Partitioning Of Bovine Serum Albumin Research Articles

Tailoring the partitioning of proteins using ionic liquids as adjuvants in polymer-polymer aqueous biphasic systems

Aqueous biphasic systems (ABS) are promising for proteins purification; however, when dealing with samples comprising several proteins, the selectivity towards a target protein is difficult to achieve. In this work, the addition of ionic liquids (ILs) as adjuvants (5 wt% and 10 wt%) in ABS composed of polyethylene glycol (PEG) and dextran was investigated to tailor proteins partitioning between the coexisting phases. The liquid-liquid phase diagrams were determined at 298 K, followed by partition studies of three proteins (bovine serum albumin (BSA), immunoglobulin G (IgG), and cytochrome C (Cyt C)). Partition coefficients of IgG and BSA indicate the preference of both proteins to the dextran-rich phase, whereas Cyt C has no preferential partitioning between the phases. The addition of chloride-based ILs as adjuvants allows to increase or decrease the partition coefficients, thus tailoring the proteins partitioning between the phases. BSA partitioning essentially depends on the IL content in each phase, whereas Cyt C and IgG partitioning is ruled by the ILs chemical structure and established interactions. Molecular docking was carried out to address the ILs effect on the proteins partitioning, supporting experimental observations, while identifying the specific interactions occurring. The partition of each protein in polymer-salt ABS with ILs as adjuvants was determined, demonstrating the higher tailoring ability of polymer-polymer ABS when adding ILs. Finally, the partition of each protein in presence of the remaining ones was determined, as well as the selectivity of the studied systems to separate each pair of proteins, paving the way for their use in liquid-liquid chromatography.

Thermodynamics of acetaminophen and bovine serum albumin partitioning in ternary aqueous solutions comprising polyethylene glycol dimethyl ether 250 and choline bitartrate: Liquid-liquid equilibria, volumetric and acoustic investigations

The present work reports the biocompatible and nontoxic aqueous biphasic system, ABS, for acetaminophen and bovine serum albumin, BSA, separation. First, the binodal curves and tie-lines for {polyethylene glycol dimethyl ether 250 (PEGDME250) + choline bitartrate ([Ch][Bit]) + water} system was measured at different temperatures under atmospheric pressure (≈ 85 kPa). The phase separation in this ABS was studied by the salting-out coefficents obtained from tie-lines correaltion with setschenow equation. The results indicated that the biphasic region was enhanced by enhancing temperatre. The experimental data were fitted by two semi-empirical equations including Marchuk and Zafarani-Moattar equations for binodals and Othmer-Tobias, Bancroft, Osmotic virial equations, and e-or m-NRTL models for the tie-lines. Later, the performance of this ABS in acetaminophen and BSA partitioning was examined via calculation of partition coefficients and extraction efficiencies at different tie-lines and temperatures, the trend showed that the acetaminophen could be partitioned preferentially into the polymer-rich top phase with a single step extraction efficiency up to 80%, in where the BSA was effectively transferred into the [Ch][Bit]-rich bottom phase. In order to evaluate the driving-force for acetaminophen preferentially transferring to top phase, density and sound speed for systems (acetaminophen + PEGDME250 + water) and (acetaminophen + [Ch][Bit] + water) were determined at 298.15 K and the obtained results utilized for calculation the infinite molar volume and compressibility, and the transfer molar volume and compressibility. From density and sound speed results, it may be concluded that the favorable interactions between acetaminophen and PEGDME250 is responsible for preferentially acetaminophen transferring into top phase.

