State Of Bovine Serum Albumin Research Articles

Influence of aluminum and iron chlorides on the parameters of zigzag patterns on films dried from BSA solutions

The relationships between the structural and aggregational state of bovine serum albumin (BSA) and the specific length and total number of zigzag pattern segments of the film textures formed upon drying biopolymer solutions with aluminum and iron chlorides have been shown. To obtain films, saline solutions of BSA were dried in a glass cuvette under thermostatically controlled conditions. It is shown that the formation of zigzag structures is sensitive to the influence of aluminum chlorides Al3+ and iron chlorides Fe3+ and depend on the concentration of AlCl3 and FeCl3. This may be due to a change in the charge and size of BSA particles and due to a change in conformation or a violation of the structure of BSA. These factors, in turn, affect the hydration of the solution components and the structural state of free water in solution, which presumably also affects the formation of zigzag structures. It is established that the analysis of the specific length and the number of segments of zigzag patterns makes it possible to evaluate changes in the state of biopolymers in the initial solution during structural changes and aggregation.

A good and bad aggregation: Effect of imidazolium- and cholinium-based ionic liquids on the thermal stability of bovine serum albumin

The present work describes the effect of imidazolium- and cholinium-based ionic liquids (ILs) on the thermal stability, binding and aggregation of bovine serum albumin (BSA). The studied ILs include choline chloride [Ch][Cl], choline dihydrogenphosphate [Ch][DHP], choline acetate [Ch][OAc], 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF4], 1-octyl-3-methylimidazolium tetrafluoroborate [OMIM][BF4] 1-butyl-3-methylimidazolium trifluoromethanesulfonate [BMIM][TfO], 1-butyl-3-methylimidazolium acetate [BMIM][OAc], and 1-butyl-3-methylimidazolium methanesulfonate [BMIM][CH3SO3]. The state of BSA in presence of ILs was assessed by circular dichroism (CD), temperature dependent circular dichroism, fluorescence spectroscopy, and dynamic light scattering (DLS). The thermal stability of BSA in presence of ILs increased (above 85 °C) in all studied ILs except [OMIM][BF4]. Differences of binding ILs with BSA depending on alkyl chain length were estimated, the probable binding modes were identified using molecular docking. The influence of ILs on the aggregation of BSA at different temperatures was established. It was found that the ILs with short alkyl chain length (up to butyl) promote aggregation of the native form of BSA increasing its thermal stability.

New insights into the pH dependence of anthocyanin-protein interactions by a case study of cyanidin-3-O-glucoside and bovine serum albumin

Anthocyanins are natural pigments that can undergo structural transformations depending on pH, and their interactions with proteins have received considerable attention in recent decades. This work intended to unravel the pH-dependence of anthocyanin-protein binding mechanisms by examining interactions between cyandin-3-O-glucoside (C3G) and bovine serum albumin (BSA) at different pH. Fluorescence quenching (FQ) and microscale thermophoresis (MST) demonstrated that their binding affinity was pH 7 > pH 5 > pH 3, with dissociation constant (Kd) at pH 7 of 43.1 μM for FQ and 33.0 μM for MST. The predominant C3G forms were determined by measuring UV–Vis absorption spectra with pH-jump experiments. Additionally, circular dichroism, Trp fluorescence, zeta potential and particle size measurements demonstrated the “molten globule” state of BSA at pH 3, and C3G had a negligible effect on BSA conformation. The binding mechanisms were investigated using molecular dynamics simulation, which revealed the importance of electrostatic interaction. Flavylium cation rarely bound to BSA due to electrostatic repulsion at pH 3, whereas the uncharged forms of C3G at all pH values bound to BSA surface due to hydrophobic interactions and hydrogen bonds, but possibly in a weak and nonspecific manner. Anionic quinoidal bases, the most common C3G forms at pH 7, mostly bound to the positively charged pockets of BSA and formed several hydrogen bonds with surrounding amino acids, resulting in higher binding affinity. These findings provide insights into the interactions of proteins with different anthocyanin forms, which may guide the future research and applications in this field.

