Mechanistic insights into protein precipitation by alcohol

Ethanol is used to precipitate proteins during various processes, including purification and crystallization. To elucidate the mechanism of protein precipitation by alcohol, we have investigated the solubility and structural changes of protein over a wide range of alcohol concentrations. Conformation of hen egg-white lysozyme was changed from native to α-helical rich structure in the presence of ethanol at concentrations above 60%. The solubility of lysozyme was reduced with increasing ethanol concentration, although gel formation at ethanol concentrations between 60% and 75% prevented accurate solubility measurements. SH-modified lysozyme showed largely unfolded structure in water and α-helical structure in the presence of ethanol. More importantly, solubility of the chemically modified lysozyme molecules decreased with increasing ethanol concentration. There is no indication of increased solubility upon unfolding of the lysozyme molecules by ethanol, indicating that any favorable interaction of ethanol with the hydrophobic side chains, if indeed occuring, is offset by the unfavorable interaction of ethanol with the hydrophilic side chains and peptide bonds.     

Purification and properties of buffalo serum albumin

Based on a detailed study of the solubility of serum albumin, a procedure for its purification by selective ammonium sulphate precipitation has been developed. Using buffalo serum, first extraneous proteins were precipitated by making the serum 2.26 M saturated with ammonium sulphate at pH 7.0 and then albumin was precipitated from the supernatant at 1.9 M ammonium sulphate concentration at pH 4.2. The overall yield of serum albumin thus isolated was about 55% with a purity of 97%. The protein preparation gave a single nearly symmetrical peak on Sephadex G-100 column and virtually a single band on polyacrylamide gel electrophoresis in the presence and absence of SDS. Buffalo serum albumin has a molecular weight of 69,000 Da. The hydrodynamic properties such as Stoke's radius (3.70 nm), diffusion coefficient (6.03 X 10(-7) cm2/s) and frictional ratio (1.36) obtained by analytical gel chromatography, bilirubin binding characteristics and its interaction with anti-bovine serum albumin antiserum suggest that buffalo serum albumin is very similar to BSA in its molecular properties.     

Do properties of bovine serum albumin at fluid/electrolyte interface follow the Hofmeister series?--An analysis using Langmuir and Langmuir-Blodgett films

Folding and solubility of proteins are dependent on their state of hydration. How does a protein-bovine serum albumin (BSA) behave in the presence of Hofmeister electrolytes, especially at interfaces? Langmuir films of bovine serum albumin (BSA) in the presence of different Hofmeister electrolytes at air/solution interface and as Langmuir-Blodgett films (LB films) at solid/solution interface have been studied using the surface pressure-molecular area (pi-A) isotherms and surface energy parameters. Changes in secondary structure have been analyzed using circular dichroism (CD) and fluorescence spectroscopy. Hydrodynamically coupled water fraction of BSA in different environments has been estimated using quartz crystal microbalance (QCM) and related to the secondary structural changes. Molecular modeling of BSA in different environments showed that the protein has a compact structure at the interface compared to vacuum. The contact areas estimated using molecular modeling agreed with the experimental results. The results show that the properties of BSA at the interface follow the Hofmeister series with NaF leading to maximum compaction in the protein. Further, in addition to ion specific solvation and different ion size, water structure alteration and the bound water fractions contribute importantly to the Hofmeister effect.     

Exploring the differences and similarities between urea and thermally driven denaturation of bovine serum albumin: intermolecular forces and solvation preferences

The interactions of bovine serum albumin (BSA) with urea/water were investigated by computer simulation. It was revealed that the BSA-hydrophobic residues in urea solutions favored contact with urea more than with water. Energy decomposition analysis showed that van der Waals energy was the dominant driving force behind urea affinity for hydrophobic residues, whereas coulombic attraction was largely responsible for water affinity for these residues. Meanwhile, urea–BSA hydrogen bond energies were found to be weaker than water–BSA hydrogen bond energies. The greater strength of water–BSA hydrogen bonds than urea–BSA hydrogen bonds, and the opposing preferential interaction between the BSA and urea suggest that disruption of hydrophobic interaction predominates urea–protein denaturation. In pure water, hydrophobic residues showed aggregation tendencies at 323 K, suggesting an increase in hydrophobicity, while at 353 K the residues were partly denatured due to loss of hydrogen bonds; thus, disruption of hydrophobic interactions appeared to contribute less to thermal denaturation.     

