this study in aqueous solutions. Dynamic light scattering (DLS) indicates the absence of BSA-polymer binding in micellar systems of copolymers with lengthy hydrophilic blocks. On the contrary, stable protein-polymer aggregates were observed in the case of E 18B 10 block copolymer. Results from DLS and SAXS suggest the dissociation of E/B copolymer micelles in the presence of protein and the absorption of polymer chains to BSA surface. At high protein loadings, bound BSA adopts a more compact conformation in solution. The secondary structure of the protein remains essentially unaffected even at high polymer concentrations. Raman spectroscopy was used to give insight to the configurations of the bound molecules in concentrated solutions. In the vicinity of the critical gel concentration of E 18B 10 introduction of BSA can dramatically modify the phase diagram, inducing a gel-sol-gel transition. The overall picture of the interaction diagram of the E 18B 10-BSA reflects the shrinkage of the suspended particles due to destabilization of micelles induced by BSA and the gelator nature of the globular protein. SAXS and rheology were used to further characterize the structure and flow behavior of the polymer-protein hybrid gels and sols.

Monitoring the Binding Processes of Black Tea Thearubigin to the Bovine Serum Albumin Surface Using Quartz Crystal Microbalance with Dissipation Monitoring

The binding processes of thearubigin, which is one of the two major polyphenols (the other one is theaflavin) that gives black tea its characteristic color and taste, to the bovine serum albumin (BSA) surface have been investigated by quartz crystal microbalance with dissipation monitoring (QCM-D). The mass and thickness of the thearubigin adlayer on BSA surfaces at various thearubigin concentrations, salt concentrations, and pH values have been determined by QCM-D using the Voigt model. Our results show that the adsorption isotherm of thearubigin on the BSA surface can be better described by the Langmuir model than the Freundlich model, suggesting that the thearubigin adsorption on the BSA surface is dominated by specific interactions, such as electrostatic interaction and hydrogen bonding, as evidenced by the stronger thearubigin adsorption at pH below the isoelectric point (pI) of BSA and shifts in the positions of both amide bands in the FTIR spectra of the BSA surface with and without thearubigin adsorption. The addition of salt can also influence the thearubigin binding to BSA surfaces. The salt concentration-enhanced effect at a salt concentration lower than 0.1 M is explained as that an increase of salt concentration can screen the electrostatic repulsion to a larger extent than the electrostatic attraction between thearubigin and BSA. On the other hand, when the salt concentration is higher than 0.1 M, both electrostatic repulsion and attraction can be significantly screened by the higher salt concentration, resulting in the salt concentration-reduced effect. However, when the salt concentration is further increased to 0.4 M, the addition of thearubigin may promote the formation of a certain type of complex with BSA, resulting in the increases of both thickness and mass of the thearubigin adlayer.

Interfacial Interactions of Pectin with Bovine Serum Albumin Studied by Quartz Crystal Microbalance with Dissipation Monitoring: Effect of Ionic Strength

The effect of ionic strength ( I) on the interfacial interactions between pectin and the bovine serum albumin (BSA) surface has been investigated using the quartz crystal microbalance with dissipation monitoring (QCM-D). As I increases from 0.01 to 0.02 M, the frequency shift (Delta F) decreases, whereas the energy dissipation shift (Delta D) changes toward a higher value. Further increase of I from 0.02 to 0.5 M causes both Delta F and Delta D to gradually return to almost zero. The adsorbed mass and thickness of the pectin adlayer estimated from the Voigt model confirm that the adsorption of pectin and the formation of thicker pectin adlayers on a BSA surface are favored by the increase of ionic strength at I = 0.01 approximately 0.02 M. An increase of I above 0.02 M hinders pectin adsorption and causes the formation of a thinner pectin adlayer. The ionic strength-enhanced effect at I values lower than 0.02 M is explained as an increase of ionic strength that can screen the electrostatic repulsion to a larger extent than the electrostatic attraction between pectin and BSA. However, when I is higher than 0.02 M, both electrostatic repulsion and attraction can be significantly screened by the increasing ionic strength, resulting in the ionic strength-reduced effect. On the other hand, the high viscoelasticity of the pectin adlayer revealed by the Voigt model suggests the formation of a network-structured pectin adlayer on the BSA surface, which contains two steps for higher pectin adsorptions at I = 0.0125 approximately 0.1 M by the indication of two slopes in Delta D-Delta F plots.