Agarose native gel electrophoresis analysis of thermal aggregation controlled by Hofmeister series

The effects of salting-in and salting-out salts defined by Hofmeister series on the solution state of bovine serum albumin (BSA) in 50 mM Tris-HCl buffer at pH 7.4 before and after thermal unfolding at 80 °C for 5 min were examined using agarose native gel electrophoresis and mass photometry. Gel electrophoresis showed that salting-in MgCl2, CaCl2 and NaSCN resulted in formation of intermediate structures of BSA upon heating on native gel, while heating in buffer alone resulted in aggregated bands. Mass photometry showed large loss of monomer and oligomers when heated in this buffer, but retaining these structures in the presence of 1 M MgCl2 and NaSCN. To our surprise, salting-out MgSO4 also showed a similar effect on gel electrophoresis and mass photometry. Salting-out NaCl and (NH4)2SO4 resulted in smearing and aggregated bands, which were supported by mass photometry. Aggregation-suppressive ArgHCl also showed oligomer aggregates upon gel electrophoresis and mass photometry.

Influence of amino acids on alkaline pH induced partially folded molten globule like intermediate of bovine serum albumin: Conformational and thermodynamic insights

Partially folded states of proteins can misfold or aggregate leading to various neurodegenerative diseases or disorders. Therefore stabilization of the partially folded states of proteins towards native conformation is important. Here, we investigate the effects of glycine, l-alanine, DL- α – aminobutyric acid, l-lysine, l-leucine and l-valine on the stability of molten globule like partially folded state of bovine serum albumin (BSA) at pH 11.2. We report the changes in the Trp microenvironment of the BSA at pH 11.2 by these amino acids. Except for l-leucine and l-valine, the other four amino acids impart thermal stabilization to partially folded MG like intermediate of BSA at pH 11.2. CD spectroscopy is employed to analyse tertiary conformation of BSA. We perform the ITC to study the interaction of BSA with these amino acids at pH 11.2. The spectroscopic in combination with calorimetric observations have enabled monitoring amino acid-induced conformational changes in BSA at pH 11.2 suggesting their osmolyte behavior. The results suggest significance of preferential hydration of partially folded proteins and the stability offered by the amino acids.

Temperature, pH and additives effects on the binding of Caffeic acid phenethyl ester to the native state of bovine serum albumin

Caffeic acid phenethyl ester (CAPE, phenethyl 3-(3,4-dihydroxyphenyl)acrylate), an important anti-tumor components of Beehive Propolis, has many pharmacological activities. The interaction of CAPE with bovine serum albumin (BSA) was thus investigated using UV-visible spectroscopy, fluorescence spectroscopy, circular dichroism (CD), dynamic light scattering (DLS), dynamic fluorescence spectrum and molecular docking under physiological conditions. The intrinsic fluorescence quenching of BSA by CAPE was a static process, confirmed by the dynamic Fluorescence Spectrum. The negative values of ΔHmo and ΔSmo indicated that the hydrogen bond and van der Walls force played a predominant role in the enthalpy-driven binding process. The CD spectrum showed that after adding CAPE, the α-helix content structure of BSA decreased, and the secondary structure changed. At pH 6.0 and 8.5, the binding affinity of BSA to CAPE decreased. After adding metal ions, the binding affinity of BSA and CAPE increased. However, the presence of biosurfactants [sodium cholate (NaC) and sodium deoxycholate (NaDC)] decreased the binding affinity of BSA to CAPE because they all bound in the same subdomain of BSA.

Ion trap operational modes for ion/ion reactions yielding high mass-to-charge product ions

To better probe large biomolecular complexes, developments in mass spectrometry (MS) have focused on improving technologies used to generate, transmit, and measure high m/z ions. The additional tandem-MS (MSn) capabilities of ion trap mass spectrometers (ITMS) facilitate experiments that facilitate probing complex biomolecular ions. In particular, charge reduction using gas-phase ion/ion reactions increase separation of charge states generated via electrospray ionization (ESI), which increases confidence in charge state assignments and therefore masses determined from the observed charge states. Current ITMS technologies struggle to generate and measure low charge states of large (>50 kDa) proteins and complexes because of power limitations associated with conventional high-frequency sine wave operation. Other approaches, including frequency scanning techniques and use of digital waveforms, reduce or eliminate some of these limitations. The work presented here studies five different operational modes for a quadrupole ion trap (QIT) mass spectrometer used to generate and measure low charge states of bovine serum albumin (BSA), pyruvate kinase (PK), and GroEL. While digital operation of a QIT presents limitations during the ion/ion reaction period of the experiment, it generally provided the best spectra in terms of resolution and signal at m/z > 50,000.

