Water Bovine Serum Albumin Research Articles

Recovering What Matters: High Protein Recovery after Endotoxin Removal from LPS-Contaminated Formulations Using Novel Anti-Lipid A Antibody Microparticle Conjugates.

Endotoxins or lipopolysaccharides (LPS), found in the outer membrane of Gram-negative bacterial cell walls, can stimulate the human innate immune system, leading to life-threatening symptoms. Therefore, regulatory limits for endotoxin content apply to injectable pharmaceuticals, and excess LPS must be removed before commercialization. The majority of available endotoxin removal systems are based on the non-specific adsorption of LPS to charged and/or hydrophobic surfaces. Albeit effective to remove endotoxins, the lack of specificity can result in the unwanted loss of essential proteins from the pharmaceutical formulation. In this work, we developed microparticles conjugated to anti-Lipid A antibodies for selective endotoxin removal. Anti-Lipid A particles were characterized using flow cytometry and microscopy techniques. These particles exhibited a depletion capacity > 6 ×103 endotoxin units/mg particles from water, as determined with two independent methods (Limulus Amebocyte Lysate test and nanoparticle tracking analysis). Additionally, we compared these particles with a non-specific endotoxin removal system in a series of formulations of increasing complexity: bovine serum albumin in water < insulin in buffer < birch pollen extracts. We demonstrated that the specific anti-Lipid A particles show a higher protein recovery without compromising their endotoxin removal capacity. Consequently, we believe that the specificity layer integrated by the anti-Lipid A antibody could be advantageous to enhance product yield.

Catechol-Based Polymers with High Efficacy in Cytosolic Protein Delivery

Catechol-Based Polymers with High Efficacy in Cytosolic Protein Delivery

Aspects of green chemistry toward synthesis of benzazoles and quinoxalines: Bovine serum albumin-water system for C–N/C–S bond formation

In this report, a biocatalytic divergent approach of therapeutically important five-membered benzazole heterocyclic compounds such as benzothiazoles and benzimidazoles has been described wherein Bovine serum albumin (BSA) and water play cooperative role for oxidative cyclo-condensation reaction between 2-mercapto/amino anilines and aromatic aldehydes under air at room temperature. Further, this green strategy also extended for the synthesis of privileged and biologically important six membered quinoxalines, 2-phenylimidazo [1,2-a]pyridine and benzo[d]imidazo[2,1-b]thiazoles. Moreover, the present biocatalytic protocol for the synthesis of such five- and six-membered heterocyclic compounds also have the added benefits of high atom economy, operational simplicity, excellent yields, recyclability (up to four cycles), and gram scalability.

Dielectric relaxations of ice and uncrystallized water in partially crystallized bovine serum albumin-water mixtures.

To investigate the dielectric relaxations of ice in low-concentration protein-water mixtures, broadband dielectric spectroscopy measurements were performed on partially crystallized bovine serum albumin (BSA)-water mixtures with BSA concentrations of 1-10 wt% at temperatures in the range of 123-298 K. The temperature dependence of the relaxation time of ice observed in all these mixtures changes twice at TC1 (∼240 K) and TC2 (200-160 K) (TC1 > TC2), i.e., at which the apparent activation energy, Ea, changes. Below 200 K, the relaxation of ice separates as 3-4 relaxations with different TC2 and Ea values. The presence of the multiple ice relaxations is the same as that observed for the gelatin-water mixtures (T. Yasuda, K. Sasaki, R. Kita, N. Shinyashiki and S. Yagihara, J. Phys. Chem. B, 2017, 121, 2896), but the concentration dependences of TC1 and TC2 are different. The relaxation interpreted to be due to uncrystallized water in 20 wt% and 40 wt% BSA-water mixtures reported (N. Shinyashiki, W. Yamamoto, A. Yokoyama, T. Yoshinari, S. Yagihara, R. Kita, K. L. Ngai and S. Capaccioli, J. Phys. Chem. B, 2009, 113, 14448) was re-examined and concluded to be due to one of the multiple relaxations of ice.

Development of Water Solubility of 2-Phenylsulfanylhydroquinone Dimer Dye.

