Separation Of Human Serum Albumin Research Articles

Macromolecular Crowding-Induced Unusual Liquid-Liquid Phase Separation of Human Serum Albumin via Soft Protein-Protein Interactions.

Macromolecular crowding has a profound impact on the conformational dynamics and intermolecular interactions of biological macromolecules. In this context, the role of inert synthetic crowders in the protein-protein interactions of globular proteins is poorly understood. Here, using native human serum albumin (HSA) under physiological conditions, we show that macromolecular crowding induces liquid-liquid phase separation (LLPS) via liquid-like membrane-less droplet formation in a concentration- and time-dependent manner. Circular dichroism measurements reveal significant alteration in the secondary structure of HSA inside the droplet during aging. In contrast, at a high protein concentration, a liquid-to-solid-like phase transition has been observed upon maturation. Our findings reveal that the LLPS of HSA is mainly driven by enthalpically controlled intermolecular protein-protein interactions via hydrophobic contacts involving aromatic and/or nonaromatic residues. Moreover, modulation of LLPS of HSA has been demonstrated upon denaturation and ligand binding. This study highlights the importance of soft protein-protein interactions of globular proteins in a crowded cellular environment in driving the LLPS.

Mixed Macromolecular Crowding: A Protein and Solvent Perspective.

In the living cell, biomolecules perform their respective functions in the presence of not only one type of macromolecules but rather in the presence of various macromolecules with different shapes and sizes. In this study, we have investigated the effects of five single macromolecular crowding agents, Dextran 6, Dextran 40, Dextran 70, Ficoll 70, and PEG 8000 and their binary mixtures on the modulation in the domain separation of human serum albumin using a Förster resonance energy transfer-based approach and the translational mobility of a small fluorescent probe fluorescein isothiocyanate (FITC) using fluorescence correlation spectroscopy (FCS). Our observations suggest that mixed crowding induces greater cooperativity in the domain movement as compared to the components of the mixtures. Thermodynamic analyses of the same provide evidence of crossovers from enthalpy-based interactions to effects dominated by hard-sphere potential. When compared with those obtained for individual crowders, both domain movements and FITC diffusion studies show significant deviations from ideality, with an ideal solution being considered to be that arising from the sum of the contributions of those obtained in the presence of individual crowding agents. Considering the fact that domain movements are local (on the order of a few angstroms) in nature while translational movements span much larger lengthscales, our results imply that the observed deviation from simple additivity exists at several possible levels or lengthscales in such mixtures. Moreover, the nature and the type of deviation not only depend on the identities of the components of the crowder mixtures but are also influenced by the particular face of the serum protein (either the domain I–II or the domain II–III face) that the crowders interact with, thus providing further insights into the possible existence of microheterogeneities in such solutions.

Separation of human serum albumin and polyethylene glycol by electro-ultrafiltration

For the purpose of residual PEG removal after protein purification and PEGylation, electro-ultrafiltration (EUF) was used for separation of human serum albumin (HSA) from polyethylene glycol (PEG) with different molecular weights. The results clearly demonstrated the effectiveness of EUF for achieving the desired objectives. Higher flux and lower HSA transmission were observed in EUF process with the feed consisting of a mixture of 1g/L PEG and 1g/L HSA, compared to conventional ultrafiltration (UF) process, while the PEG transmission was nearly the same. When the concentration of PEG increased from 1g/L to 10g/L while keeping the HSA concentration the same, more HSA passed through the membrane in the UF process due to reduction in protein diffusion and hydration by PEG. When the HSA concentration increased from 1g/L to 10g/L with the PEG concentration being 1g/L, the PEG transmission decreased significantly. The application of electric field resulted in high flux and PEG transmission and low protein transmission through the membrane. The effect of electrolyte (i.e., ionic strength and type) on the efficiency of the EUF process was also investigated.

