Bovine Serum Albumin Surface Research Articles

Mechanism of interaction of vincristine sulphate and rifampicin with bovine serum albumin: A spectroscopic study

The mechanism of interaction of vincristine sulphate (VS) and rifampicin (RF) with bovine serum albumin (BSA) has been studied by quenching of BSA fluorescence by RF/VS. The Stern-Volmer plot indicates the presence of a static component in the quenching mechanism. Results also show that both the tryptophan residues of BSA are accessible to VS and RF. The high magnitude of rate constant of quenching indicates that the process of energy transfer occurs by intermolecular interaction and VS/RF-binding site is in close proximity to the tryptophan residues of BSA. Binding studies in the presence of a hydrophobic probe, 8-anilino-1-naphthalene-sulphonic acid sodium salt (ANS) indicate that the VS and RF compete with ANS for hydrophobic sites on the surface of BSA. Small decreases in critical micellar concentrations (CMC) of anionic surfactants in presence of VS/ RF show that the ionic character of VS/RF also contributes to binding. The temperature dependence of the association constant is used to estimate the values of the thermodynamic parameters involved in the interaction of VS/RF with BSA and the results indicate that hydrophobic forces play a significant role in the binding. Circular dichroism studies reveal that the change in helicity of BSA are due to binding of VS/RF to BSA.

Determination of bovine serum albumin by a resonance light-scattering technique with the mixed-complex La(Phth)(phen) 3+

A mixed-complex of La(Phth)(phen) 3+ was synthesized. Resonance light-scattering characteristics of interaction between La(Phth)(phen) 3+ with bovine serum albumin (BSA) were studied. When BSA was added, aggregation of La(Phth)(phen) 3+ on the molecular surface of bovine serum albumin occurred in the pH 5.5–6.3, resulted in an enhanced resonance light-scattering (RLS) peak at 360 nm. The intensity of resonance light-scattering was found to be proportional to the concentration of BSA.

Residence Time, Loading Force, pH, and Ionic Strength Affect Adhesion Forces between Colloids and Biopolymer-Coated Surfaces

Exopolymers are thought to influence bacterial adhesion to surfaces, but the time-dependent nature of molecular-scale interactions of biopolymers with a surface are poorly understood. In this study, the adhesion forces between two proteins and a polysaccharide [Bovine serum albumin (BSA), lysozyme, or dextran] and colloids (uncoated or BSA-coated carboxylated latex microspheres) were analyzed using colloid probe atomic force microscopy (AFM). Increasing the residence time of an uncoated or BSA-coated microsphere on a surface consistently increased the adhesion force measured during retraction of the colloid from the surface, demonstrating the important contribution of polymer rearrangement to increased adhesion force. Increasing the force applied on the colloid (loading force) also increased the adhesion force. For example, at a lower loading force of approximately 0.6 nN there was little adhesion (less than -0.47 nN) measured between a microsphere and the BSA surface for an exposure time up to 10 s. Increasing the loading force to 5.4 nN increased the adhesion force to -4.1 nN for an uncoated microsphere to a BSA surface and to as much as -7.5 nN for a BSA-coated microsphere to a BSA-coated glass surface for a residence time of 10 s. Adhesion forces between colloids and biopolymer surfaces decreased inversely with pH over a pH range of 4.5-10.6, suggesting that hydrogen bonding and a reduction of electrostatic repulsion were dominant mechanisms of adhesion in lower pH solutions. Larger adhesion forces were observed at low (1 mM) versus high ionic strength (100 mM), consistent with previous AFM findings. These results show the importance of polymers for colloid adhesion to surfaces by demonstrating that adhesion forces increase with applied force and detention time, and that changes in the adhesion forces reflect changes in solution chemistry.