With the aim of developing a new fluorescence dye with enhanced photophysical properties, this study describes the modification of the 2-phenylsulfanylhydroquinone dimer to realize a new bioimaging molecule. The characteristics of the dimer were advanced by introducing tetraethylene glycol side chains to provide sufficient water solubility and a tether consisting of an N-hydroxysuccinimide-terminated C6-carbon chain to attach bioactive molecules. Two derivatives containing two or three tetraethylene glycol side chains were designed and prepared, and the latter showed sufficient water solubility for biochemical applications. Both compounds exhibited similar photophysical properties and blue fluorescence under UV light irradiation. The dye containing three tetraethylene glycol units reacted with bovine serum albumin in water to give fluorescent derivatives.

Experimental Validation of Mass Balance Models for in Vitro Cell-Based Bioassays.

The freely dissolved concentration in the assay medium (Cfree) and the total cellular concentration (Ccell) are essential input parameters for quantitative in vitro-to-in vivo extrapolations (QIVIVE), but available prediction tools for Cfree and Ccell have not been sufficiently validated with experimental data. In this study, medium-water distribution ratios (DFBS/w) and cell-water distribution ratios (Dcell/w) for four different cells lines were determined experimentally for 12 neutral and five ionizable chemicals. Literature data for seven organic acids were added to the dataset, leading to 24 chemicals in total. A mass balance model based on bovine serum albumin-water (DBSA/w) and liposome-water distribution ratios (Dlip/w) of the chemicals was used to calculate DFBS/w and Dcell/w. For all neutral and basic test chemicals, the mass balance model predicted DFBS/w and Dcell/w within a factor of 3 and 3.4, respectively, indicating that existing models can reliably predict Cfree and Ccell for these chemicals. For organic acids, a further refinement of the model will be required as large deviations between modeled and measured binding to assay medium and cells of up to a factor of 370 were found. Furthermore, saturation of medium proteins should be further explored for organic acids and neutral chemicals with moderate hydrophobicity.

Protein Resistance Driven by Polymer Nanoarchitecture.

We report that the nanometer-scale architecture of polymer chains plays a crucial role in its protein resistant property over surface chemistry. Protein-repellent (noncharged), few nanometer thick polymer layers were designed with homopolymer chains physisorbed on solids. We evaluated the antifouling property of the hydrophilic or hydrophobic adsorbed homopolymer chains against bovine serum albumin in water. Molecular dynamics simulations along with sum frequency generation spectroscopy data revealed the self-organized nanoarchitecture of the adsorbed chains composed of inner nematic-like ordered segments and outer brush-like segments across homopolymer systems with different interactions among a polymer, substrate, and interfacial water. We propose that this structure acts as a dual barrier against protein adsorption.

Aluminum film over nanosphere surface for Deep Ultraviolet plasmonic nanosensors

Deep ultraviolet (DUV) plasmonic nanosensors are emerging as potential candidates to detect biomolecules with enhanced sensitivity, as these molecules strongly absorb light in the DUV region. In the present work, we fabricated aluminium film over nanospheres surface (AlFON) and successfully demonstrated, for the first time, an AlFON based DUV plasmonic nanosensor for detecting volatile organic compounds (VOCs) such as ethanol, isopropyl alcohol (IPA) and toluene, and bovine serum albumin (BSA) protein. The AlFON was fabricated by thermally evaporating aluminium thin film onto a two-dimensional photonic crystal (2DPC). The 2DPC was prepared by drop casting colloidal polystyrene spheres (PSS) on quartz substrates that self-assembled to form a monolayer of hexagonally close packed PSS with periodic interstices. The optical reflection spectrum of 70 nm AlFON exhibited a sharp minimum at 289 nm in the DUV wavelength range which corresponds to strong absorption in V-shaped nanocavities due to excitation of localized cavity plasmons. The reflection minimum—or LSPR wavelength—is sensitive to the changes in the refractive index of the surrounding medium. The LSPR mode of the V-shaped nanocavities in 70 nm AlFON was exploited to sense VOCs and BSA. The 70 nm AlFON exhibited 244 nm/RIU sensitivity in detecting VOCs and was capable of sensing 1 mM concentration of BSA in water. The AlFON is physically stable and has a self-limiting Al2O3 oxide layer of less than 5 nm thickness as elucidated from x-ray photoelectron spectroscopy measurements. The effect of higher aluminum film thickness, i.e. 100 nm and 120 nm on the sensitivity of AlFON also was studied. The performance of AlFON based DUV plasmonic nanosensors is better than the previously reported Al nanostructure based nanosensors and can still be enhanced by optimizing the structure of 2DPC and AlFON.

