Mechanism of bovine serum albumin aggregation during ultrafiltration

Protein fouling is a critical problem for ultrafiltration. In this study, we adopted bovine serum albumin (BSA) as a model protein and polysulfone membrane as a typical ultrafiltration membrane. We then investigated the factors of the protein denaturation and aggregation, such as stirring shear stress and intermolecular exchange of disulfide during ultrafiltration, and discussed the BSA fouling mechanism. Fourier transform-infrared analysis revealed that magnetic stirring did not cause any difference in the secondary structural change of BSA gel-like deposits on the ultrafiltration membrane. BSA aggregates were collected from BSA gel-like deposits on the ultrafiltration membrane by centrifugation. Polyacrylamide gel electrophoresis in SDS analysis of BSA aggregates proved that the major binding of the BSA aggregates involved intermolecular disulfhydryl binding and that capping the free thiol group in BSA molecules with cysteine induced a remarkable decrease in the amount of the BSA aggregates during ultrafiltration. We concluded that one of the main factors in the BSA aggregation during ultrafiltration is the intermolecular exchange of disulfide through cysteinyl residue. We also found that the BSA aggregation caused a decrease in alpha-helix from 66% to 50% and an increase in beta-sheet from 20% to 36%, which was presumably because the cysteine residues associated with the intermolecular disulfide bonds had been located in alpha-helices. Copyright John Wiley & Sons, Inc.     

Application of a pulsed electric field to cross-flow ultrafiltration of protein solution

The application of pulsed electric field was investigated in the crossflow ultrafiltration of BSA (bovine serum albumn) to economize the application time of electric current as well as to avoid inherent problems of long-term application of electric field. During the application of various cyclic patterns of pulsed electric current, the averaged filtration flowrate and the degree of concentration were maintained higher than those obtained in the absence of electric current application. The temperature increase, pH change, and BSA loss by electrodeposition were all negligible during the operation. The averaged filtration flowrate increased as the ON/OFF duration ratio of electric current was higher and as the period of ON/OFF cycle was shorter. The re-establishment of concentration polarization was dependent to the duration of current OFF state and, therefore, a longer duration of OFF state was not favorable in maintaining higher filtration flow rate. Although the averaged filtration flowrate was enhanced as the magnitude of electric current increased, the flowrate enhancement became smaller as the magnitude of current increased because there exists a current value above which the degree of electrokinetic depolarization is no further improved.     

Effects of protein-protein interaction in ultrafiltration based fractionation processes

This paper discusses the use of pulsed sample injection ultrafiltration (UF) for investigating protein-protein interaction, particularly its effect on protein transmission through UF membranes. Several binary protein mixtures were investigated; the proteins in each mixture being selected such that one of the proteins in the pair would be preferentially transmitted while the other would be either totally or substantially retained. The "retained" protein either decreased or increased or did not affect the sieving coefficient of the "transmitted" protein, this depending the type of protein-protein interaction, that is, associative, repulsive, or neutral. The type of protein-protein interaction depended on the particular protein pair under investigation as well as on the operating conditions used (pH and salt concentration). The magnitude of either decrease or increase in transmission of a preferentially transmitted protein due to the presence of a retained protein was found to be independent of the manner in which the proteins were injected into the system, that is, simultaneous or sequential. These magnitudes however correlated well with the ratio of the two proteins present in the feed.     

Bovine serum albumin-bovine hemoglobin conjugate as a candidate blood substitute

A bovine serum albumin-bovine hemoglobin conjugate was prepared using 3-maleimidobenzoic acid N-hydroxysuccinimide ester as cross-linker. The conjugate was purified using DEAE Sepharose. It had an M _r of 127 kDa. Its P₅₀ (half-saturated O₂ pressure) value and Hill coefficient were 27 mm Hg and 2, respectively.     