Effects of collagen matrix on Sindbis virus infection of BHK cells

The effects of extracellular matrix (ECM) components on the outcomes of alphavirus interaction with cells are not known. Studies that address such interactions have to address several methodological difficulties, including: the survival of the cells within the matrix; the passage of the virus through the matrix to infect embedded cells; and the dissociation of cells and matrix into single-cell suspension, before and after virus infection, for quantitative analysis. Herein, these issues were addressed in the context of a model system of collagen as the ECM component, baby hamster kidney (BHK) cells, and Sindbis virus. The outcomes of Sindbis virus infection of BHK cells, grown in three-dimensional (3D) collagen gel versus on plastic, and on two-dimensional (2D) collagen versus bovine serum albumin (BSA)-coated surfaces were compared. Cell morphology was more slender in 3D and on 2D collagen than on plastic or BSA-coated surfaces. The cells were able to survive in the 3D environment. Using Sindbis virus carrying the green fluorescent protein gene, the virions were found to be capable of penetrating the 3D collagen matrix and infecting the cells. There was more infectious virus in cultures of cells in 3D and on 2D collagen than on plastic or BSA-coated surfaces, respectively. Higher virus titers from cells on 2D collagen compared to BSA-coated surfaces was not associated with uninfected cell number or viability but with increased cell survival after infection. Infected cells on BSA surfaces became detached, while those on 2D collagen remained attached. These experiments establish procedures for analysis of interaction of collagen, BHK cells, and Sindbis virus and suggest that collagen increases infectious Sindbis virus titers from BHK cells by enhancing post-infection cell survival.

Effect of albumin on the kinetics of ascorbate oxidation

The fluorescence intensity of the fluorophore in dansyl piperidine-nitroxide is intramolecularly quenched by the nitroxyl fragment. Therefore, the oxidation of ascorbic acid by the fluorophore-nitroxide (FN) probe can be monitored by two independent methods: steady-state fluorescence and electron paramagnetic resonance. Bovine serum albumin (BSA) affects the rate of this reaction. The influence of BSA on the rate is attributed to the adsorption of both ascorbate and the probe to BSA. Adsorption of ascorbate to BSA is confirmed by NMR relaxation experiments. The spatial distribution of the molecules on the BSA surface changes the availability of ascorbate and FN to each other. The results also point out that, in the presence of BSA, the autoxidation of ascorbate is significantly slowed down. The effect is studied at different pH values and explained in terms of the electrostatic interaction between the ascorbate anion and the BSA molecule.

Vapor pressure osmometry studies of osmolyte-protein interactions: implications for the action of osmoprotectants in vivo and for the interpretation of "osmotic stress" experiments in vitro.

To interpret or to predict the responses of biopolymer processes in vivo and in vitro to changes in solute concentration and to coupled changes in water activity (osmotic stress), a quantitative understanding of the thermodynamic consequences of interactions of solutes and water with biopolymer surfaces is required. To this end, we report isoosmolal preferential interaction coefficients (Gamma(mu1) determined by vapor pressure osmometry (VPO) over a wide range of concentrations for interactions between native bovine serum albumin (BSA) and six small solutes. These include Escherichia coli cytoplasmic osmolytes [potassium glutamate (K(+)Glu(-)), trehalose], E. coli osmoprotectants (proline, glycine betaine), and also glycerol and trimethylamine N-oxide (TMAO). For all six solutes, Gamma(mu1) and the corresponding dialysis preferential interaction coefficient Gamma(mu1),(mu3) (both calculated from the VPO data) are negative; Gamma(mu1), (mu3) is proportional to bulk solute molality (m(bulk)3) at least up to 1 m (molal). Negative values of Gamma(mu1),(mu3) indicate preferential exclusion of these solutes from a BSA solution at dialysis equilibrium and correspond to local concentrations of these solutes in the vicinity of BSA which are lower than their bulk concentrations. Of the solutes investigated, betaine is the most excluded (Gamma(mu1),(mu3)/m(bulk)3 = -49 +/- 1 m(-1)); glycerol is the least excluded (Gamma(mu1),(mu3)/m(bulk)3 = -10 +/- 1 m(-1)). Between these extremes, the magnitude of Gamma(mu1),(mu3)/m(bulk)3 decreases in the order glycine betaine >> proline >TMAO > trehalose approximately K(+)Glu(-) > glycerol. The order of exclusion of E. coli osmolytes from BSA surface correlates with their effectiveness as osmoprotectants, which increase the growth rate of E. coli at high external osmolality. For the most excluded solute (betaine), Gamma(mu1),(mu3) provides a minimum estimate of the hydration of native BSA of approximately 2.8 x 10(3) H(2)O/BSA, which corresponds to slightly less than a monolayer (estimated to be approximately 3.2 x 10(3) H(2)O). Consequently, of the solutes investigated here, only betaine might be suitable for use in osmotic stress experiments in vitro as a direct probe to quantify changes in hydration of protein surface in biopolymer processes. More generally, however, our results and analysis lead to the proposal that any of these solutes can be used to quantify changes in water-accessible surface area (ASA) in biopolymer processes once preferential interactions of the solute with biopolymer surface are properly taken into account.