Influence of bovine serum albumin on the secondary structure of interferon alpha 2b as determined by far UV circular dichroism spectropolarimetry

Many therapeutic biologics are formulated with excipients, including the protein excipient human serum albumin (HSA), to increase stability and prevent protein aggregation and adsorption onto glass vials. One biologic formulated with albumin is interferon alpha-2b (IFN α-2b). As is the case with other therapeutic biologics, the increased structural complexity of IFN α-2b compared to a small molecule drug requires that both the correct chemical structure (amino acid sequence) and also the correct secondary and tertiary structures (3 dimensional fold) be verified to assure safety and efficacy. Although numerous techniques are available to assess a biologic's primary, secondary and tertiary structures, difficulties arise when assessing higher order structure in the presence of protein excipients. In these studies far UV circular dichroism spectropolarimetry (far UV-CD) was used to determine the secondary structure of IFN α-2b in the presence of a protein excipient (bovine serum albumin, BSA). We demonstrated that the secondary structure of IFN α-2b remains mostly unchanged at a variety of BSA to IFN α-2b protein ratios. A significant difference in alpha helix and beta sheet content was noted when the BSA to IFN α-2b ratio was 5:1 (w/w), suggesting a potential conformational change in IFN α-2b secondary structure when BSA is in molar excess.     

BSA structural changes during homomolecular exchange between the adsorbed and the dissolved states

The secondary structure and the thermostability of bovine serum albumin (BSA), before adsorption and after homomolecular displacement from silica and polystyrene particles, are studied by circular dichroism spectroscopy and differential scanning calorimetry. The structural perturbations induced by the hydrophilic silica surface are reversible, i.e. BSA completely regains the native structure and stability after being exchanged. On the other hand, the adsorption on, and subsequent desorption from, polystyrene particles causes irreversible changes in the stability and (secondary) structure of BSA. The exchanged proteins have a higher denaturation temperature and a lower enthalpy of denaturation than native BSA. The alpha-helix content is reduced while the beta-turn fraction is increased in the exchanged molecules. Both effects are more pronounced when the protein is displaced from less crowded sorbent surfaces. The irreversible surface-induced conformational change may be related to some aggregation of BSA molecules after being exposed to a hydrophobic surface.     

Toxic effects of ethanol on bovine serum albumin

The toxic effects of ethanol on bovine serum albumin (BSA) were measured by resonance light scattering (RLS), fluorescence spectroscopy, ultraviolet spectrophotometry (UV), circular dichroism (CD), and transmission electron microscopy (TEM). The results indicated that ethanol had toxic effects on BSA, which led to protein denaturation and the effects increased with the ethanol dose. By means of RLS, BSA was found to aggregate in the presence of ethanol and particles smaller than 100 nm were observed from TEM. The fluorescence spectra showed that the intensity of the characteristic peak of BSA decreased and blue shifted, because of changes in the BSA skeleton structure, as well as alteration of the microenvironment of tryptophan (Trp) residues. The conformation changes of BSA were also shown by UV and CD spectrometry. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:66–71, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20314     

Al (III) metal augment thermal aggregation and fibrillation in protein: Role of metal toxicity in neurological diseases