A method of chemiluminescence coupled with ultrafiltration for investigating the interaction between ibuprofen and human serum albumin

In acidic media, ibuprofen substantially enhanced the weak chemiluminescence (CL) produced by sodium sulfite and potassium permanganate. The increased signals were linearly correlated with ibuprofen concentrations ranging from 1.2 × 10^‐3 to 4.8 μM, with a detection limit of 4.8 × 10^‐4 μM. Two ultrafiltration (UF) membranes were used to construct a unit for trapping 0.15 and 0.75 μM human serum albumin (HSA) and coupled online with the CL system. At low HSA concentrations, the numbers of bound molecules per binding site were calculated to be 0.9 for Sudlow site I and 6.2 for Sudlow site II. The association constants on these binding sites were 5.9 × 10⁵ and 3.4 × 10⁴ M^‐1, respectively. Our CL–UF protocol presents a rapid and sensitive method for studies on drug–protein interaction. Copyright © 2012 John Wiley & Sons, Ltd.     

Fractionation of bovine serum albumin and monoclonal antibody alemtuzumab using carrier phase ultrafiltration

Protein transmission and hence selectivity of separation can be significantly affected by solution pH and ionic strength in protein fractionation using ultrafiltration. Using parameter scanning ultrafiltration, the transmission of bovine serum albumin (BSA) and monoclonal antibody alemtuzumab (Campath-1H) through 300 kDa polyethersulfone (PES) ultrafiltration membranes were studied over a range of pH and salt concentrations, with focus on the likely conditions for achieving "reverse selectivity," i.e., obtaining purified alemtuzumab (approximately 155 kDa) in the permeate. Experimental results demonstrate that the reverse selectivity could be obtained by manipulating the operating conditions such as the solution pH, ionic strength, permeate flux, and system hydrodynamics. With a two-stage batch ultrafiltration process under suitable conditions, the monoclonal antibody alemtuzumab with a purity of > 98% was obtained in the permeate from a feed solution initially containing 0.50 g/l each of BSA and alemtuzumab. Further purity can be expected by selecting more suitable membranes and optimizing operating conditions.     

Separation albumin-PEG: transmission of PEG through ultrafiltration membranes

Transmission of polyethylene glycol (PEG) through ultrafiltration membranes has been studied under various operating conditions of pressure, crossflow, and concentration, using different membranes cut-offs and two module designs with the aim of understanding the separation of PEG from BSA. The influence of protein adsorption and fouling of the choice of a membrane has also been considered. Retention depends in general on the molecule to average pore size ratio, as expected, but also on concentration polarization. Accordingly, all operating and design parameters favoring concentration polarization lead to higher transmission. At high fluxes, flexible macromolecules can pass through the membrane, even if the random coil is larger than the apparent average pore. From a process selectivity point of view, the best way to separate PEG from BSA would be to use a membrane totally retaining BSA and to enhance concentration polarization of PEG. Unfortunately, such conditions also increase fouling and concentration polarization by BSA, which limits flux and thus PEG concentration polarization and transmission. Consequences of such conditions on separation efficiency are discussed. (c) 1993 Wiley & Sons, Inc.     

Bovine serum albumin‐confined silver nanoclusters as fluorometric probe for detection of biothiols

Fluorescent bovine serum albumin‐confined silver nanoclusters (BSA–AgNCs) were demonstrated to be a novel and environmentally friendly probe for the rapid detection of biothiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH). The sensing was ascribed to the strong affinity between the mercapto group of the biothiols and the silver nanoclusters. The fluorescence intensity of BSA–AgNCs was quenched efficiently on increasing the concentration of biothiol, corresponding with a red‐shift in emission wavelength. However, the fluorescence of the silver nanoclusters was almost unchanged in the presence of other α‐amino acids at 10‐fold higher concentrations. By virtue of this specific response, a new, simple and rapid fluorescent method for detecting biothiols has been developed. The linear ranges for Cys, Hcy and GSH were 2.0 × 10^‐6 to 9.0 × 10^‐5 M (R² = 0.994), 2.0 × 10^‐6 to 1.2 × 10^‐4 M (R² = 0.996) and 1.0 × 10^‐5 to 8.0 × 10^‐5 M (R² = 0.980), respectively. The detection limits were 8.1 × 10^‐7 M for Cys, 1.0 × 10^‐6 M for Hcy and 1.1 × 10^‐6 M for GSH. Our proposed method was successfully applied to the determination of thiols in human plasma and the recovery was 94.83–105.24%. It is potentially applicable to protein‐stabilized silver nanoclusters in a chemical or biochemical sensing system. Copyright © 2014 John Wiley & Sons, Ltd.     