Thermal stability modulation of the native and chemically-unfolded state of bovine serum albumin by amino acids.

Cells are crowded with various cosolutes including salts, osmolytes, nucleic acids, peptides and proteins. These cosolutes modulate the protein folding equilibrium in different ways, however, a unifying concept remains elusive. To elucidate the cosolute size-effect, macromolecular crowders are commonly compared to their monomeric building blocks (e.g. dextran vs. glucose or polyethylene glycol with different degrees of polymerization). To the best of our knowledge, such studies do not exist for protein crowders, raising the question of how single amino acids modulate the folding equilibrium. Therefore, we investigate the effect of glycine, alanine, proline and arginine on the stability of a model globular protein bovine serum albumin (BSA) upon thermal and urea-induced unfolding. We use three complementary techniques, fluorescence spectroscopy (as a local site-specific probe), circular dichroism (as a global probe for α-helical structure) and differential scanning calorimetry (to probe the energetics of unfolding). We find that the amino acids modulate BSA stability and unfolding, however, without following a particular trend with either the hydrophobicity scale or the solvent accessible surface area (SASA) of the added amino acids. Our data rather suggest that solvation effects play a role in understanding the cosolute effect.

Alkali induced unique partially folded state of bovine serum albumin: qualitative and quantitative insights

Qualitative and quantitative understanding of partially folded states of protein is essential in gaining deeper insights into folding pathways. We have observed a partially folded state of bovine serum albumin (BSA) in the presence of 2,2,2-trifluoroethanol at pH11.2 which does not resembles the properties of the molten globule state. ThT, a frequently used marker for protein fibrils have two order of greater affinity towards the intermediate state at pH11.2 compared to native BSA at pH7.4. Surprisingly, the binding of ANS with this partially folded state is weaker than that of native state of BSA. Combined fluorescence, circular dichroism spectroscopy and isothermal titration calorimetric studies indicate that for such partially folded state, ThT is a better marker compared to ANS. The results have highlighted the importance of dyes like ThT in characterizing partilally folded states of protein which might appear as intermediates in the funnel moldel describing the protein folding pathway.

Effect of trehalose on structural state of bovine serum albumin adsorbed onto mesoporous silica and the protein release kinetics in vitro

Protein – silica composites are promising platform for development of novel formulations of protein drugs with improved pharmacological and consumer properties. Adsorption is simple and frequently used method of preparation of the composites. However often proteins undergo significant conformation changes upon interaction with silica surface leading to decrease or loss of their biological functionality. The aim of this study was to elucidate the ability of disaccharide trehalose to protect structure of bovine serum albumin (BSA) as model protein drug from unfolding in the protein – silica composites prepared by adsorption as well as an effect of the disaccharide on release kinetics of the protein from the composites. For this purpose, BSA was adsorbed onto presinthesized mesoporous silica from solutions containing trehalose and without it. The adsorbent was thoroughly characterized by X-ray diffraction, nitrogen adsorption/desorption, FTIR spectroscopy, dynamic light scattering. The influence of different amounts of trehalose on the protein loading, structural alterations of BSA in solutions and in the composites was studied. The protein release behavior from the composites was analyzed in vitro. It was found that trehalose promotes a higher protein loading in mesoporous silica, stabilization of BSA structure in the solid BSA- silica composites and the protein structure released from the composites. Trehalose also promotes a higher release rate of the protein from the synthesized composites. The effects increase with increasing amount of the disaccharide in the adsorption medium. Thus, use of trehalose can help to prepare high loaded BSA-silica composite with minimal structural alteration of the protein by adsorption and to regulate the protein release rate.