Protein fibrillation is a leading cause of innumerable neurodegenerative diseases. The exact underlying mechanism associated with the formation of fibrils is yet to be known. Recently, the role of metal ions resulting into fibrillation of proteins has gained attention of the scientific community. In this piece of work, we have investigated the effect of the aluminum (Al) metal ion on the kinetics of aggregation of bovine serum albumin (BSA) protein under physiological conditions by employing several biophysical and microscopic techniques. Quenching of tryptophan fluorescence was observed along with 9 nm blue shift, demonstrating BSA becomes more hydrophobic during unfolding pathway of thermal denaturation. Moreover, ANS (8-Anilino-1-naphthalene sulfonic acid) binding shows quenching in fluorescence intensity with increasing time of incubation at 65 °C, suggesting unfolding leading to the disruption of hydrophobic patches in BSA. Besides, Thioflavin T intensity indicated a significant acceleration in BSA fibrillation at a ratio of 1:1 and 1:2 of BSA and Al (III) metal ion respectively. In addition, circular dichroism (CD) spectroscopy study revealed the transition of BSA from α-helical conformation to the β-sheet rich structure. Molecular docking analysis demonstrated significant binding affinity (−1.2 kcal/mol) of Al (III) with BSA involving Phe501, Phe506, Val575, Thr578, Gln579, Leu531 residues. Transmission electron microscopy (TEM) reaffirm augmentation of thermal-induced BSA fibril formation in the presence of Al (III) metal ions. This study highlights the metal chelating potency as the possible therapeutic target for neurological diseases.     

Effects of formaldehyde fixation on protein secondary structure: a calorimetric and infrared spectroscopic investigation

We investigated the effects of formaldehyde fixation on the secondary structure of isolated proteins (bovine serum albumin, ribonuclease A, and hemoglobin) using high-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy. Whereas thermograms obtained by scanning calorimetry on unfixed purified proteins demonstrated denaturation transitions in the 70-90 degrees C temperature range, the thermograms showed no denaturation transitions in this temperature range when the proteins had been placed in formaldehyde solutions. Thus, fixation destroyed the denaturation transition of bovine serum albumin, ribonuclease A, and hemoglobin. Infrared spectra obtained on the unfixed and fixed proteins were essentially identical. This demonstrates that the "fixed" proteins retain the secondary structure present before fixation. We therefore conclude that the cross-linking of proteins that occurs in the process of formaldehyde fixation "locks in" the secondary structure of these protein molecules.     

Simultaneous purification and polymerization method for bovine serum albumin preparation

Bovine serum albumin (BSA) has various applications in blood group serology and different research purposes. In this study purification of BSA has been compared with human serum albumin (HSA) using modified ethanol precipitation method based on the method of Cohn. The purification process was carried out under controlled conditions, particularly of ethanol concentration, pH, ionic strength and temperature. It was revealed that the produced BSA and HSA have purity more than 95%. It is obvious that HSA can be used, as a drug when the amount of its polymers is less than 5% whereas polymer generation is required in order to enhance the potentiating properties of BSA in agglutination of red cells. We propose here a simple and rapid two-step method for simultaneously purification and polymerization of BSA. By this method simply BSA with desired amount of polymers was obtained by 40% ethanol concentration.     

Structure characterization of human serum proteins in solution and dry state.

The present report describes application of advanced analytical methods to establish correlation between changes in human serum proteins of patients with coronary atherosclerosis (protein metabolism) before and after moderate beer consumption. Intrinsic fluorescence, circular dichroism (CD), differential scanning calorimetry and hydrophobicity (So) were used to study human serum proteins. Globulin and albumin from human serum (HSG and HSA, respectively) were denatured with 8 m urea as the maximal concentration. The results obtained provided evidence of differences in their secondary and tertiary structures. The thermal denaturation of HSA and HSG expressed in temperature of denaturation (Td, degrees C), enthalpy (DeltaH, kcal/mol) and entropy (DeltaS kcal/mol K) showed qualitative changes in these protein fractions, which were characterized and compared with fluorescence and CD. Number of hydrogen bonds (n) ruptured during this process was calculated from these thermodynamic parameters and then used for determination of the degree of denaturation (%D). Unfolding of HSA and HSG fractions is a result of promoted interactions between exposed functional groups, which involve conformational changes of alpha-helix, beta-sheet and aperiodic structure. Here evidence is provided that the loosening of the human serum protein structure takes place primarily in various concentrations of urea before and after beer consumption (BC). Differences in the fluorescence behavior of the proteins are attributed to disruption of the structure of proteins by denaturants as well as by the change in their compactability as a result of ethanol consumption. In summary, thermal denaturation parameters, fluorescence, So and the content of secondary structure have shown that HSG is more stable fraction than HSA.     