Phase and structural transitions in vicinal water on protein surfaces by means of the DSC

This paper presents investigations of phase and structural transitions occurring in water adsorbed on the surface of bovine serum albumin (BSA) and on the so-called 'intelligent' or 'smart' silica gel surface covered with a chemically bonded BSA phase. Cyclic changes of heat flow (HF) were observed in the samples studied during cooling and heating of the measuring cell of the differential scanning calorimetry (DSC) apparatus. These cyclic changes reflect structural transitions occurring in the water adsorbed on the surface at subambient and elevated temperatures. This is connected with cyclic changes (decay and reproduction) of ice-like structures existing in the adsorbed water layers. On the basis of quantitative investigations it appears that, depending on the direction of the cooling or heating process of the samples studied, the number of ice-like water structures in the surface film increases or decreases. It has been stated that the observed fluctuations occur spontaneously and suddenly in the whole volume of adsorbed water in different and not regular temperature ranges, especially at the 'paradoxical effect' temperatures.

The Osmotic Pressure of Electrostatically Stabilized Colloidal Dispersions

A mathematical model for predicting the osmotic pressure of electrostatically stabilized colloids has been developed. The model is based on detailed descriptions of the colloidal interactions within an electrostatically stabilized dispersion. Electrostatic interactions are accounted for by a Wigner–Seitz cell approach including a numerical solution of the nonlinear Poisson–Boltzmann equation. London–van der Waals forces are calculated using a computationally efficient means of approximating screened, retarded Lifshitz–Hamaker constants. Configurational entropy effects are calculated using an equation of state giving excellent agreement with molecular dynamic data. These descriptions of colloidal interactions are used to develop ana priorimodel, with no adjustable parameters, that allows quantitative prediction of the osmotic pressure of colloidal dispersions as a function of ζ potential (and hence pH), colloid size, ionic strength, and colloid concentration. The model shows good agreement with literature experimental data for the osmotic pressure of the protein bovine serum albumin (BSA). A charge regulation model for the BSA surface has also been developed from knowledge of the amino acid groups giving rise to the protein charge. A comparison of ζ potentials calculated from this model with experimentally determined values for dilute BSA dispersions shows good agreement for a wide range of conditions. The charge regulation model has also been incorporated into the osmotic pressure prediction, resulting in excellent agreement between theory and experiment.

Covalent complexes between serum albumin and 7-hydroxycoumarin-4-acetic acid: synthesis and applications in the spectrophotometric detection of long-chain fatty acids