Transforming diatomaceous earth into sensing devices by surface modification with gold nanoparticles

Abstract Diatomaceous earth, or diatomite, is produced through the accumulation of diatom (Bacillariophyceae) skeletons (i.e. cell walls called frustules) made of amorphous silica. The porous, highly symmetrical structure and microscopic size of diatom cell walls make them ideal constituents of sensing devices and analytical chips. Here, we propose chemical methods to purify diatom frustules extracted from diatomaceous earth. Using photo deposition techniques, we grow gold nanoparticles on the surface of diatom skeletons and within the pores of the skeletons, where the size and density of nanoparticles can be controlled by changing the parameters of the synthesis. Resulting devices have an internal porous structure that can harvest molecules from a solution, and an external shell of gold nanoparticles that amplifies the electromagnetic field generated by the measurement laser in Raman or other spectroscopies. The combination of these effects enables the analysis of biological specimens, chemical analytes and pollutants in extremely low abundance ranges. The devices were demonstrated in the analysis of Bovine Serum Albumin in water with a concentration of 100 aM, and mineral oil with a concentration of 50 ppm.

Calorimetric studies of interactions between low molecular weight salts and bovine serum albumin in water at pH values below and above the isoionic point

Isothermal titration calorimetry was used to determine the temperature and salt concentration dependence of the enthalpy of mixing, ΔmixH, of bovine serum albumin (BSA) in aqueous buffer solutions with several low molecular weight salts. Three buffers were used: acetate (pH=4.0), MOPS (7.2), and borate (9.2). Since the isoionic point of BSA is at pI ≈ 4.7, the net charge of BSA in acetate buffer was positive (≈ +20), while in the other two buffer solutions it was negative (≈−15 in MOPS and ≈−25 in borate). The majority of the recorded heat effects were exothermic, while only at pH=9.2 a weak endothermic effect upon mixing BSA with LiCl, NaCl, and KCl was observed. For all buffer solutions the absolute values of ΔmixH of sodium salts followed the order: NaCl < NaBr < NaNO3< NaI < NaSCN, which is the reverse Hofmeister series for anions. The magnitude of the effects was the largest in acetate buffer and decreased with an increasing pH value of the solution. While the effect of varying the anion of the added salts was strongly pronounced at all pH values, the effect of the cation (LiCl, NaCl, KCl, RbCl and CsCl salts) was weak. The most interesting feature of the results obtained for pH >pI was the fact that ΔmixH were considerably more sensitive to the anion (co-ion to the net BSA charge) than to the cation species. This indicated that anions interacted quite strongly with the BSA even at pH values where the net charge of the protein was negative. We showed that ΔmixH at high addition of salts correlated well with the enthalpy of hydration of the corresponding salt anion. This finding suggested, consistently with some previous studies, that a part of the exothermic contribution to ΔmixH originated from the hydration changes upon the protein–salt interaction. Theoretical analysis, based on the primitive model of highly asymmetric electrolyte solutions solved within the mean spherical approximation, was used to estimate Coulomb effects upon mixing.

Competitive binding affinity of two lanthanum(III) macrocycle complexes toward DNA and bovine serum albumin in water

In the present study, two water-soluble lanthanum(III) hexaaza Schiff base complexes were synthesized and characterized and also theoretically investigated. The interactions of these complexes with DNA and bovine serum albumin (BSA) were studied using different spectroscopic assessments and docking simulation analysis. The DNA docking studies suggested that these two complexes are able to interact with DNA through the minor groove, and also the binding affinity is in the order of La(L1) > La(L2). Furthermore, the spectral titration was carried out and viscosity measurements were taken. In this regard, protein-binding studies revealed that these complexes quench the intrinsic fluorescence of BSA, and indicated that the possible binding site is located on the vicinity of Trp 213, which is further validated by docking simulation analysis. The in vitro anticancer activities of these complexes indicated that the La(L1) complex is more effective than the other one and also exhibits a better interaction with DNA.