Robust and selective nano cavities for protein separation: An interpenetrating polymer network modified hierarchically protein imprinted hydrogel

In the present work, we report a novel method for the reinforcement of hierarchically structured molecularly imprinted polymer (MIP) for the separation of human serum albumin (HSA) and immunoglobulin G (IgG) proteins under pressure driven flow conditions. The template proteins (HSA or IgG) were first physically adsorbed at their isoelectric point on the surface of wide pore silica particles. Thereafter, the pore system was filled with a monomer solution and polymerized to form a lightly crosslinked polyacrylamide network covering the protein template. In order to enhance the rigidity of the hydrogels, different type of crosslinkers such as ethylene glycol dimethacrylate (EGDMA), 3,4-dihydroxyphenethylamine (DA) and methylene-bis-acrylamide (MBA), at least partially interpenetrated into the initially made acrylamide base hydrogel leading to formation of an interpenetrating polymer network (IPN). Then the silica matrix was removed to leave highly porous and reinforced MIP. TGA together with FT-IR and TEM analysis supported the interpenetration of the secondary crosslinkers in the initially formed polymer matrix. The compression property of the modified hydrogels as a function of degree of swelling was in the following order: DA>EGDMA>MBA-IPN-modified hydrogel. Batch binding assay verified the capability of the IPN modified MIPs to capture the target proteins. Moreover, solid phase extraction, HPLC and SDS-PAGE revealed that the EGDMA modified MIP could selectively capture and separate the proteins from human serum or fermentation broth.

A Crosslinked Carboxylic Acid Containing Cation Exchange Monolithic Cryogel for Human Serum Albumin Separation

For this work, we synthesized poly(N-isopropylacrylamide-acrylamide)-acrylic acid (poly(NIPAM-Am)-AAc) monolithic cryogel for a human serum albumin separation (HSA) from a protein mixture (human serum immunoglobulin, human serum albumin and lysozyme) and performed HSA adsorption studies using the cryogel to do continuous system experiments in a syringe column connected by a peristaltic pump. Poly(NIPAM-Am)-AAc with a pore size of 10–100 μm was produced by free radical polymerization that proceeded in an aqueous solution of monomers frozen inside a syringe column. The monolithic poly(NIPAM-Am)-AAc cryogel was characterized by performing swelling studies, FTIR and SEM that showed a swelling ratio of 6.2 g H2O/g dry cryogel. The maximum HSA adsorption by the cryogel was 42.5 mg/g polymer at pH 4.0 in a 50 mM acetate buffer. We also studied the effect of two different temperatures (25 and 40°C). The higher temperature increased the adsorption capacity of the cryogel. HSA molecules could be reversibly adsorbed and desorbed five times with the same poly(NIPAM-Am)-AAc cryogel without a noticeable loss of their HSA adsorption capacity. The synthesized cryogel was used to separate albumin from the protein mixture. Adsorbed albumin was eluted by changing the pH of the buffer (pH 7.0 and 25°C). Poly(NIPAM-Am)-AAc monolithic cryogel behaved as a cation exchange column because of its functional carboxylic group.

Selective separation of human serum albumin with copper(II) chelated poly(hydroxyethyl methacrylate) based nanoparticles

Poly(hydroxyethyl methacrylate) (PHEMA) nanoparticles with an average size of 300 nm in diameter and with a polydispersity index of 1.156 were produced by surfactant free emulsion polymerization. Specific surface area of the PHEMA nanoparticles was found to be 996 m 2/g. Metal-chelating ligand 3-(2-imidazoline-1-yl)propyl(triethoxysilane) (IMEO) was covalently attached to the PHEMA nanoparticles. IMEO content was 0.97 mmol IEMO/g. The morphology and properties of these nanoparticles were characterized with scanning electron microscopy, Fourier transform infrared spectroscopy and atomic force microscopy. The Cu 2+-chelated PHEMA–IMEO nanoparticles were used in the adsorption-elution studies of human serum albumin (HSA) in a batch system. Maximum HSA adsorption amount of the Cu 2+ chelated nanoparticles was 680 mg HSA/g. The PHEMA–IMEO–Cu 2+ nanoparticles exhibited a quite high adsorption capacity and fast adsorption rate due to their high specific surface area and the absence of internal diffusion resistance.

Predicting the binding properties of cibacron blue F3GA in affinity separation systems