Using a hydrophobic 8-aminooctanoic acid cross-linker, the pH-indicator dye 7-hydroxycoumarin-4-acetic acid (7-HCA) is covalently bound to bovine serum albumin (BSA) at the positions of reactive amino groups. A highly stable and water-soluble complex (BSA-HCA) with a 1:4 molar stoichiometry is synthesized. Appearance of a strong absorption band at λ max = 372 nm is associated to ionization of the 7-HCA chromophore when it is transferred from water into a basic microenvironment on the BSA surface. This particular surface site is related to the region(s) for high-affinity binding of long-chain fatty acids (FA). BSA-HCA responds to binding of FA (14–20 carbons) with immediate spectral changes and a decrease in 372 nm absorption. BSA-HCA provides an indicator-protein having a range of practical applications for the quantitative determination of long-chain FA in biochemical studies. The lower detection limit in a spectrophotometric method is ≈ 1 μM FA. BSA-HCA is usable both in various buffers and in the presence of detergents such as n-octylglucoside, Triton X-100 and CHAPS. A novel method for continuous assay of phospholipase A 2 activity with BSA-HCA and a mixed phosphatidylcholine/CHAPS micellar substrate is reported.

Thermodynamic characterization of interactions of native bovine serum albumin with highly excluded (glycine betaine) and moderately accumulated (urea) solutes by a novel application of vapor pressure osmometry.

The thermodynamic consequences of interactions of native bovine serum albumin (BSA) with two smaller solutes (glycine betaine or urea) in aqueous solution are characterized by a novel application of vapor pressure osmometry (VPO), which demonstrates the utility of this method of investigating preferential interactions involving solutes that are either accumulated or excluded near the surface of a protein. From VPO measurements of osmolality (water activity) as a function of the solute concentration in the presence and absence of BSA, we determine the dependence of the solute molarity (C3) on that of BSA (C2) at fixed temperature (37 degrees C), pressure (approximately 1 atm), and osmolality (over the range 0-1.6 molal). After some thermodynamic transformations, these results yield values of [formula: see text] which characterizes the interdependence of solute molalities when temperature, pressure, and the chemical potential of solute 3 are fixed. This form of the preferential interaction coefficient can be interpreted directly in terms of the molecular exclusion or accumulation of the solute (relative to water) near the protein surface. Within experimental uncertainty, [formula: see text] is proportional to m3 both for glycine betaine (0-0.9 m) and for urea (0-1.6 m). For glycine betaine [formula: see text] = -49 +/- 4, a value consistent with the interpretation that this solute is completely excluded from the hydrated surface of BSA, whereas for urea [formula: see text] = 6 +/- 1, which indicates a moderate extent of accumulation at the surface of native BSA. The preferential accumulation of solutes (e.g., urea) that have some binding affinity for a protein can be quantified and interpreted using the two-domain model if the extent of hydration of the protein has been determined using a completely excluded solute (e.g., glycine betaine). Complete exclusion from the local hydration domain surrounding proteins, if general, justifies the use of glycine betaine as a thermodynamic probe of the changes in hydration that accompany protein folding, protein association, and protein-ligand binding interactions.

Binding of rose bengal onto bovine serum albumin

The binding of rose bengal (RB) to bovine serum albumin (BSA) occurs with both the folded (atpH 7·4) and the unfolded (pH 12·7) forms of BSA. Absorption spectroscopy has revealed an identical red-shift of 15nm in λmax of RB in presence of BSA both atpH 7·4 and 12·7. The affinity constants (K) atpH 12·7 have been reduced only by 50% in magnitude from those atpH 7·4. These lead us to infer that neither disulphide loops nor buried residues are involved but that the binding of RB occurs at the sites near the surface of BSA. Moreover, the drastic alterations in the near-UV circular dichroism suggest tertiary structural changes induced by RB on binding to BSA. The conformational changes at the binding sites of BSA atpH 7·4 and the affinity of RB particularly towards the exposed residues in BSA atpH 12·7 are the significant factors in the binding of RB to BSA.

Surface Diffusion of Adsorbed Proteins in the Vicinity of the Substrate Glass Transition Temperature

We report an investigation of the role of substrate thermal motions in the mechanism of adsorbed protein surface diffusion on polymeric materials. Bovine serum albumin (BSA) surface diffusion coefficients (Ds) are measured by fluorescence recovery after photobleaching. Polymeric substrate mobility is controlled via temperature changes in the vicinity of the glass transition temperature (Tg) of polybutylmethacrylate (PBMA). Several empirical surface energy parameters of PBMA are shown to be similar to those of polyhexylmethacrylate (PHMA). As the glass transition of PHMA occurs well below the experimental temperature range, the temperature dependence of the BSA surface diffusion coefficient on PHMA serves as a transition-free baseline for comparison to that on PBMA as it passes through its glass transition. The temperature dependences of Ds on PBMA and PHMA implicate substrate polymer chain motions in the mechanism of protein transport.