QSPR models for prediction of bovine serum albumin-water partition coefficients of organic compounds and drugs based on enhanced replacement method and support vector machine

A quantitative structure property relationship (QSPR) study based on enhanced replacement method (ERM) and support vector machine (SVM) was used to correlate molecular structures to their bovine serum albumin water partition coefficients (KBSA/W). A wide variety of natural organic compounds and drugs were selected as a dataset and suitable sets of molecular descriptors were calculated using Dragon package. ERM was used as variable selection method. The nonlinear-support vector machine models were applied to correlate the ERM-selected molecular descriptors with the experimental values of KBSA/W. Results obtained demonstrate the reliability and good predictability of support vector machine model to predict KBSA/W of organic compounds and drugs. Satisfactory results demonstrate that the ERM approach is a very powerful method for variable selection and the predictive ability of the SVM model is superior to those acquired by ERM.

Development of bovine serum albumin-water partition coefficients predictive models for ionogenic organic chemicals based on chemical form adjusted descriptors

The partition coefficients between bovine serum albumin (BSA) and water (KBSA/w) for ionogenic organic chemicals (IOCs) were different greatly from those of neutral organic chemicals (NOCs). For NOCs, several excellent models were developed to predict their logKBSA/w. However, it was found that the conventional descriptors are inappropriate for modeling logKBSA/w of IOCs. Thus, alternative approaches are urgently needed to develop predictive models for KBSA/w of IOCs. In this study, molecular descriptors that can be used to characterize the ionization effects (e.g. chemical form adjusted descriptors) were calculated and used to develop predictive models for logKBSA/w of IOCs. The models developed had high goodness-of-fit, robustness, and predictive ability. The predictor variables selected to construct the models included the chemical form adjusted averages of the negative potentials on the molecular surface (Vs−adj−), the chemical form adjusted molecular dipole moment (dipolemomentadj), the logarithm of the n-octanol/water distribution coefficient (logD). As these molecular descriptors can be calculated from their molecular structures directly, the developed model can be easily used to fill the logKBSA/w data gap for other IOCs within the applicability domain. Furthermore, the chemical form adjusted descriptors calculated in this study also could be used to construct predictive models on other endpoints of IOCs.

Development and evaluation of predictive model for bovine serum albumin-water partition coefficients of neutral organic chemicals

The binding of organic chemicals to serum albumin can significantly reduce their unbound concentration in blood and affect their biological reactions. In this study, we developed a new QSAR model for bovine serum albumin (BSA) – water partition coefficients (KBSA/W) of neutral organic chemicals with large structural variance, logKBSA/W values covering 3.5 orders of magnitude (1.19–4.76). All chemical geometries were optimized by semi-empirical PM6 algorithm. Several quantum chemical parameters that reflect various intermolecular interactions as well as hydrophobicity were selected to develop QSAR model. The result indicates the regression model derived from logKow, the most positive net atomic charges on an atom, Connolly solvent excluded volume, polarizability, and Abraham acidity could explain the partitioning mechanism of organic chemicals between BSA and water. The simulated external validation and cross validation verifies the developed model has good statistical robustness and predictive ability, thus can be used to estimate the logKBSA/W values for chemicals in application domain, accordingly to provide basic data for the toxicity assessment of the chemicals.

Equilibrium Sorption of Structurally Diverse Organic Ions to Bovine Serum Albumin.