PM-IRRAS investigation of the interaction of serum albumin and fibrinogen with a biomedical-grade stainless steel 316LVM surface

Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) was applied to investigate the interaction of bovine serum albumin (BSA) and fibrinogen with a biomedical-grade 316LVM stainless steel surface, in terms of the adsorption thermodynamics and adsorption-induced secondary structure changes of the proteins. Highly negative apparent Gibbs energy of adsorption values revealed a spontaneous adsorption of both proteins onto the surface, accompanied by significant changes in their secondary structure. It was determined that, at saturated surface coverages, lateral interactions between the adsorbed BSA molecules induced rather extensive secondary structure changes. Fibrinogen's two coiled coils appeared to undergo negligible secondary structure changes upon adsorption of the protein, while large structural rearrangements of the protein's globular domains occurred upon adsorption. The secondary structure of adsorbed fibrinogen was not influenced by lateral interactions between the adsorbed fibrinogen molecules. PM-IRRAS was deemed to be viable for investigating protein adsorption and for obtaining information on adsorption-induced changes in their secondary structures.     

Vibrational circular dichroism spectra of protein films: thermal denaturation of bovine serum albumin

Vibrational circular dichroism (VCD) spectroscopy has been used for the first time to investigate the thermal denaturation of proteins in H(2)O solutions. Films prepared from heated aqueous solutions were used for these investigations. A well-known alpha-helical protein, bovine serum albumin (BSA), is used for this first study. Both VCD and infrared absorption results obtained for BSA films indicate that the heat treatment of BSA induces significant amounts of beta-sheet structure and that the denaturation process is irreversible. To verify the irreversible nature of thermal denaturation, optical rotation was also measured as a function of temperature in both heating and cooling cycles. These results also indicate that thermal denaturation of BSA in solution is irreversible. This study establishes the usefulness of films for VCD investigations and offers new avenues for VCD studies on biologically important systems.     

Chitosan–bovine serum albumin complex formation: A model to design an enzyme isolation method by polyelectrolyte precipitation

Interactions between a model protein (bovine serum albumin—BSA) and the cationic polyelectrolyte, chitosan (Chi), have been characterized by turbidimetry, circular dichroism and fluorescence spectroscopy. It has been found that the conformation of the BSA does not change significantly during the chain interaction between BSA and chitosan forming the non-covalently linked complex. The effects of pH, ionic strength and anions which modify the water structure around BSA were evaluated in the chitosan–BSA complex formation. A net coulombic interaction force between BSA and Chi was found as the insoluble complex formation decreased after the addition of NaCl. Around 80% of the BSA in solution precipitates with the Chi addition. A concentration of 0.05% (w/v) Chi was necessary to precipitate the protein, with a stoichiometry of 6.9 g BSA/g Chi. No modification of the tertiary and secondary structure of BSA was observed when the precipitate was dissolved by changing the pH of the medium. Chitosan proved to be a useful framework to isolate proteins with a slightly acid isoelectrical pH by means of precipitation.     

Studies on the antigenic properties of serum albumins

Bovine serum albumin (BSA) is one ofthe most widely studied proteins; its structure iswell known and its antigenic properties have beendescribed in animal models. The aimof our work was to evaluate the role of conformationon antigenicity of serum albumins. This study was performed using electrophoresisassociated with the immunoblotting technique, wheresera from children allergic to BSA were used.Data obtained in this research indicatethat serum albumin antigenicity is only partiallycorrelated to its native three-dimensional structure.Heat treatment and chemical denaturation(SDS treatment) are not able to significantly decrease its capability to bind circulating IgEs. Only thereducing treatment is able to modify the antigenicityof this protein. Moreover, a direct correlationbetween the cross-reactivity observed inimmunoblotting between different serum albumins andthe percentage of their sequence identity(phylogenetic similarity of the species) was shown.     