Thermodynamic study of aqueous two-phase systems containing biocompatible cholinium aminoate ionic-liquids and polyethylene glycol di-methyl ether 250 and their performances for bovine serum albumin separation

Aqueous two-phase systems, ATPSs, are known as an interesting media for the separation and purification of vital biomolecules in biotechnology applications. From this viewpoint, the ambition of this work is investigating ability of cholinium aminoate ([Ch][AA]) as novel, low toxic and high biodegradable ionic liquids, including of cholinium glycinate ([Ch][Gly]), cholinium prolinate ([Ch][l-Pro]) and cholinium valinate ([Ch][l-Val]) for inducing ATPSs with polyethylene glycol di-methyl ether 250, PEGDME250, and then evaluating their performances for partitioning of bovine serum albumin, BSA, as a model protein. For achieving this purpose, binodal curves and tie-lines were experimentally measured at T = (298.15, 308.15 and 318.15) K under atmospheric pressure (≈85 kPa) for all of the aforementioned systems. The influences of ionic liquid anion and temperature were searched systematically on the binodal curve and tie-lines. The phase splitting ability in these systems was assessed by utilizing the effective exclude volume theory from which the locations of binodals may be predicted and the Setschenow-type equation which provides salting-out coefficients by fitting the experimental tie-lines to this equation. Two empirical equations including of Merchuk equation were applied in order to represent the binodal data. Moreover, the Othmer-Tobias and Bancraft, Osmotic virial equations and two versions of NRTL models (m- or e-NRTL) were selected to correlate the tie-lines. In addition, the partition behaviour of BSA was studied by measuring of partition coefficient and extraction efficiency in the mentioned systems. The measured partition coefficients for BSA were discussed on the basis of physiochemical properties of amino acids involved in ionic liquid structure.

Reverse micellar partitioning of Bovine Serum Albumin with novel system

To overcome the difficulties associated with the conventional extraction process like poor selective extrac- tion of biomolecule and scale up of the process, the reverse micellar system consist of AOT/n-heptanol was considered to extract Bovine Serum Albumin (BSA) as a model biomolecule. The maximum forward extraction of BSA from aqueous phase to micelle phase was observed at AOT concentration 160 mM, aque- ous phase pH value of 4, NaCl concentration 0.8 M and 95% back extraction of BSA from micelle phase to stripping phase was obtained at 1 M NaCl concentration with the pH of 7.5. HPLC analysis confirmed the stability of BSA during extraction. The size and water content of the reverse micelle was also reported. The obtained results emphasize the application of the AOT/n-heptanol reverse micellar system for the extraction of BSA and may be utilized for the selective extraction of similar hydrophilic proteins from the complex sources.

Reverse micellar partitioning of Bovine Serum Albumin with novel system

Abstract To overcome the difficulties associated with the conventional extraction process like poor selective extraction of biomolecule and scale up of the process, the reverse micellar system consist of AOT/n-heptanol was considered to extract Bovine Serum Albumin (BSA) as a model biomolecule. The maximum forward extraction of BSA from aqueous phase to micelle phase was observed at AOT concentration 160 mM, aqueous phase pH value of 4, NaCl concentration 0.8 M and 95% back extraction of BSA from micelle phase to stripping phase was obtained at 1 M NaCl concentration with the pH of 7.5. HPLC analysis confirmed the stability of BSA during extraction. The size and water content of the reverse micelle was also reported. The obtained results emphasize the application of the AOT/n-heptanol reverse micellar system for the extraction of BSA and may be utilized for the selective extraction of similar hydrophilic proteins from the complex sources.

Study of the applicability of non-conventional aqueous two-phase systems in counter-current and centrifugal partition chromatography

Aqueous two-phase systems composed of imidazolium-based ionic liquids and phosphate salts were evaluated for their applicability in liquid–liquid chromatography. The influence of the nature of ionic liquid anion and cation on the partitioning of bovine serum albumin, lysozyme and myoglobin was investigated. A mixture of K2HPO4 and KH2PO4 in a ratio of 1.82:1wt/wt was used in all of the tested biphasic systems to adjust the pH to a range of 7–8. The results show that more hydrophobic cations decrease the partition coefficients of the proteins in the biphasic systems and outweigh the effect of the anion on the distribution of the macromolecules. Viscosities and densities of the biphasic systems were in a suitable range for liquid–liquid chromatography. Even though the partition coefficients were too high for a conventional batch operation mode, these aqueous two-phase systems show favorable properties for protein capturing in liquid–liquid chromatographic columns. Additionally, the possible application of ionic liquids as modifiers in polyethylene glycol (PEG)-based aqueous two-phase systems was investigated. It could be demonstrated that ionic liquids alter the partition coefficients of the proteins.