The binding properties of cibacron blue F3GA (CB-F3GA) bound to a model NAD(P)H/FAD(H 2)-dependent protein system, namely cytosolic quinone reductase (QR), was characterized by AMBER in an attempt to address the binding properties of immobilized CB-F3GA used in the separation of serum albumin. A favorable binding free energy of −4.52 kcal/mol ( K D = 5.09 × 10 −4 kcal/mol) was determined for CB-F3GA binding by MM-PBSA method, which was found to be a ballpark estimate of empirical values reported in literature (Δ G ≈ −6 kcal/mol). We propose that CB-F3GA primarily follows a class III binding motif in presence of FAD in the binding site of QR in solution, while a class II binding motif is observed in the crystal form. It was found that favorable van der Waals/hydrophobic interactions take place in the binding site making a major contribution to a favorably dominating enthalpy of binding (Δ H tot = −25.87 kcal/mol) as compared to a disfavorable binding entropy term ( TΔ S tot = −21.35 kcal/mol). Additional MM-PBSA experiments in the absence of FAD gave rise to a disfavorable binding free energy for CB in complex with QR, suggesting that FAD is an essential determinant of CB-F3GA binding. This is in contrast to an earlier observation of Denizli et al. on separation of human serum albumin (HSA) by immobilized CB-F3GA in the absence of FAD. Therefore, a class I binding model for CB-F3GA is proposed here to account for the efficient separation of HSA in affinity chromatography systems.

Synthesis and spectroscopic characterization of superparamagnetic beads of copolymers of methacrylic acid, methyl methacrylate and ethylene glycol dimethacrylate and their application to protein separation

AbstractNovel magnetic beads were prepared from methacrylic acid, methyl methacrylate and ethylene glycol dimethacrylate in the presence of ferric ions via suspension polymerization. After polymerization, thermal treatment of the Fe(III)‐ion containing beads with Fe(II) ions under alkaline condition resulted encapsulation of Fe3O4 crystals within the polymer matrix. The magnetic beads were characterized by surface area measurement, swelling tests, electron spin resonance (ESR), differential scanning calorimetry (DSC), differential thermal analysis (DTA) and scanning electron microscopy (SEM). To evaluate the magnetic properties of the composite beads, Mössbauer spectra measurements were carried out. ESR and Mössbauer spectroscopy data revealed that the beads were highly superparamagnetic. The DSC data of the magnetic beads also showed an enhanced glass transition temperature owing to the presence of the Fe3O4 crystals within the polymer network. In addition, the presence of functional carboxyl groups on the surface permits further modifications in future applications. The magnetic beads were used for separation of human serum albumin and cytochrome c from aqueous solution in a batch system. Copyright © 2007 Society of Chemical Industry

Separation of human plasma proteins HSA and HIgG using high-capacity macroporous gel-filled membranes

This paper discusses the separation of human serum albumin (HSA), the most abundant protein present in plasma from human immunoglobulin G (HIgG) by membrane chromatography using a novel macroporous gel-filled membrane (designated Q Type 2). The membrane was prepared by anchoring a quaternary ammonium salt macroporous gel within the pores of a non-woven, polypropylene fabric. Factors affecting HSA binding were examined and operating conditions suitable for separating it from human plasma were identified. At an optimized condition, the HSA binding capacity of this novel membrane under saturating conditions was in the range of 290–300 mg/ml. This was not only significantly higher than binding capacities reported for other chromatographic membranes, but also higher than binding capacities of conventional gel based chromatographic media. The protein binding capacity was also largely insensitive to the superficial velocity, indicating the dominance of convective protein transport to and from the binding sites. The suitability of using this membrane for plasma fractionation was demonstrated by the separation of a simulated feed solution consisting of HSA and HIgG.

Separation of human serum albumin and human immunoglobulins using carrier phase ultrafiltration.

The fractionation of the plasma proteins human serum albumin (HSA) and human immunoglobulins (HIgG) using the combination of two newly developed techniques, pulsed sample injection technique and carrier phase ultrafiltration (CPUF), is discussed in this paper. The effects of pH and ionic strength on the transmission of a single protein (i.e., either HSA or HIgG) through 100 and 300 kDa MWCO polyethersulfone (PES) membranes were quantified using the pulsed sample injection technique. The experimental results thus obtained suggested that it would be possible to fractionate these proteins by optimizing the solution pH and ionic strength. With 100 and 300 kDa PES membranes, effective separation of HSA and HIgG was achieved by CPUF using suitable conditions, i.e., pH 4.7 and low salt concentration. The fractionation of HSA and HIgG by "reverse selectivity" using 300 kDa membranes was also examined.