Bovine serum albumin partitioning in an aqueous two-phase system: Effect of pH and sodium chloride concentration

The partitioning of bovine serum albumin (BSA) in a polyethylene glycol 3350 (8% w/w)–dextran 37 500 (6% w/w)–0.05 M phosphate aqueous two-phase was investigated at different pHs, at varying concentrations of sodium chloride at 20°C. The effect of NaCl concentration on the partition coefficient of BSA was studied for the PEG–dx systems with initial pH values of 4.2, 5.0, 7.0, 9.0, and 9.8. The NaCl concentrations in the phase systems with constant pH value were 0.06, 0.1, 0.2, 0.3, and 0.34 M. It was observed that the BSA partition coefficient decreased at concentrations smaller than 0.2 M NaCl and increased at concentrations greater than 0.2 M NaCl for all systems with initial pHs of 4.2, 5.0, 7.0, 9.0, and 9.8. It was also seen that the partition coefficient of BSA decreased as the pH of the aqueous two-phase systems increased at any NaCl salt concentration studied.     

Rapid protein separation and diffusion coefficient measurement by frit inlet flow field-flow fractionation.

In this study three flow field-flow fractionation (flow FFF) channels are utilized for the separation of proteins and for the simultaneous measurement of their translational diffusion coefficients, D. One channel has a traditional sample inlet, whereas the other two incorporate a frit inlet design that permits more convenient and rapid sample introduction. The dependence of retention time on D, which leads to differential elution and the opportunity to measure D for protein peaks purified by the flow FFF process, is described theoretically and examined experimentally. Factors affecting band broadening, resolution, and optimization are also examined. The separation of proteins is achieved in the time range 4-20 min. Partial resolution is achieved in multiple runs requiring 2 min each. Values of D calculated from retention times are reported for 15 proteins. These include two protein dimers (bovine serum albumin and gamma-globulin) not ordinarily accessible to measurement. The D values from the three channels are compared with one another and with literature data. Reasonable consistency (within 3-4%) is found. High-speed repetitive runs can be used to acquire multiple values of D in time intervals as short as 1 min.     

Albumin denaturation during ultrafiltration: effects of operating conditions and consequences on membrane fouling

Ultrafiltration of high-purity grade bovine serum albumin has been carried out under various temperature between 5 and 30 degrees C and at various cross-flow velocities, pressures, and concentrations with the aim of studying protein denaturation and its consequences on the process. Three different pump heads have been tested. Denaturation of proteins in solution has been monitored by laser light scattering and size exclusion chromatography. The rate of protein denaturation increases with temperature, cross-flow, and time. It is observed that membrane fouling is different whether denaturation has occurred or not. Under high-concentration polarization, denaturation can occur in the boundary layer if the wall concentration exceeds 400 g/L. It is shown how the residence time, operating temperature, and pressure play an important part in membrane fouling. This can provide guidelines for process design and control.     

羧甲基化壳聚糖季铵盐与牛血清白蛋白的相互作用研究

应用荧光光谱研究了羧甲基化壳聚糖季铵盐(CMCQA)与牛血清白蛋白(BSA)的相互作用.研究表明:CMCQA对BSA内源性荧光猝灭机制属于CMCQA和BSA形成复合物所引起的静态猝灭.在室温下,二者的结合常数为2.45×104 L/mol,结合位点数为1.04.二者主要靠静电引力相互作用.     

Structural studies of bovine,equine, and leporine serum albumin complexes with naproxen