Spectroscopic studies of interaction between CuO nanoparticles and bovine serum albumin

Recently, the great interests in manufacturing and application of metal oxide nanoparticles in commercial and industrial products have led to focus on the potential impact of these particles on biomacromolecules. In the present study, the interaction of copper oxide (CuO) nanoparticles with bovine serum albumin (BSA) was studied by spectroscopic techniques. The zeta potential value for BSA and CuO nanoparticles with average diameter of around 50 nm at concentration of 10 μM in the deionized (DI) water were ?5.8 and ?22.5 mV, respectively. Circular dichroism studies did not show any changes in the content of secondary structure of the protein after CuO nanoparticles interaction. Fluorescence data revealed that the fluorescence quenching of BSA by CuO nanoparticles was the result of the formed complex of CuO nanoparticles – BSA. Binding constants and other thermodynamic parameters were determined at three different temperatures. The hydrogen bond interactions are the predominant intermolecular forces to stabilize the CuO nanoparticle – BSA complex. This study provides important insight into the interaction of CuO nanoparticles with proteins, which may be of importance for further application of these nanoparticles in biomedical applications.     

Binding interaction of a biological photosensitizer with serum albumins: a biophysical study

A photophysical study on the binding interaction of an efficient cancer cell photosensitizer, norharmane (NHM), with model transport proteins, bovine serum albumin (BSA) and human serum albumin (HSA), has been performed using a combination of steady-state and time-resolved fluorescence techniques. The emission profile undergoes a remarkable change upon addition of the proteins to the buffered aqueous solution of the photosensitizer. The polarity-dependent prototropic transformation is responsible for the remarkable sensitivity of this biological fluorophore to the protein environments. A marked increase in the fluorescence anisotropy in the proteinous environments indicates that the albumin proteins introduce motional restriction on the drug molecule. Light has been thrown on the denaturing action of urea on the probe-bound protein. The probable binding site of the drug in proteins has also been assessed from the combination of denaturation study, micropolarity measurement, and fluorescence resonance energy transfer (FRET) study. The present study suggests that the stability of serum albumins is enhanced upon binding with the drug.     

Secondary structural changes of large and small fragments of bovine serum albumin in thermal denaturation and in sodium dodecyl sulfate denaturation

The helicities in various fragments of bovine serum albumin (BSA) were examined in the thermal denaturation and in sodium docecyl sulfate (SDS) denaturation. The thermal denaturation was examined in a temperature range between 2 and 65°C. The helicity decreased with a rise of temperature and it recovered to some degree upon cooling temperature. A rather high reversibility was observed in the BSA fragments, which were located in the N-terminal of the parent protein and then contained the first large loop with no disulfide bridge. The high reversibility was available also for the helicity in the first large loop of the fragment, disulfide bridges of which were reduced. The fragments, which were smaller than one domain, became unstable in the SDS denaturation. The helicities of such fragments decreased in lower SDS concentrations compared with those of the intact BSA and the large fragments, which contained one or more domains. A resistance to the SDS denaturation appeared in the helices of every large loop even after the fragmentation. On the other hand, helicities of the fragments decreased to 20–25% upon the reduction of disulfide bridges. However, the helicities of these fragments increased to 35–40% in the SDS denaturation.     

Studies on the antigenic properties of serum albumins

Summary Bovine serum albumin (BSA) is one of the most widely studied proteins; its structure is well known and its antigenic properties have been described in animal models. The aim of our work was to evaluate the role of conformation on antigenicity of serum albumins. This study was performed using electrophoresis associated with the immunoblotting technique, where sera from children allergic to BSA were used. Data obtained in this research indicate that serum albumin antigenicity is only partially correlated to its native three-dimensional structure. Heat treatment and chemical denaturation (SDS treatment) are not able to significantly decrease its capability to bind circulating IgEs. Only the reducing treatment is able to modify the antigenicity of this protein. Moreover, a direct correlation between the cross-reactivity observed in immunoblotting between different serum albumins and the percentage of their sequence identity (phylogenetic similarity of the species) was shown.