Reliable partitioning data are essential for assessing the bioaccumulation potential and the toxicity of chemicals. In contrast to neutral organic chemicals, the partitioning behavior of ionogenic organic chemicals (IOCs) is still a black box for environmental scientists. Partitioning to serum albumin, the major protein in blood plasma, strongly influences the freely dissolved concentration of many chemicals (including IOCs), which affects their transport and distribution in the body. Because consistent data sets for partitioning of IOCs are rarely available, bovine serum albumin-water partition coefficients (KBSA/w) were measured in this study for 45 anionic and 4 cationic organic chemicals, including various substituted benzoic and naphthoic acids, sulfonates and several pesticides and pharmaceuticals. The results of this study suggest that binding to BSA is substantially influenced by the three-dimensional structure of the chemicals and the position of substitutions on the sorbing molecules. For example, we found a difference of >1.5 log units between isomeric chemicals such as 3,4-dichlorobenzoic acid and 2,6-dichlorobenzoic acid, and 1-naphthoic acid and 2-naphthoic acid. Conventional modeling approaches (e.g., based on octanol-water partition coefficients) poorly predict log KBSA/w of organic ions (R(2) ≤ 0.5), partially because they do not capture the observed steric effects. Hence, alternative modeling strategies will be required for accurate prediction of serum albumin-water partition coefficients of organic ions.

Synthesis, characterization and DFT study of new water-soluble aluminum(III), gallium(III) and indium(III) Schiff base complexes: effect of metal on the binding propensity with bovine serum albumin in water

In this work, three new water-soluble aluminum(III), gallium(III) and indium(III) Schiff base complexes; [M(L)NO3], where L denotes; N,N′-bis(5-sulfosalicyliden)-1,2-phenylendiamin (5-SO3-1,2-salophen); were synthesized and characterized by UV–Vis spectroscopy, 1H NMR, FT-IR, thermal gravimetry and C.H.N analyses. Also, the optimized structure and the energy level of the frontier orbitals for the complexes were computed using B3LYP/LANL2DZ method and 6–311g basis set. The interaction of these complexes with bovine serum albumin (BSA) was studied by fluorescence and UV–Vis techniques. The results showed that the intrinsic fluorescence of BSA was quenched by adding the complexes, which was rationalized in terms of the dynamic quenching mechanism. Binding constants, Stern–Volmer constants, number of binding sites and binding stoichiometry were determined by fluorescence method. Furthermore, the thermodynamic parameters calculating by van’t Hoff equation, indicated that the binding mode is entropy driven and enthalpically disfavored. Site marker competitive experiments revealed that these complexes can bind to the subdomain IIIA (site II) of BSA. Based on the Forster theory of non-radiation energy transfer, the efficiency of energy transfer and the distance between the donor (BSA) and the acceptor (complex) were obtained. Also, the synchronous fluorescence spectra displayed that the microenvironment of the tyrosine residues was not apparently changed. Finally, the binding of the complexes to BSA was modeled by molecular docking method. Excellent agreement was found between the experimental and theoretical results with respect to the mechanism of binding and binding constants. Results indicated that the order of propensity of the complexes to BSA was as follows: [In(L)NO3] > [Ga(L)NO3] > [Al(L)NO3].

Binding Modes of Cabergoline to Bovine Serum Albumin in Free- and β-Cyclodextrin-Encapsulated Forms: Differences in Quenching Behavior and Förster Resonance Energy Transfer

We report, in this paper, the interaction of cabergoline in the free form and β-cyclodextrin-bound form with bovine serum albumin. The stoichiometry and the binding constant of the Cabergoline–β-cyclodextrin inclusion complex are reported based on UV–Vis absorption and fluorescence spectroscopic studies and the structure of the 1:1 inclusion complex is proposed using two-dimensional rotating-frame Overhauser effect spectroscopy. Molecular docking is used to propose the mode of interaction of cabergoline with bovine serum albumin. The apparent binding constants for the interaction of cabergoline with bovine serum albumin in water and in aqueous β-cyclodextrin solution are compared. The average distance between donor and acceptor is altered in the presence of β-cyclodextrin, as indicated by Forster resonance energy transfer. The influence of β-CD on the binding of the small molecule with bovine serum albumin is discussed.