Direct fluorimetric determination of γ-globulin in human serum with organic nanoparticle biosensor

This paper describes the development of organic fluorescence nanoparticles. The nanoparticles have a narrow, tunable, symmetric emission spectrum and a broad, continuous excitation spectrum. The nanoparticles have high room-temperature fluorescence quantum yields and long fluorescence lifetime. They are also photochemically stable and water-soluble. They were used as fluorescence biosensor in the determination of proteins, which was proved to be a simple, rapid and specific method. In comparison with single organic fluorephores, these nanoparticles are brighter, more stable against photobleaching, and do not suffer from blinking. Under optimal conditions, the linear ranges of the calibration curves were 0.1–4.5 μg ml −1 for human serum albumin (HSA), 0.2–3.5 μg ml −1 for bovine serum albumin (BSA) and 0.04–0.8 μg ml −1 for gamma globulin (γ-IgG), respectively. The detection limits were 0.062 μg ml −1 for HSA, 0.036 μg ml −1 for BSA and 0.022 μg ml −1 for γ-IgG, respectively. However, when the content of HSA is lower than 0.8 μg ml −1, HSA makes little contribution to the fluorescence quenching. So, the method was applied to direct selective quantification of γ-IgG in human blood serum without separation of HSA. The results were in good agreement with these reported by the hospital, indicating that the method presented here is not only sensitive, selective and simple, but also reliable and suitable for practical applications.

Preparation of an iminodiacetic acid-modified capillary and its performance in capillary liquid chromatography and immobilized metal chelate affinity capillary electrophoresis

We prepared iminodiacetic acid (IDA)-modified and Cu(II)–IDA-modified capillaries through polymerization of N-(vinylbenzylimino)diacetic acid. The fundamental performance of these capillaries was examined in capillary liquid chromatography (LC) and immobilized metal chelate affinity capillary electrophoresis (IMACE). Copper(II), cobalt(II), and hematin were detected at different retention times by means of capillary LC with a chemiluminescence detector, during which the IDA-modified capillary was used. The difference in the retention times was attributed to the difference in the interaction between metal ions or complex and IDA moieties on the inner wall of the capillary. In addition, human serum albumin (HSA) and human serum γ-globulin (HγG) were separated and detected using IMACE with an absorption detector, during which the Cu(II)–IDA-modified capillary was used. The separation of HSA and HγG was achieved through the interaction between proteins and Cu(II) chelate moieties on the inner wall of this capillary.

Determination of γ-globulin at nanogram levels by its enhancement effect on the resonance light scattering of functionalized HgS nanoparticles

A novel assay of γ-globulin (γ-IgG) with a sensitivity at the nanogram level is proposed based on the measurement of enhanced resonance light-scattering (RLS) signals resulting from the interaction of functionalized nano-HgS with γ-globulin. At pH 5.03, the RLS signals of functionalized nano-HgS were greatly enhanced by γ-globulin in the region of 200–700 nm characterized by the peak around 362 nm. Linear relationship can be established between the enhanced RLS intensity and γ-globulin concentration in the range of 10–140 ng ml −1. The limit of detection is 2.71 ng ml −1. Based on this, a new direct quantitative determination method for γ-globulin in blood serum samples without separation of human serum albumin (HSA) is established. The contents of γ-IgG in blood serum samples were determined with recovery of 95.7–102.5% and R.S.D. of 1.6–2.4%. This method is proved to be very sensitive, rapid, simple and tolerance of most interfering substances.

Direct quantification of γ-globulin in human blood serum by resonance light scattering techniques without separation of human serum albumin

We describe the preparation and characterization of functionalized nano-PbS. The functionalized nanoparticles are water-soluble. Reaction of mercaptoacetic acid functionalized nano-PbS with γ-globulin (γ-IgG) results in an enhanced resonance light scattering (RLS) at 385 nm. Based on this, a new direct quantitative method for γ-globulin in blood serum samples without separation of human serum albumin is established. Under optimal conditions, the enhanced RLS intensity is in proportion to the γ-IgG concentration in the range 10–500 ng ml −1. The 3 σ limit of detection is 2.75 ng ml −1. The contents of γ-IgG in blood serum samples were determined with a recovery of 97–104% and R.S.D. of 1.5–2.1% ( n=6). This method proved to be very sensitive, rapid, simple and tolerant of most interfering substances.