Serum albumin, a protein naturally abundant in blood plasma, shows remarkable ligand binding properties of numerous endogenous and exogenous compounds. Most of serum albumin binding sites are able to interact with more than one class of ligands. Determining the protein‐ligand interactions among mammalian serum albumins is essential for understanding the complexity of this transporter. We present three crystal structures of serum albumins in complexes with naproxen (NPS): bovine (BSA‐NPS), equine (ESA‐NPS), and leporine (LSA‐NPS) determined to 2.58 Å (C2), 2.42 Å (P6₁), and 2.73 Å (P2₁2₁2₁) resolutions, respectively. A comparison of the structurally investigated complexes with the analogous complex of human serum albumin (HSA‐NPS) revealed surprising differences in the number and distribution of naproxen binding sites. Bovine and leporine serum albumins possess three NPS binding sites, but ESA has only two. All three complexes of albumins studied here have two common naproxen locations, but BSA and LSA differ in the third NPS binding site. None of these binding sites coincides with the naproxen location in the HSA‐NPS complex, which was obtained in the presence of other ligands besides naproxen. Even small differences in sequences of serum albumins from various species, especially in the area of the binding pockets, influence the affinity and the binding mode of naproxen to this transport protein. Proteins 2014; 82:2199–2208. © 2014 Wiley Periodicals, Inc.     

Monitoring of adsorption behaviors of bovine serum albumin onto a stainless steel surface by the quartz crystal microbalance based on admittance analysis

The adsorption process of bovine serum albumin (BSA) onto a stainless steel surface was investigated using the quartz crystal microbalance based on admittance analysis. The adhered mass change ?m increased with time as a result of contacting the BSA solution, and considerably long period (>2 h) was required for the attainment of the asymptotic values of ?m as well as dissipation factor ?D. The relation between ΔD and Δm suggested that the layer of adsorbed BSA molecules became stiffer with increasing time at higher BSA concentration. The relation between Δm after 2 h and the final BSA concentration was described well by the Langmuir adsorption isotherm. However, the time course of Δm clearly deviated from the Langmuir adsorption model. The stretched exponential function model described the time course of Δm well although it was an empirical one.     

Separation of monoclonal antibody alemtuzumab monomer and dimers using ultrafiltration

This article examines the feasibility of using ultrafiltration to separate the monomer of the monoclonal antibody alemtuzumab (Campath or Campath-1H) from a mixture of dimer and higher-order oligomers (collectively called "dimers" here). Using parameter scanning ultrafiltration, we initially assessed the suitability of the following membranes: 100 kDa and 300 kDa polyethersulfone (PES) membranes, and a 100 kDa polyvinylidene fluoride (PVDF) membrane. A detailed study was then carried out to examine the effects of operating conditions (such as solution pH, ionic strength, stirring speed, and permeate flux) on the separation of the monomer from the dimers using 300 kDa PES and 100 kDa PVDF membranes. Results of the experiments carried out in the carrier phase ultrafiltration (CPUF) mode indicate that the size-based protein-protein separation critically depends on the membrane used as well as the system hydrodynamics. The separation of the monoclonal antibody monomer and dimers using 100 kDa PVDF membranes in the diafiltration mode was also examined. Experimental results demonstrate that under suitable conditions, it is feasible to obtain the alemtuzumab monomer with a purity of more than 93% and a yield of more than 85% (from a mixture of 75% monomer and 25% dimers, which is the typical composition obtained after affinity chromatography). Simulation study indicates that this could be further improved to a purity of more than 96% and a monomer yield of more than 96% by increasing the selectivity of separation or by employing a two-stage diafiltration process.     

Concentration of Marine Birnavirus from Seawater with a Glass Fiber Filter Precoated with Bovine Serum Albumin

In the present study an efficient method for sampling the marine birnavirus (MABV) gene from seawater was developed. MABV gene was monitored by a specific polymerase chain reaction. When Millipore filters were used, MABV was efficiently collected on a filter with 0.05-µm pore size. When both millipore and glass fiber filters were used, MABV was recovered from both filters. Use of plain glass fiber filters resulted in poor recovering efficiency. However, coating the glass fiber filters with 1% bovine serum albumin trapped MABV efficiently. Combining concentration on glass fiber filters with polymerase chain reaction is quantitative, economic and fast, suggesting that this method can be used to detect genetically identified fish disease viruses, algal viruses, and phages.     