Thermodynamics investigation of a series of metalloporphyrazine-bovine serum albumin complexes

The equilibrium binding of the tetra-cationic complexes ( N , N ′, N ″, N ‴-tetra-methyltetra-2,3-pyridinoporphyrazinato)copper(II), ([ Cu (2,3- TMTPPA )]4+), ( N , N ′, N ″, N ‴-tetra-methyltetra-3,4-pyridinoporphyrazinato)copper(II), ([ Cu (3,4- TMTPPA )]4+), (( N , N ′, N ″, N ‴-tetra-methyltetra-3,4-pyridinoporphyrazinato)cobalt(II), ([ Co (3,4- TMTPPA )]4+) and (( N , N ′, N ″, N ‴-tetra-methyltetra-3,4-pyridinoporphyrazinato)zinc(II), ([ Zn (3,4- TMTPPA )]4+) with bovine serum albumin (BSA) has been studied in phosphate buffer pH = 7.0 and at various temperatures using multi-spectroscopy techniques. The results of resonance light scattering (RLS) studies represent no aggregate formation of porphyrazine in the surface of BSA and low tendency of these porphyrazine for aggregate formation. The binding constants and binding stoichiometries were determined by analyzing of optical absorption spectra of porphyrazine complexes at various concentration of BSA using SQUAD software. The results show that the best fitting corresponds to a 1:1 complex model between BSA and porphyrazines. The thermodynamic parameters were calculated by van't Hoff equation at various temperatures. The data indicate that the process is entropy driven suggesting that hydrophobic interactions play a considerable role in the complex formation. The binding of porphyrazine complexes to BSA quenches fluorescence emission of BSA via a dynamic mechanism and the quenching process obeys a linear Stern-Volmer relationship. The average aggregation number of BSA, which has been calculated from the analysis of fluorescence quenching data, indicates the absence of any porphyrazine induced aggregation of BSA due to its interaction with porphyrazine complexes. Fluorescence studies also indicate that porphyrazine is bound to site I of BSA placed in sub-domain IIA, where tryptophan 214 is located.

Investigation of Adsorption Behavior of (−)-Epigallocatechin Gallate on Bovine Serum Albumin Surface Using Quartz Crystal Microbalance with Dissipation Monitoring

Quartz crystal microbalance with dissipation monitoring (QCM-D) has been employed to study the interactions between (-)-epigallocatechin gallate (EGCG) and bovine serum albumin (BSA) surface. The adsorbed mass, thickness, and viscoelastic properties of EGCG adlayer on BSA surface at various EGCG concentrations, temperatures, sodium chloride concentrations, and pH values have been determined by QCM-D in combination with the Voigt model. The adsorption isotherm of EGCG on BSA surfaces can be better described by the Freundlich model than the Langmuir model, indicating that EGCG adsorption on BSA surfaces is dominated by nonspecific hydrophobic interactions, as supported by stronger EGCG adsorption at higher temperature. Shifts in the Fourier transform infrared spectra of the BSA surface with and without EGCG adsorption disclose that hydrogen bonding might also be involved in EGCG adsorption on BSA surfaces. The addition of salt and change of pH can also influence the EGCG adsorption on BSA surfaces. Usually, higher EGCG adsorption leads to higher values of viscosity and shear elastic modulus of EGCG adlayer, which can be explained by the aggregation of BSA through EGCG bridges. Compared with EGCG, nongalloylated (+)-catechin shows much lower adsorption capacity on BSA surfaces, suggesting the importance of the galloyl group in polyphenol/protein interactions.