Characterization of bovine serum albumin partitioning behaviors in polymer-salt aqueous two-phase systems

In this paper, a linear relationship is proposed relating the natural logarithm of partition coefficient, ln K for protein partitioning in poly (ethylene glycol) (PEG)-phosphate aqueous two-phase system (ATPS) to the square of tie-line length (TLL(2)). This relationship provides good fits (r(2) > 0.98) to the partition of bovine serum albumin (BSA) in PEG (1450 g/mol, 2000 g/mol, 3350 g/mol, and 4000 g/mol)-phosphate ATPS with TLL of 25.0-50.0% (w/w) at pH 7.0. Results also showed that the plot of ln K against pH for BSA partitioning in the ATPS containing 33.0% (w/w) PEG1450 and 8.0% (w/w) phosphate with varied working pH between 6.0 and 9.0 exhibited a linear relationship which is in good agreement (r(2) = 0.94) with the proposed relationship, ln K = α' pH + β'. These results suggested that both the relationships proposed could be applied to correlate and elucidate the partition behavior of biomolecules in the polymer-salt ATPS. The influence of other system parameters on the partition behavior of BSA was also investigated. An optimum BSA yield of 90.80% in the top phase and K of 2.40 was achieved in an ATPS constituted with 33.0% (w/w) PEG 1450 and 8.0% (w/w) phosphate in the presence of 8.5% (w/w) sodium chloride (NaCl) at pH 9.0 for 0.3% (w/w) BSA load.

Interfacial partitioning behaviour of bovine serum albumin in polymer-salt aqueous two-phase system

A relationship is proposed for the interfacial partitioning of protein in poly(ethylene glycol) (PEG)-phosphate aqueous two-phase system (ATPS). The relationship relates the natural logarithm of interfacial partition coefficient, ln G to the PEG concentration difference between the top and bottom phases, Δ[PEG], with the equation ln G=AΔ[PEG]+B. Results showed that this relationship provides good fits to the partition of bovine serum albumin (BSA) in ATPS which is comprised of phosphate and PEG of four different molecular weight 1450g/mol, 2000g/mol, 3350g/mol and 4000g/mol, with the tie-line length (TLL) in the range of 44-60% (w/w) at pH 7.0. The decrease of A values with the increase of PEG molecular weight indicates that the correlation between ln G and Δ[PEG] decreases with the increase in PEG molecular weight and the presence of protein-polymer hydrophobic interaction. When temperature was increased, a non-linear relationship of ln G inversely proportional to temperature was observed. The amount of proteins adsorbed at the interface increased proportionally with the amount of BSA loaded whereas the partition coefficient, K remained relatively constant. The relationship proposed could be applied to elucidate interfacial partitioning behaviour of other biomolecules in polymer-salt ATPS.

Lysozyme and bovine serum albumin partitioning in polyethylene glycol–phenylalanine conjugate polymer/salt aqueous two-phase systems

Polyethylene glycol (PEG) with average molecular weight of 8000 was used as a model polymer and modified by introducing an amino acid (phenylalanine) in its hydroxyl terminals. The effect of different percentages of the conjugate polymer in relation to the total polymer on binodal curve of PEG-8000–potassium phosphate buffer (KPB), pH 7.2 aqueous two-phase systems and solute partitioning was explored. The partition of lysozyme (Lyz) and bovine serum albumin (BSA) and also of the conjugated polymer was evaluated in ATPS composed of 17% (w/w) total polymer and 12.5% (w/w) KPB, pH 7.2. Lyz partition profile follows the expected trend decreasing with the increase of modified polymer concentration. However BSA shows a partition profile that is approximately symmetrical to the one observed for the partition of the conjugate polymer, which depends on PEG–phenylalanine concentration in the system.Determination of the relative hydrophobicity of the equilibrium phases and of the protein surface hydrophobicity (So) delivered information that shed some light in the mechanisms controlling the partitioning behavior. Therefore, it becomes clear that the hydrophobic character of the protein taken together with the molecular weight explain the different behavior of these two proteins with the increase of PEG–phenylalanine substitution in the systems.