Role of hydrogen-bonding and photoinduced electron transfer (PET) on the interaction of resorcinol based acridinedione dyes with Bovine Serum Albumin (BSA) in water

Resorcinol based acridinedione (ADDR) dyes are a class of laser dyes and have structural similarity with purine derivatives, nicotinamide adenine dinucleotide (NADH) analogs. These dyes are classified into photoinduced electron transfer (PET) and non-photoinduced electron transfer dyes, and the photophysical properties of family of these dyes exhibiting PET behavior are entirely different from that of non-PET dyes. The PET process in ADDR dyes is governed by the solvent polarity such that an ADDR dye exhibits PET process through space in an aprotic solvent like acetonitrile and does not exhibit the same in protic solvents like water and methanol. A comparison on the fluorescence emission, lifetime and nature of interaction of various ADDR dyes with a large globular protein like Bovine Serum Albumin (BSA) was carried out in aqueous solution. The interaction of PET based ADDR dyes with BSA in water is found to be largely hydrophobic, but hydrogen-bonding interaction of BSA with dye molecule influences the fluorescence emission of the dye and shifts the emission towards red region. Fluorescence spectral studies reveal that the excited state properties of PET based ADDR dyes are largely influenced by the addition of BSA. The microenvironment around the dye results in significant change in the fluorescence lifetime and emission. Fluorescence enhancement with a red shift in the emission results after the addition of BSA to ADDR dyes containing free amino hydrogen in the 10th position of basic acridinedione dye. The amino hydrogen (N–H) in the 10th position of ADDR dye is replaced by methyl group (N–CH3), a significant decrease in the fluorescence intensity with no apparent shift in the emission maximum was observed after the addition of BSA. The nature of interaction between ADDR dyes with BSA is hydrogen-bonding and the dye remains unbound even at the highest concentration of BSA. Circular Dichroism (CD) studies show that the addition of dye to BSA results in a decrease in the alpha helical content of the protein. The shape and the pattern of CD bands after the addition of ADDR dye to BSA remain largely unaltered.

In situ ATR-IR spectroscopy study of adsorbed protein: Visible light denaturation of bovine serum albumin on TiO2

In this work in situ Fourier transform infrared-attenuated total reflection (FTIR-ATR) spectroscopy in a flow-through cell was used to study the effect of visible light irradiation on bovine serum albumin (BSA) adsorbed on porous TiO2 films. The experiments were performed in water at concentrations of 10−6mol/l at room temperature. The curve fitting method of the second derivative spectra allowed us to explore details of the secondary structure of pure BSA in water and conformation changes upon adsorption as well as during and after illumination by visible light. The results clearly show that visible light influences the conformation of adsorbed BSA. The appearance of a shift of the amide I band, in the original spectra, from 1653cm−1 to 1648cm−1, is interpreted by the creation of random coil in the secondary structure of adsorbed BSA. The second derivative analysis of infrared spectra permits direct quantitative analysis of the secondary structural components of BSA, which show that the percentage of α-helix decreases during visible light illumination whereas the percentage of random coil increases.

Interference of some aqueous two-phase system phase-forming components in protein determination by the Bradford method

The interference of some specific aqueous two-phase system (ATPS) phase-forming components in bovine serum albumin (BSA) determination by the Bradford method was investigated. For this purpose, calibration curves were obtained for BSA in the presence of different concentrations of salts and polymers. A total of 19 salts [Na2SO4, (NH4)2SO4, MgSO4, LiSO4, Na2HPO4, sodium phosphate buffer (pH 7.0), NaH2PO4, K2HPO4, potassium phosphate buffer (pH 7.0), KH2PO4, C6H8O7, Na3C6H5O7, KCHO2, NaCHO2, NaCO3, NaHCO3, C2H4O2, sodium acetate buffer (pH 4.5), and NaC2H3O2] and 7 polymers [PEG 4000, PEG 8000, PEG 20000, UCON 3900, Ficoll 70000, PES 100000, and PVP 40000] were tested, and each calibration curve was compared with the one obtained for BSA in water. Some concentrations of salts and polymers had considerable effect in the BSA calibration curve. Carbonate salts were responsible for the highest salt interference, whereas citric and acetic acids did not produce interference even in the maximum concentration level tested (5wt%). Among the polymers, UCON gave the highest interference, whereas Ficoll did not produce interference when used in concentrations up to 10wt%. It was concluded that a convenient dilution of the samples prior to the protein quantification is needed to ensure no significant interference from ATPS phase-forming constituents.