Spectroscopic investigation of the interaction between rifabutin and bovine serum albumin

The binding of rifabutin (RB) to bovine serum albumin (BSA) was investigated by fluorescence spectroscopy and circular dichroism (CD). RB effectively quenched the intrinsic fluorescence of BSA via a combination of static and dynamic quenching. The thermodynamic parameters, Δ H° and Δ S° were estimated to be −5.14 kJ mol −1, +120.44 J mol −1 K −1 according to the van’t Hoff equation. This indicates that hydrophobic interaction plays a major role in stabilizing the RB–BSA complex. The high order of magnitude of k q (10 15) for RB–BSA suggests that the process of energy transfer occurs through intermolecular interaction. Binding studies in the presence of fluorescence probe, 8-anilino-1-naphthalein-sulphonic acid (ANS), showed that RB competed with ANS for hydrophobic sites on the surface of BSA. The effect of metal ion on the binding constant of RB–BSA complex varied with metal ion. Cu 2+ and Ca 2+ increased the binding constant, whereas Zn 2+ and Mg 2+ decreased the binding efficacy of RB to BSA. Synchronous fluorescence spectra and CD data revealed that RB induced the conformational change of BSA.

Binding of the Environmental Pollutant Naphthol to Bovine Serum Albumin

The interactions between naphthol and bovine serum albumin (BSA) were investigated by spectroscopy. Our results prove the formation of complex between naphthol and BSA. Hydrophobic interaction dominates in the association reaction. The isomers stack with the aromatic residues in their binding sites with different geometries. Effects of BSA on the excited-state proton transfer and fluorescence spectra of the isomers indicate the different characters of their binding sites. 1-Naphthol inserts deeply into a hydrophobic cavity whereas 2-naphthol is in a basic environment on the surface of BSA. Naphthol statically quenches the fluorescence of BSA in a concentration-dependent manner positively deviating from the linear Stern-Volmer equation. Naphthol binds near Trp-134 in the subdomain IA of the native BSA and is accessible to Trp-212 when BSA is unfolded by naphthol. The folding pattern of the main chain is altered at high naphthol concentration as revealed by the change in the secondary structure. The binding of 1-naphthol is more cooperative than that of 2-naphthol. The extent of cooperativity was estimated by the Hill equation.

High Capacity, Charge-Selective Protein Uptake by Polyelectrolyte Brushes

Surface plasmon resonance was used to measure binding of proteins from solution to poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) brushes end-grafted from gold surfaces by atom transfer radical polymerization (ATRP). PDMAEMA brushes were prepared with a variety of grafting densities and degrees of polymerization. These brushes displayed charge selective protein uptake. The extent of uptake for net negatively charged bovine serum albumin (BSA) scaled linearly with the surface mass concentration of grafted PDMAEMA, regardless of grafting density. BSA was bound at a constant ratio of 120 DMAEMA monomer units per protein molecule for all brushes examined. The equivalent three-dimensional concentration of BSA bound in the brush (i.e., the bound BSA surface excess concentration divided by the brush thickness) decreased monotonically with decreasing grafting density. The concentration of BSA bound within brushes prepared at higher grafting densities was comparable with the aqueous protein solubility limit. BSA desorption from the brush required changes in solution pH and/or ionic strength to eliminate its net electrostatic attraction to PDMAEMA. Net positively charged lysozyme was completely rejected by the PDMAEMA brushes.