Protein adsorption on histidyl-aminohexyl-Sepharose 4B: I. Study of the mechanistic aspects of adsorption for the separation of human serum albumin from its non-enzymatic glycated isoforms (advanced glycosylated end products)

The characteristics of albumin adsorption on histidyl-aminohexyl-Sepharose 4B were investigated. In particular, the adsorption capacity of the gel was studied as a function of conductivity and pH of the running buffer. The adsorption was maximum at low salt concentration around neutral pH, involving electrostatic and hydrophobic interactions. Kinetic aspects were also investigated. Dissociation constant ( K D) and maximum capacity ( Q x ) were, respectively, estimated to be 4.5×10 −5 M (medium affinity) and 93.3 mg (high capacity) of human serum albumin per ml of adsorbent. According to these preliminary results, separation of HSA and its non-enzymatically glycated isoforms (conventionally named advanced glycated end products: AGEs) was achieved. Chromatographic potential of this separation tool is discussed.

Electrophoretic affinity chromatography: method validation.

A new method for preparative-scale separation of biomolecules, electrophoretic affinity chromatography (EAC), is proposed in this paper. Separation by EAC is carried out in a long and ribbon-like multicompartment electrolyser separated by membranes, in which the two central compartments are used for packing the gel matrix and for sample loading respectively. Next to the central compartments are the elution compartments and electrode compartments. The electric field is applied perpendicular to the fluid flow in the compartments. Adsorption and desorption steps may both be carried out in the presence of an electric field, which transports the target components into the gel compartment for adsorption and the impurities into the elution compartments for washing. After the adsorption step an elution solution is introduced and the product is released from the gel matrix and washed out. Separation of human serum albumin (HSA) from human serum gives HSA product of high purity, as demonstrated by isoelectric focusing analysis. The characteristics of electrophoretic binding of HSA on Blue Sepharose Fast Flow are examined. The preliminary results show that this new method has advantages in terms of high rate of mass transfer and ease of scaling up, which are of particular interest when large-scale separation of biomolecules is considered.

Continuous Separation of High Molecular Weight Compounds Using a Microliter Volume Free-Flow Electrophoresis Microstructure

A microliter volume free-flow electrophoresis microstructure (μ-FFE) was used to perform a continuous separation of high molecular weight compounds. The μ-FFE microstructure had a separation bed volume of 25 μL and was fabricated from silicon using standard micromachining technology. Laser-induced fluorescence was used to detect the sample components, which were labeled with fluorescein isothiocyanate (FITC) prior to analysis. The continuous separation of human serum albumin (HSA), bradykinin, and ribonuclease A demonstrated that only 25 V/cm was required to isolate HSA from bradykinin and ribonuclease A, while 100 V/cm was needed for the separation of bradykinin from ribonuclease A. Comparison of the observed band broadening with the theoretical variance indicated that dispersion due to the initial bandwidth, diffusion, and hydrodynamic broadening were the main contributors to the band broadening of HSA and bradykinin. However, the band broadening for ribonuclease A could not be sufficiently accounted for using the above contributors. Adsorption was found to be a possible contributor to the overall variance for ribonuclease A. Calculation of the theoretical variance due to Joule heating indicated that broadening due to Joule heating effects was insignificant. This was likely due to the narrow cross-sectional area of the device, which facilitated efficient cooling. Electrohydrodynamic distortion was observed for HSA as it migrated toward the side bed. Studies of the resolution of bradykinin and ribonuclease A as a function of field strength at various sample and carrier flow rates indicated that, for maximum throughput, high field strengths and high flow rates were required. However, no optimal conditions were found. The μ-FFE device has a peak capacity of ∼8 bands/cm, while for a typical separation of proteins using a commercial system, a peak capacity of 10 bands/cm is obtained. Thus, the resolving power of the μ-FFE device is similar to those of conventional systems. The continuous separation of tryptic digests of mellitin and cytochrome c demonstrated the ability to continuously separate more complex mixtures. Finally, modifications were made to the microstructure to facilitate fraction collection, and the fractionation of whole rat plasma was performed. Off-line analysis of the resulting fractions indicated that the complete isolation of serum albumin and globulins was possible using a field strength of 25 V/cm.

High-performance capillary electrophoretic separation of human serum albumin using a neutral coated capillary

A rapid, simple and highly resolving capillary electrophoresis technique is described for the separation of sub-species of clinical grade human serum albumin (HSA) using a neutral coated capillary. As the buffer pH approached the p I of HSA (5.2), eight peaks representing HSA sub-populations were resolved. On a non-coated capillary, no such resolution was obtained and buffers several pH units away from HSA's p I were required to prevent protein adsorption to the capillary wall. Some sub-species could be identified by mass spectrometric analysis as representing intact and amino-terminally damaged HSA with and without a blocked thiol at cysteine 34.