Effect of Chromatographic Conditions on Enantioseparation of Bovine Serum Albumin Chiral Stationary Phase in HPLC and Thermodynamic Studies

Twelve chiral compounds were enantiomerically resolved on bovine serum albumin chiral stationary phase (BSA‐CSP) by high‐performance liquid chromatography (HPLC) in reversed‐phase modes. Chromatographic conditions such as mobile phase pH, the percentage of organic modifier, and concentration of analyte were optimized for separation of enantiomers. For N‐(2, 4‐dinitrophenyl)‐serine (DNP‐ser), the retention factors (k) greatly increase from 0.81 to 6.23 as the pH decreasing from 7.21 to 5.14, and the resolution factor (R_s) exhibited a similar increasing trend (from 0 to 1.34). More interestingly, the retention factors for N‐(2, 4‐dinitrophenyl)‐proline (DNP‐pro) decrease along with increasing 1‐propanol in mobile phase (3%, 5%, 7% and 9% by volume), whereas the resolution factor shows an upward trend (from 0.96 to 2.04). Moreover, chiral recognition mechanisms for chiral analytes were further investigated through thermodynamic methods. Chirality 25:487–492, 2013. © 2013 Wiley Periodicals, Inc.     

The investigation of the interaction between orientin and bovine serum albumin by spectroscopic analysis

In this paper, the interaction between orientin and bovine serum albumin (BSA) was examined using fluorescence and absorbance spectroscopy. The analysis of the quenching mechanism was done using Stern–Volmer plots which exhibit upward (positive) deviation. A linear response to orientin was shown in the concentration range between 3 and 50 μM. The experimental results showed the presence of a static quenching process between orientin and BSA. The thermodynamic parameters ΔH, ΔS and ΔG were also calculated and suggested that the hydrophobic and electrostatic interactions played an important role in the interaction between orientin and BSA. Furthermore, the distances between BSA and orientin were determined according to Förster non‐radiation energy transfer theory. In addition, the results of the synchronous fluorescence obtained indicated that the binding of orientin with BSA could affect conformation in BSA. Copyright © 2013 John Wiley & Sons, Ltd.     

Study on the synthesis of sulfonamide derivatives and their interaction with bovine serum albumin

Three sulfonamide derivatives (SAD) were first synthesized from p‐hydroxybenzoic acid and sulfonamides (sulfadimidine, sulfamethoxazole and sulfachloropyridazine sodium) and were characterized by elemental analysis, ¹H NMR and MS. The interaction between bovine serum albumin (BSA) and SAD was studied using UV/vis absorption spectroscopy, fluorescence spectroscopy, time‐resolved fluorescence spectroscopy and circular dichroism spectra under imitated physiological conditions. The experimental results indicated that SAD effectively quenched the intrinsic fluorescence of BSA via a static quenching process. The thermodynamic parameters showed that hydrogen bonding and van der Waal's forces were the predominant intermolecular forces between BSA and two SADs [4‐((4‐(N‐(4,6‐dimethylpyrimidin‐2‐yl)sulfamoyl)phenyl)carbamoyl)phenyl acetate and 4‐((4‐(N‐(5‐methylisoxazol‐3‐yl)sulfamoyl)phenyl)carbamoyl)phenyl acetate], but hydrophobic forces played a major role in the binding process of BSA and 4‐((4‐(N‐(6‐chloropyridazin‐3‐yl)sulfamoyl)phenyl) carbamoyl)phenyl acetate. In addition, the effect of SAD on the conformation of BSA was investigated using synchronous fluorescence spectroscopy and circular dichroism spectra. Molecular modeling results showed that SAD was situated in subdomain IIA of BSA. Copyright © 2014 John Wiley & Sons, Ltd.     

Spectroscopic study of the interaction between lycopene and bovine serum albumin

The interaction of lycopene with bovine serum albumin (BSA) in aqueous solution was studied by fluorescence quenching, three‐dimensional fluorescence and circular dichroism spectroscopy. The data showed that the fluorescence of BSA was quenched by lycopene at different temperatures through a dynamic mechanism. The evaluation of three‐dimensional fluorescence spectra revealed a conformational modification of BSA induced by coupling with lycopene and an increase in protein diameter as a consequence of the ligand–protein interaction. Moreover, the information obtained from evaluation of the effect of lycopene on BSA conformation by circular dichroism strongly supported the existence of a slight unfolding of BSA induced by coupling to lycopene. Copyright © 2012 John Wiley & Sons, Ltd.