Adsorption of bovine serum albumin (BSA) onto lecithin studied by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy

The adsorption of bovine serum albumin (BSA) to lecithin was investigated by ATR-FTIR spectroscopy. Lecithin films were prepared by casting aliquots of 3.2 μg lecithin in methanol onto ZnSe ATR prisms. Surface morphology and the thickness of the films were investigated by laser scanning confocal electron microscopy and scanning electron microscopy and the thickness of the films used for adsorption studies was estimated to be 40 Å. The dependency of the C O peak area on the lecithin mass in the calibration curve confirms that the thickness of the film is below the penetration depth of the infrared evanescent wave. Size exclusion HPLC and fluorescence spectroscopy show that BSA conformation in up to 1 M NaCl and CaCl 2 solutions is similar to that in water with no aggregation or changes in protein conformation seen over 4 h. The kinetics of BSA adsorption on the lecithin film from water, NaCl and CaCl 2 solutions demonstrates that ions promote the protein adsorption. BSA bound more in the presence of NaCl compared to CaCl 2 at equivalent concentrations. The adsorption appeared greatest at a 0.1 M concentration for both NaCl and CaCl 2. The results are explained in terms of absorptive reactivity of BSA and lecithin surfaces upon salt addition.

Molecular Characteristics of Bovine Serum Albumin-Dextran Conjugates

Bovine serum albumin (BSA)-dextran conjugates were prepared by using the Maillard reaction; depending on the ratio of dextran to BSA used, about 0.5-1 mol of dextran could be bound to 1 mol of native BSA. SDS-PAGE patterns revealed that BSA and dextran had been covalently bonded. Structural analyses by fluorescence spectroscopy and circular dichroism indicated that the BSA surface in each conjugate was covered with dextran without any great disruption of the native conformation. The conjugates could be grouped into two fractions on the basis of the weight-average molecular mass measured: the main fraction at 1.95-2.35 x 10(5) g/mol and a less-abundant fraction with aggregates greater than 1.50 x 10(6) g/mol. High-performance size-exclusion chromatography in conjunction with multi-angle laser light scattering detection revealed that the BSA-dextran conjugates prepared by using the Maillard reaction had various molar masses and radii.

Spectroscopic Study on the Interaction between Bovine Serum Albumin and Tween‐20

The interaction between nonionic surfactant Tween-20 and bovine serum albumin (BSA) is studied in Tris‐HCl buffer solution by spectroscopic methods. The intrinsic fluorescence of BSA is quenched by the addition of Tween‐20. The UV‐visible absorption spectra and the synchronous fluorescence spectra show that the addition of Tween‐20 changes the polarity of the environment around tryptophan (trp) residues of BSA. The fraction of trp residues on the surface of BSA with and without Tween- 20 is calculated via I− quenching experiments.

Protein adsorption properties on a microfiltration membrane: A comparison between static and dynamic adsorption methods

The effects of volumetric flow on the adsorption properties of bovine serum albumin (BSA) on the pore surface of sirasu porous glass (SPG) membrane having a pore size of 100 nm were studied by comparing the static adsorption method with the dynamic adsorption method, i.e. membrane filtration. The adsorption isotherm for the static adsorption was a Langmuir type while that for the dynamic adsorption was a multilayer type. The dynamic adsorption behavior was observed with the membrane pre-adsorbed by the static adsorption and it was shown that the flow in pore would cause the multilayer adsorption. The effects of the pre-adsorption on the dynamic adsorption behavior could be explained in terms of the surface coverage of pore surface by BSA and the electrostatic interaction between BSA and pore surface, which can be estimated by the zeta potential measurement. The change ratio of the zeta potential, | ζ stat − ζ 0|/| ζ dyn − ζ 0|, after the static adsorption was directly proportional to the surface coverage of BSA.