Discrimination between Single Protein Conformations Using Dynamic SERS

In biomedicine and biophysics, the discrimination of protein conformations is of critical importance for identifying the unfolding states in the diagnosis of neurodegenerative diseases. We develop a dynamic Raman spectroscopic approach based on a statistical analysis of the time series of spectral fingerprints of single protein. We show that the unfolded state of bovine serum albumin can be identified in the time series using the fluctuations of the Raman bands of some amino acids, tryptophan, tyrosine, leucine, and histidine, acting as biomarkers. The statistical analysis induces also the sorting between physisorption and chemisorption events. This is confirmed by the spectral analysis of the different characteristic spectra highlighted based on the amino acids fingerprints following, notably, the hydrophobicity

MR imaging of protein folding in vitro employing Nuclear‐Overhauser‐mediated saturation transfer

MR Z-spectroscopy allows enhanced imaging contrast on the basis of saturation transfer between the proton pools of cellular compounds and water, occurring via chemical exchange (chemical exchange saturation transfer, CEST) or dipole-dipole coupling (nuclear Overhauser effect, NOE). In previous studies, signals observed in the aliphatic proton region of Z-spectra have been assigned to NOEs between protons in water molecules and protons at the surface of proteins. We investigated a possible relationship between the signal strength of NOE peaks in Z-spectra obtained at B0 = 7 T and protein structure. Here, we report a correlation of NOE-mediated saturation transfer with the structural state of bovine serum albumin (BSA), which was monitored by fluorescence spectroscopy. Encouraged by CEST signal changes observed in tumor tissue, our observation also points to a possible contrast mechanism for MRI sensitive to the structural integrity of proteins in cells. Therefore, protein folding should be considered as an additional property affecting saturation transfer between water and proteins, in combination with the microenvironment and physiological quantities, such as metabolite concentration, temperature and pH.

Binding of antioxidant flavonol morin to the native state of bovine serum albumin: Effects of urea and metal ions on the binding

In consideration of the various medicinal aspects of the flavonoid polyphenols, the interaction of morin with bovine serum albumin (BSA) has been investigated using multi-spectroscopic approaches. The pKa1 of morin being 5.09, which is below physiological pH, binding studies provide important insights into its potential use as a biotherapeutic. The binding was performed under different pH (5, 7 and 9) conditions and in absence and presence of Cu(II) and Fe(III) ions. It is observed that the presence of metal ions affect the binding of morin towards BSA. The binding with BSA results in a motional restriction of morin in solution that causes an increase in anisotropy (r), rotational correlation time (tr) and steady-state lifetime (tav) of the ligand. Urea causes denaturation of BSA resulting in the release of morin from the protein core as determined from both the steady-state fluorescence and anisotropy (r) measurements. The possibility of non-radiative energy transfer from the donor tryptophan to the acceptor morin is detected following the Förster's theory. The site marker displacement studies along with the molecular docking results indicated that morin binds to the hydrophobic pocket of site 1 (subdomain IIA) near Trp 213 of BSA.

The Importance of Protein-Protein Interactions on the pH-Induced Conformational Changes of Bovine Serum Albumin: A Small-Angle X-Ray Scattering Study

The combined effects of concentration and pH on the conformational states of bovine serum albumin (BSA) are investigated by small-angle x-ray scattering. Serum albumins, at physiological conditions, are found at concentrations of ∼35–45 mg/mL (42 mg/mL in the case of humans). In this work, BSA at three different concentrations (10, 25, and 50 mg/mL) and pH values (2.0–9.0) have been studied. Data were analyzed by means of the Global Fitting procedure, with the protein form factor calculated from human serum albumin (HSA) crystallographic structure and the interference function described, considering repulsive and attractive interaction potentials within a random phase approximation. Small-angle x-ray scattering data show that BSA maintains its native state from pH 4.0 up to 9.0 at all investigated concentrations. A pH-dependence of the absolute net protein charge is shown and the charge number per BSA is quantified to 10(2), 8(1), 13(2), 20(2), and 26(2) for pH values 4.0, 5.4, 7.0, 8.0, and 9.0, respectively. The attractive potential diminishes as BSA concentration increases. The coexistence of monomers and dimers is observed at 50 mg/mL and pH 5.4, near the BSA isoelectric point. Samples at pH 2.0 show a different behavior, because BSA overall shape changes as a function of concentration. At 10 mg/mL, BSA is partially unfolded and a strong repulsive protein-protein interaction occurs due to the high amount of exposed charge. At 25 and 50 mg/mL, BSA undergoes some re-folding, which likely results in a molten-globule state. This work concludes by confirming that the protein concentration plays an important role on the pH-unfolded BSA state, due to a delicate compromise between interaction forces and crowding effects.