Phase equilibrium and protein partitioning in aqueous micellar two-phase system composed of surfactant and polymer

The phase equilibrium and protein partitioning in the aqueous micellar two-phase system composed of nonionic surfactant polyoxyethylene octyl phenyl ether (Triton X-100) and polymer polyethylene glycol (PEG) with different low molecular weights are studied. The phase diagrams under various PEG molecular weight, salt concentration and pH value are determined at 298K. The results show that the biphase areas of the aqueous micellar two-phase system are expanded with an increase in PEG molecular weight or salt concentration, while the biphase areas and binodal curves are nearly unchanged by varying pH values. With raising PEG molecular weight, the tie lines lengths increase, and the tie lines slopes decrease first and then increase. Using lysozyme and bovine serum albumin (BSA) as model proteins, protein partitioning in the aqueous micellar two-phase system is investigated. As the hydrophilic proteins, both lysozyme and BSA partition preferably to the PEG-rich phases. The partition coefficients of proteins increase with increasing tie line length, or with decreasing PEG molecular weight and protein molecular size. These results indicate that the excluded volume interactions play an important role in protein partitioning in the aqueous micellar two-phase system composed of surfactant and polymer.

Extraction Separation of BSA in Aqueous Two-Phase Systems of Anionic and Cationic Surfactant Mixtures

Aqueous two-phase systems (ATPS) are formed in mixtures of sodium dodecylsulfate (SDS), cetyltrimethyl ammonium bromide (CTAB), and sodium bromide (NaBr). Two different kinds of ATPS appear when CTAB and SDS surfactants are in excess, respectively. Such ATPS may provide a new, useful partitioning system for separation of bovine serum albumin (BSA). The partitioning of BSA was studied in these systems. The results show that the partitioning is affected by the electrical property, the hydrophobicity and the structure of liquid crystal (LC), which exists in the top phase of the ATPS. BSA is extracted into the top phase with higher distribution coefficient when LC and BSA are oppositely charged. The hydrophobicity of LC, which can be improved by increasing the length of alkyl group, enhances the distribution coefficient. The hydrophobicity of lamellar LC and hexagonal LC is stronger than that of cubic LC, which causes extractive capability of the former is higher than the latter.

Synergistic extraction of bovine serum albumin using polyethylene glycol based aqueous biphasic system

An attempt was made to study the partitioning behaviour of bovine serum albumin (BSA) in the PEG (polyethylene glycol)–sodium citrate based aqueous two-phase system. The investigation focus on the factors that influence partitioning of BSA, including PEG molar mass (4000, 6000 and 8000), and concentration, phase forming salt concentration, pH and temperature. The concentration of the BSA was measured by Bradford method. The partition of BSA to the salt rich (bottom) phase is strongly favoured in systems with higher molar mass of PEG, due to increase in hydrophobic nature of PEG with molar mass. The partition coefficient value tends to decrease with increase in PEG concentration, due to diminution of free volume available for the protein to accommodate in the top PEG rich phase. The enhanced affinity of BSA for the PEG phase increases with increase in sodium citrate concentration, due to an increase of the salting-out effect in the bottom phase. The results indicates that BSA partitioning equilibrium is displaced to the citrate rich-phase as the system pH increases, therefore the partition coefficient value changes depending upon the isoelectric point and protein charge. Furthermore, it is observed that the BSA partitions to the bottom phase at higher temperature.