Rotational reorientation kinetics of dansylated bovine serum albumin

Time-resolved fluorescence intensity and anisotropy decays of dansylated bovine serum albumin (BSA) are investigated by multifrequency phase and modulation fluorescence spectroscopy. We found that a double exponential decay law best describes the fluorescence intensity decay of covalently attached dansyl to BSA. The short lifetime component is attributed to dansyl located at the exterior surface of BSA. The longer-lived component reflects dansyl at the interior of BSA. This result indicates that there are two dansyl-BSA populations in the ground state. An associated model is then found to best describe the anisotropy decay kinetics of dansylated BSA. This result is consistent with the observed ground-state heterogeneity

Protein Adsorption to HEMA/EMA Copolymers Studied by Integrated Optical Techniques

Adsorption of chromophore-labeled bovine serum albumin (BSA) to homopolymers and copolymers of hydroxyethyl methacrylate (HEMA) and ethyl methacrylate (EMA) was measured using integrated optical waveguide attenuated total reflection (IOW-ATR) spectrometry. In IOW-ATR, the intrinsic propagation loss of the waveguide, usually on the order of 1 dB/cm, is increased by the adsorption of a chromophoric molecule to the waveguide/solution interface. Measuring the difference in propagation losses before and after protein adsorption allows one to calculate the amount of protein adsorption (e.g., ng/cm2) directly from the Beer-Lambert law without calibration. Adsorbed amounts ranged from 35 ± 2 to 684 ± 55 ng/cm2 for the HEMA and EMA homopolymers, respectively. Radiolabeling experiments of 125I-BSA adsorption to the HEMA/EMA polymers performed in tandem correlated strongly with the IOW-ATR results (R2 = 0.934). Except for the EMA homopolymer, both techniques showed BSA surface densities that increased linearly with decreasing mol% HEMA of the polymer.

Excited state proton transfer in 2-naphthol derivatives bound to selected sites of proteins

Derivatives of 2-naphthol-6-sulphonic acid were anchored by covalent bonding to the active site of papain and to the surface of bovine serum albumin. The rate constants of excited state proton transfer for the introduced fluorophoric groups were determined by steady state fluorescence spectroscopy and compared with the data for low-molecular-weight analogues of 2-naphthol. In the light of these results and other data published previously, it is concluded that the fluorophores bound inside macromolecules are, in most cases, characterized by a higher rate of excited state proton transfer than groups situated on the surface. The unique physicochemical properties of the enzyme active site are taken into consideration in the case of the papain conjugate.

The elasticities of spread monolayers of bovine serum albumin and of ovalbumin.

The elasticities of monolayers of bovine serum albumin (BSA) and of ovalbumin (OA) have been measured directly by determining the changes in surface tension caused by sinusoidal compression-expansion cycles of small amplitude. Below a surface pressure (π) of about 5 dynes/cm, the initial elasticities of both monolayers agree with the values determined from the slope of the BSA surface pressure-area isotherm. At higher pressures, the measurements are erratic, although they are still in the same range as the results from the isotherm. Continued deformation at small amplitude of both BSA and OA monolayers results in no changes in elasticity for films below π = 5 dynes/cm, whereas above this pressure the elasticity of BSA increases and that of OA decreases. Deformation at higher amplitude of BSA monolayers causes the elasticity to increase more markedly as a function of time, at all surface pressures. The presence of (0.1%) 1-butanol in the subphase results in a decrease of the elasticity of BSA films at all pressures. These results provide new evidence regarding intermolecular interactions in protein films, specifically with regard to the questions of reversibility and collapse.