Native and denatured bovine serum albumin. D.c. polarography, stripping voltammetry and constant current chronopotentiometry

Native and denatured states of bovine serum albumin (BSA) were studied by d.c. polarographic and voltammetric Brdicka catalytic responses (BCR) in cobalt-containing solution and by constant current chronopotentiometric stripping analysis (CPSA) in borate buffer, pH 9.3. We found that 90 nM denatured BSA produced catalytic peak H (around − 1.8 V vs. Ag/AgCl/3 M KCl). This peak was about 50-fold higher than the native protein under the same conditions. Qualitatively similar results were obtained also with other proteins in native and denatured states, such as human serum albumin, γ-globulin, myoglobin and α-crystallin. 2 nM denatured BSA produced a well-developed CPS peak (at accumulation time 5 min) while native BSA yielded almost no signal under the same conditions.

Perturbation of Lipid and Protein Structure by General Anesthetics: How Little Is Too Little?

Assistant Professor of Anesthesia Harvard Medical SchoolAssistant AnesthetistMassachusetts General HospitalDepartment of AnesthesiaBoston, Massachusetts 02114Raines@etherdome.MGH.harvard.edu.To the Editor:—Lipid-based theories of general anesthetic action have long endured because numerous studies have shown that the in vivo pharmacology of an anesthetic correlates remarkably well with its ability to perturb the structural properties of simple lipid bilayers. The Meyer-Overton correlation between anesthetic potency and hydrophobicity, the inactivity of nonanesthetic long chain alcohols and highly halogenated volatile compounds (nonimmobilizers), and pressure reversal have all been demonstrated in studies using protein-free lipid bilayers. 1–6Nevertheless, a most persuasive and often mentioned argument against lipid-based theories is that at clinically relevant concentrations, anesthetics induce only small perturbations in lipid bilayer structure. 7,8For example, halothane reduces the order parameter (increase the “fluidity”) of lipid bilayers by only 1% at clinically relevant concentrations. 9An equivalent reduction in order parameter may be obtained by raising the temperature of the bilayer by less than 1°C. Similarly, halothane reduces the transition temperature between a lipid bilayer’s liquid and gel phases by only 0.5°C at anesthetic concentrations and by only 5°C even at 10 times the minimum aveolar concentration (MAC). 10I was, therefore, very interested to read the study by Johansson et al. quantifying the effects of isoflurane and halothane on structural properties of bovine serum albumin, a lipid-free protein model used in mechanistic studies of anesthetic action. 11What did their studies show? At approximately 1 MAC, isoflurane and halothane increased the fluorescence anisotropy of bovine serum albumin by 1%. An equivalent reduction was obtained by raising the temperature of bovine serum albumin by approximately 1° C. Similarly, isoflurane and halothane raised the transition temperature between the folded and unfolded states of bovine serum albumin by less than 1°C at anesthetic concentrations and by only 3–4°C even at 10 times MAC. Studies of anesthetic binding to other protein models have been similarly unable to demonstrate significant anesthetic-induced changes in protein structure. 12,13Thus, anesthetics induce similarly small changes in the structural properties of lipids and proteins. For consistency, shouldn’t we now conclude that such insensitivity argues strongly against a protein site of anesthetic action?The inability to detect significant anesthetic-induced structural changes in either lipid or protein model systems highlights the practical (and obvious) limitations of such studies: we can only measure what we can measure. Fluorescence anisotropy, denaturation temperature, phase transition temperature, and order parameter have been used by biophysicists for many years as indicators of lipid bilayer and protein structure in large part because they are easily quantitated. There is no compelling theoretical reason to believe that changes in these properties directly accounts for the functional effects of anesthetics on relevant targets in the central nervous system. In fact, it seems quite likely that the anesthetic state results from changes in other lipid and/or protein physical properties that are not so easily measured.

