Blue Sepharose Research Articles

A mammalian protein homologous to fructosamine-3-kinase is a ketosamine-3-kinase acting on psicosamines and ribulosamines but not on fructosamines.

Fructosamine-3-kinase (FN3K) is an enzyme that appears to be responsible for the removal of fructosamines from proteins. In this study, we report the sequence of human and mouse cDNAs encoding proteins sharing 65% sequence identity with FN3K. The genes encoding FN3K and FN3K-related protein (FN3K-RP) are present next to each other on human chromosome 17q25, and they both have a similar 6-exon structure. Northern blots of mouse tissues RNAs indicate a high level of expression of both genes in bone marrow, brain, kidneys, and spleen. Human FN3K-RP was transfected in human embryonic kidney (HEK) cells, and the expressed protein was partially purified by chromatography on Blue Sepharose. Unlike FN3K, FN3K-RP did not phosphorylate fructoselysine, 1-deoxy-1-morpholino-fructose, or lysozyme glycated with glucose. In a more systematic screening for potential substrates for FN3K-RP, we found, however, that both enzymes phosphorylated ketosamines with a D-configuration in C3 (psicoselysine, 1-deoxy-1-morpholino-psicose, 1-deoxy-1-morpholino-ribulose, lysozyme glycated with allose-the C3 epimer of glucose, or with ribose). Tandem mass spectrometry and nuclear magnetic resonance analysis of the product of phosphorylation of 1-deoxy-1-morpholino-psicose by FN3K-RP indicated that this enzyme phosphorylates the third carbon of the sugar moiety. These results indicate that FN3K-RP is a ketosamine-3-kinase (ketosamine-3-kinase 2). This enzyme presumably plays a role in freeing proteins from ribulosamines or psicosamines, which might arise in a several step process, from the reaction of amines with glucose and/or glycolytic intermediates. This role is shared by fructosamine-3-kinase (ketosamine-3-kinase 1), which has, in addition, the unique capacity to phosphorylate fructosamines.

Identification of peptides that mimic the pertussis toxin binding site on bovine fetuin.

The introduction of acellular pertussis vaccines has greatly enhanced the safety profile of vaccines to prevent whooping cough. Pertussis toxin (Ptx) is one component produced by Bordetella pertussis that is contained in all of these vaccines, either in combination with other known pertussis virulence factors or as the sole pertussis component, combined with tetanus and diphtheria toxoids. A hydrogen peroxide toxoid of Ptx has been shown to be efficacious in preventing pertussis infections in a mass vaccination trial and is presently licensed in the United States and Europe (B. Trollfors, J. Taranger, T. Lagergard, L. Lind, V. Sundh, G. Zackrisson, C. U. Lowe, W. Blackwelder, and J. B. Robbins, N. Engl. J. Med. 333:1045-1050, 1995). The industrial production of Ptx can be performed through the cultivation of B. pertussis in well-defined growth media, in which the components can be well characterized and their origins can be documented. Once the bacteria are removed from the culture, Ptx can be isolated from the supernatant and purified by using the technique described by Sekura et al. (R. D. Sekura, F. Fish, C. R. Manclark, B. Meade, and Y. L. Zhang, J. Biol. Chem. 258:14647-14651, 1983). The only drawback of this procedure, which combines two affinity chromatography steps, one with Blue Sepharose and a second with matrix-bound bovine fetuin (BF), is the source and purity of the BF. Concern about vaccine preparations that may possibly risk contamination by material associated with bovine spongioform encephalopathy has continued to increase. We thus sought a replacement for the BF affinity chromatography and, more specifically, for the glycosidic moiety on BF. We describe here the identification of a seven-amino-acid peptide that mimics the glycosidic moiety on BF to which Ptx binds. Furthermore, we have constructed an affinity column containing this peptide that can be used to replace BF in Ptx purification. Finally, we used the X-ray crystallographic structure of Ptx bound to the oligosaccharide moiety of BF as a scaffold and replaced the oligosaccharide with the peptide.

Purification and characterization of Moschatin, a novel type I ribosome-inactivating protein from the mature seeds of pumpkin (Cucurbita moschata), and preparation of its immunotoxin against human melanoma cells.

A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin (Cucurbita moschata) has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 column chromatography, Blue Sepharose CL-6B Affinity column chromatography and FPLC size-exclusion column chromatography. Moschatin is a type 1 RIP with a pI of 9.4 and molecular weight of approximately 29 kD. It is a rRNA N-glycosidase and potently blocked the protein synthesis in the rabbit reticulocyte lysate with a IC50 of 0.26 nM. Using the anti-human melanoma McAb Ng76, a novel immunotoxin Moschatin-Ng76 was prepared successfully and it efficiently inhibited the growth of targeted melanoma cells M21 with a IC50 of 0.04 nM, 1500 times lower than that of free Moschatin. The results implied that Moschatin could be used as a new potential anticancer agent.

Improved resolution of human cerebrospinal fluid proteins on two-dimensional gels.

Proteomics combines two-dimensional gel electrophoresis and peptide mass fingerprinting and can potentially identify a protein(s) unique to disease. Such proteins can be used either for diagnosis or may be relevant to the pathogenesis of disease. Because patients with multiple sclerosis (MS) have increased amounts of immunoglobulin (Ig) G in their cerebrospinal fluid (CSF) that is directed against an as yet unidentified protein, we are applying proteomics to MS CSF, studies that require optimal separation of proteins in human CSF. We found that recovery of proteins from CSF of MS patients was improved using ultrafiltration, rather than dialysis, for desalting. Resolution of these proteins was enhanced by acetone precipitation of desalted CSF before electrophoresis and by fractionation of CSF using Cibacron Blue sepharose affinity chromatography. Improved protein recovery and resolution will facilitate excision from gels for analysis by peptide mass fingerprinting.