Influence of Phase Compositions, pH and Temperature on Bovine Serum Albumin Partitioning in Aqueous Biphasic System

The partitioning of bovine serum albumin (BSA) was investigated in polyethylene glycol (PEG) 4000-sodium citrate system at 308K and 318K with varying pHs and tie line lengths (TLL). The systems were prepared at a pH of 5.2, 7.1 and 9.2 with different TLL compositions. The affinity of the BSA for the lower phase increases with an increase in pH, which is due to an increase of trivalent to divalent citrate ions in the two phase system. The affinity of the BSA to the upper phase increases with an increase in TLL, which is due to the increase in available free volume in the upper phase. As the temperature increases, the partition coefficient of BSA shows increment.

Application of aqueous mixtures of polypropylene glycol or polyethylene glycol with salts in proteomic analysis

Partitioning of Bovine serum albumin (BSA), β-lactoglobulin (β-LG) and zein as model proteins in aqueous two-phase systems (S) containing polypropylene glycol (PPG425) or polyethylene glycol (PEG 6000) and salts (MgSO4, (NH4)2SO4, Na2SO4) is presented in this paper. The effects of different factors such as tie-line length, salt type and polymer type on the partition coefficient and recovery percent of proteins were analysed. The model proteins were separated by these systems (S) and directly used for gel electrophoresis without separating the target proteins from phase-forming reagents. The results revealed that the S, studied in this work could be used as a novel prefractionation method in proteomic analysis and could separate proteomic proteins in multigroup by one step extraction.

Bovine serum albumin partitioning in polyethylene glycol (PEG)/potassium citrate aqueous two-phase systems

The extraction and back-extraction of bovine serum albumin (BSA) have been studied by liquid–liquid extraction with poly(ethylene glycol) (PEG)/potassium citrate aqueous two-phase system (ATPS). In this work, the ATPS was examined with regard to the effects of PEG molecular weight (PEG 1000, 2000, 4000 and 6000), PEG and potassium citrate concentration, BSA concentration ( C BSA) and pH on BSA partition. The pH was found to have significant effects on BSA partition with low molecular weight PEG 1000. The yield of the BSA, 99%, was obtained in the top phase under the following conditions: 19% (w/w) PEG 1000, 20% (w/w) potassium citrate and 0.75 mg/g C BSA at pH 7.0 and 30 °C. BSA can be re-extracted to a new citrate phase by decreasing the pH of the system with a 92% yield. The back-extraction not only separates the BSA from the polymer, but also allows the polymer to be recycled. The global yield ( Y e + Y be) is up to 91%.

Vinyl-2-pyrrolidone derivatized guar gum based aqueous two-phase system

Aqueous two-phase systems (ATPs) are formed when certain combinations of two polymers or polymer and salt are added to water. Phase separation occurs at certain specific concentrations that have similar densities and low interfacial tensions. The present study involves synthesis and application of a novel derivatized guar in aqueous two-phase system. The derivation of guar gum was performed by grafting of 1-vinyl-2-pyrrolidone by a redox reaction in aqueous medium ceric ammonium nitrate (CAN) and potassium peroxydisulfate (K 2S 2O 8). The ratio of grafting efficiency (%GE) and ratio of grafting (%G) was 56% and 70.1% respectively. The grafted copolymers were characterized for percent yield and FTIR absorbance. A new aqueous two-phase extraction protocol was established using a mixture of derivatized guar and dextran. A phase diagram was constructed based on the phase separation exhibited by the system at 25 °C. The partition coefficient was determined for standard bovine serum albumin (BSA), lactate oxidase (LO) and phytase. The partitioning coefficients were 0.479, 1.981, and 2.586 respectively in the Dextran (2 million)/V-GG/H 2O system. The influence of an ionic salt sodium chloride (NaCl) was investigated on the partitioning of BSA.