Interactions of adsorbed albumin with underpotentially deposited copper on gold piezoelectrodes

The adsorption of a model protein, bovine serum albumin (BSA), on Au electrodes was investigated using the Cu adatom probe method and Electrochemical Quartz Crystal Nanobalance (EQCN) technique. The adsorption of BSA was confirmed by AFM imaging and has been found to be controlled by kinetics. Using the Cu adatom probe method, we were able to reconstruct the entire BSA adsorption transient Θ BSA vs. t. The adsorption rate constant k 1, determined from this transient is k 1 = 2.45 × 10 5 L mol − 1 s − 1 . We have found that the bulk Cu 0 deposition process is blocked by BSA adsorption and it decays exponentially with time during BSA adsorption. It ceases completely when a full monolayer of BSA is formed. In contrast to that, the mass associated with Cu-u.p.d. decreases only to ca. 50% of that in the absence of BSA, indicating that Cu adatoms can penetrate (wedge) into the space between the surface Au atoms and the adsorbed BSA molecules. In addition to that, we have found that the degree of penetration of Cu adatoms can be controlled by the applied deposition potential. By selecting a sufficiently cathodic potential, we were able to deposit a full Cu-u.p.d. monolayer, independent of the BSA surface coverage extending from Θ BSA = 0 to Θ BSA ≈ 1. The positive shift of Cu ad desorption peak potential E p, observed in the presence of adsorbed BSA, has been interpreted in terms of Frumkin exchange interaction forces between Cu ad and BSA ad, on the basis of our earlier theoretical model, expanded here to include adsorbed species in two monolayers. This expansion is possible owing to the fast rate of Cu adatom penetration in the interfacial region. From the plots of E p vs. Θ BSA, the presence of strong attractive interactions between Cu ad and BSA ad was deduced. These interactions result in a super-shift of the Cu-u.p.d. desorption peak potential, corresponding to the exchange interaction coefficient g M,X < − 4, indicating on a possibility of the formation of a stable interface complex.

Properties of protein adsorption onto pore surface during microfiltration: Effects of solution environment and membrane hydrophobicity

Properties of bovine serum albumin (BSA) adsorption onto pore surface during the filtration of BSA containing solution with the Sirasu porous glass membrane with a pore size of 0.1 μm were studied. The effects of pH, ionic strength, and surface modification on the flux decline and breakthrough curves were observed. The adsorption properties of BSA were estimated quantitatively by using the internal fouling model, which relates the filtration performance to the adsorption interaction, the adsorption capacity, and the thickness of the adsorption layer. The electrostatic interaction between BSA and pore surface was estimated by the streaming potential measurement. The BSA adsorption involved a rapid adsorption in the early stage of filtration followed by a slow multilayer adsorption that dominates the long-term filtration performance. The electrostatic repulsive force reduced the overall adsorption interaction but the electrostatic attractive force did not affect the adsorption interaction. The effect of ionic strength on the BSA adsorption could be explained in terms of the shift of the IEP of BSA toward lower pH with the increase in ionic strength. The hydrophobicity of membrane did not affect the adsorption properties except for the adsorption interaction in the early stage of the filtration.

Dielectric film for biosensor application

The surface of dielectric barium strontium titanate (BST) film was modified by a self-assembled monolayer (SAM) of mono-dodecyl phosphate (DDPO 4) from aqueous solutions of alkyl phosphate ammonium salts. The phosphate terminal groups were believed to anchor to the Ti atoms through oxygen bridges with the terminal methyl groups exposed outwardly, resulting in a highly hydrophobic surface with an advancing water contact angle (CA) of 99.8°, compared with the original CA 40.8° of the bare BST surface. X-ray photoelectron spectroscopy (XPS) showed the dense coverage of the SAM and immobilized BSA layer. The microscopic features and the biosensing properties were characterized by atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS) in the presence of [Fe(CN) 6] 3−/4− as a redox couple. AFM images revealed that bovine serum albumin (BSA) was well-immobilized on the DDPO 4-modified BST surface. The EIS showed a significant increase in the electron-transfer resistance. Specific binding of anti-BSA on the immobilized BSA surface was evidenced by AFM imaging and EIS. It was demonstrated that the specific binding of anti-BSA can be quantitatively detected by measuring the electron-transfer resistance. The investigation indicated the potential application of the dielectric film for electrochemical biosensors.