Investigation of Slow Dynamics of the Sulfhydryl in the Solution and Gel States of Bovine Serum Albumin: A Vector Electron Paramagnetic Resonance Study.

Serum albumin has one reactive sulfhydryl (Cys-34) that is one of the important binding sites. Cys-34 is located in the crevice on the surface of the albumin molecule and is therefore restricted in its motion. Bovine serum albumin (BSA) Fraction V forms a transparent gel at pD 4.0 (F-form) in D2O at protein concentrations above 7% (BSA*-gel). We studied the molecular motion of Cys-34 on BSA in the solution and gel states by the vector electron paramagnetic resonance (EPR) method using a maleimide spin label. The rotational correlation times of the spin label bound to Cys-34 in the BSA solution and BSA*-gel were in the order of 10(-6) and 10(-5) s, respectively. A longer rotational correlation time of the Cys-34 spin label in the BSA*-gel suggested that the gel network formed in BSA may drastically slow the motion of Cys-34. The integrated value obtained from the vector EPR spectra also showed an extremely dramatic slowing of the Cys-34 spin label during the gel formation. On the other hand, the values for order parameter and the inclination of the principal axis (z) of the Cys-34 spin label to the rotational axis (mu) were the same in the BSA solution and BSA*-gel.

Molecular Association State of Bovine Serum Albumin and Its Effect on Emulsifying Activity

Oil-in-water (4:6) emulsions stabilized with bovine serum albumin (BSA) (0.225 mM), at pH 7, were used to study the effects of molecular association on the viscosity of emulsions and emulsifying activity (EA). Anions -TCA– and -S04 had different effects on the intermolecular association of BSA. Aggregates (A) of BSA comprising A1, A2, A3, A4, A5, A6 and A7 were detected in the control and the anion (100 mM) containing emulsions. The extent of association of BSA was in the order; control >-SO4>-TCA–. The EA also showed the same order whereas the viscosity of emulsions showed the reverse order. High energy input per unit volume (>684 106 J.m-3) caused molecular dissociation and a concomitant decrease in EA. Apparently, a certain degree of association is required for EA of BSA.

Molecular association state of bovine serum albumin and its effect on emulsifying activity.

Oil-in-water (4:6) emulsions stabilized with bovine serum albumin (BSA) (0.225 HIM), at pH 7, were used to study the effects of molecular association on the viscosity of emulsions and emulsifying activity (EA). Anions -TCA- and -SO4- had different effects on the intermolecular association of BSA. Aggregates (A) of BSA comprising A1, A2, A3, A4, A5, A6 and A7 were detected in the control and the anion (100 mM) containing emulsions. The extent of association of BSA was in the order; control > -SO4- > -TCA-. The EA also showed the same order whereas the viscosity of emulsions showed the reverse order. High energy input per unit volume (>684 106 J•m-3) caused molecular dissociation and a concomitant decrease in EA. Apparently, a certain degree of association is required for EA of BSA.

Bovine serum albumin in lithium chloride solutions. Changes in conformation.

The physical state of bovine serum albumin in LiCl solutions at the pH near isoelectric point and room temperature has been studied with intrinsic viscosity, optical rotatory dispersion, circular dichroism and ultraviolet absorption difference spectra. The results indicate that the effects of Li+ take place in two processes. 1. At the concentration of LiCl below 4.0 M, only the general electrostatic effect occurs. There is no conformational change in the protein. 2. At the concentration of LiCl above 4.0 M, a specific effect of the Li+ occurs in which both the secondary and tertiary structures of the protein change. In addition, the protein molecules aggregate. In correspondence with the two processes, two different possible mechanisms are discussed.