Partition Behavior of Bovine Serum Albumin in PEG2000‐Sodium Citrate‐Water Based Aqueous Two‐Phase System

The effect of phase forming components, pH, neutral salt (NaCl) addition on partitioning of bovine serum albumin (BSA) in PEG2000‐sodium citrate based aqueous two‐phase system was investigated at 25°, 35°, and 45°C. The systems were prepared by varying the phase forming components concentration at a pH of 5.0, 6.0, 7.0, 8.0, and 9.0 with different NaCl salt concentrations of 0.05 M, 0.1 M, 1.5 M, 0.2 M, 0.3 M, and 0.4 M. The affinity of the BSA for the lower phase increases with increase in pH, due to increase in ratio of trivalent to divalent citrate ions in the two‐phase system. It was confirmed that the partition of BSA depends on the relative hydrophobicity of the compounds as well as their charge. Optimum level of pH and NaCl concentration for the partitioning of bovine serum albumin was determined.

Partitioning of model proteins in aqueous two-phase systems containing polyethylene glycol and ammonium carbamate

The phase equilibrium and the partitioning of bovine serum albumin, trypsin and lysozyme in aqueous two-phase systems containing ammonium carbamate and polyethylene glycol 1500, 4000 and 6000 are presented in this communication. Partition coefficients, defined as the ratio of protein concentration in the polymer phase to that in the salt phase, vary in the range of 0.1–0.8 for bovine serum albumin, 1.0–2.4 for trypsin and from 2.3 to 9.0 for lysozyme, depending on the polymer chain size and on the tie-line length. The analysis of the separation factor, defined as the ratio of partition coefficients of two proteins, shows that high degrees of separation can be achieved. These results indicate that aqueous two-phase systems containing ammonium carbamate and polyethylene glycol can be an alternative to be considered for protein downstream processing.

Study of the serum albumin-polyethyleneglycol interaction to predict the protein partitioning in aqueous two-phase systems

The theoretical framework based only on the excluded volume forces is not enough to explain the bovine serum albumin partitioning behaviour in aqueous biphasic systems. The goal of this work is to look at the phase separation via the polymer effect on the water structure. Our findings suggest that polyethyleneglycol 600-protein interaction is conducted by van der Waals forces between the hydrophobic surfaces from PEG and protein molecules, which implies the rupture of hydrogen bonds from the structured water in their neighbours. Therefore, the protein will concentrate in the most water-structured phase (polyethyleneglycol) in order to reach the minimal free energy condition. When polyethyleneglycol molecular weight increases, its exclusion from protein surface prevails, thus pushing the bovine serum albumin to the bottom phase.

Protein partitioning in thermoseparating systems of a charged hydrophobically modified ethylene oxide polymer

The phase behavior of a thermoseparating cationic hydrophobically modified ethylene oxide polymer (HM-EO) containing tertiary amines has been investigated at different pH, salt and sodium dodecyl sulfate (SDS) concentrations, in order to find a water/HM-EO two-phase system suitable for protein partitioning. The used polymer forms micellar aggregates that can be charged. By changing pH and SDS concentrations the netcharge of the SDS/HM-EO aggregate can be shifted from positive to negative. Bovine serum albumin (BSA) and lysozyme were partitioned in the thermoseparated two-phase systems of the cationic polymer at different pH, salt and SDS concentrations. The dominant attractive interactions between the polymer aggregates and the studied proteins were shown to be of electrostatic (Coulomb) nature rather than hydrophobic interaction. At low ionic strength the positively charged polymeric aggregates attracted negatively charged BSA and repelled positively charged lysozyme. Upon addition of SDS the negatively charged aggregates attracted lysozyme and repelled BSA. Thus, it was possible to direct proteins with different charges to the polymeric phase and redirect them to a polymer-depleted phase by changing the netcharge of the polymeric aggregates. The effect of different salts on the partitioning of BSA in a system of slightly positively charged HM-EO was studied. NaCl and KBr have a significant effect on driving the BSA to the polymer-depleted phase, whereas KF and K 2SO 4 have a smaller effect on the partitioning. The cloud point temperature of the charged polymer decreased upon addition of SDS near the isoelectric molar ratio of SDS to polymer and also upon salt addition. In the latter case the decrease was smaller than expected from model calculations based on Flory–Huggins theory, which were performed for a charged thermoseparating polymer at different charges and salt concentrations.