Comparison of pyruvate kinase variants from breast tumor and normal breast

Background. Pyruvate kinase isozymes in human breast tumor tissue were compared in this study with normal human breast tissue. Two forms of pyruvate kinase present in normal and tumor human breast were purified by ammonium sulfate precipitation, dialysis, gel filtration, ion exchange, and affinity chromatography. Molecular weight of the native enzyme was determined. Methods. Presence of pyruvate kinase activity was examined in normal and tumor breast tissues. Pyruvate kinase was purified with Sephadex DEAE-50, Sepharyl S-200, and Blue Sepharose CL-6B chromatography. Spectrophotometric methods were used to determine activities of pyruvate kinase. Results. Molecular weights of fractions I and II as determined by gel filtration on Sepharyl S-200 were 135,000 Da, 260,000 Da in normal breast tissue, and 72,000 Da, 250,000 Da in tumor breast tissue, respectively. Fractions I and II of pyruvate kinase may be purified approximately 1,591-fold, 636.4-fold in normal breast tissue and 219-fold, 318-fold in tumor breast tissue, respectively. Pyruvate kinase activity in tumor tissue was found higher than in normal tissue. Only tumor fraction II showed tumor-specific sensitivity to L-cysteine. L-phenylalanine inhibited both fractions I and II of normal breast and fraction I of tumor breast, but not fraction II of pyruvate from tumor. ATP inhibited normal and tumor fraction I of pyruvate kinase. The influence of ATP on enzyme activity from normal and tumor fraction II depended upon its concentration. Conclusions. It was thought that isozymes of pyruvate kinase from human breast tissue might be M1 and M2 isozymes when compared with those of other tissue pyruvate kinase isoenzymes. Fraction II from breast tumor represented different sensitivity to L-cysteine, L-phenylalanine, and specific activity in comparison with fraction II from normal breast. Different kinetic behavior of fractions in the human breast tumors may support the concept of an isozyme shift.

Albumin activates the AKT signaling pathway and protects B-chronic lymphocytic leukemia cells from chlorambucil- and radiation-induced apoptosis

AbstractActivation of the phosphatidylinositol 3- kinase/AKT pathway antagonizes apoptosis in diverse cellular systems. We previously showed that human plasma activated AKT and potently blocked the ability of chlorambucil or gamma radiation to induce apoptosis of B-chronic lymphocytic leukemia (CLL) cells. Here we report experiments that identify albumin as the major component of plasma that blocks CLL cell killing by chlorambucil or radiation. Intact plasma depleted of albumin by chromatography on Cibacron blue–Sepharose or plasma from a subject with analbuminemia failed either to activate AKT or to protect CLL cells from chlorambucil-induced apoptosis. Both functions were restored by re-addition of albumin. The protective action of albumin as well as AKT activation was compromised by the binding of lipids. Fluorescence-activated cell sorter (FACScan) analysis demonstrated the uptake of fluoresceinated albumin by CLL cells. Accumulation of albumin in intracellular vesicles was also shown by confocal microscopy. Indirect inhibition of AKT activation by the phosphatidylinositol 3-kinase inhibitor LY294002 reversed the blockade of chlorambucil-induced killing by plasma albumin. The data suggest that activation of AKT consequent to binding of albumin by CLL cells blocks chlorambucil- and radiation-induced apoptosis. Strategies designed to block albumin-induced antiapoptotic signaling may, therefore, be of value in enhancing cytotoxic drug action on CLL cells.

Expression, purification, and characterization of arginine kinase from the sea cucumber Stichopus japonicus

The arginine kinase gene of sea cucumber Stichopus japonicus was cloned and inserted into the prokaryotic expression plasmid pET-21b. The protein was expressed in a soluble and functional form in Escherichia coli and purified by Blue Sepharose CL-6B, DEAE-32, and Sephadex G-100 chromotography with a final yield of 83 mg L −1 of LB medium. The specific activity, electrophoretic mobility, and isoelectric focusing were all identical with those of arginine kinase that was purified from sea cucumber muscle. The fluorescence emission spectrum of arginine kinase had a maximum fluorescence at a wavelength of 330 nm upon excitation at 295 nm. These results are the first report of this purified protein.

Purification of native dehydrin from Glycine Max cv., Pisum sativum, and Rosmarinum officinalis by affinity chromatography

An improved method for the purification of dehydrin from soy ( glycine max) is described. Acidic extraction of soy whey was followed by a three step chromatographic process: capture on copper charged Chelating Sepharose Big Beads, intermediate hydrophobic interaction chromatography on Source 15 PHE, and a polishing step on blue Sepharose. The 32-kDa native soy dehydrin was purified to a purity of greater than 98.5% with an overall recovery of 63%. When compared to a previously published purification procedure, recovery, time requirements, and sample preparation steps were improved. The developed method is readily scaleable. Preliminary results show that the process can be used for dehydrins from rosemary ( Rosmarinum officinalis) and pea ( Pisum sativum).

Affinity adsorption and hydrodynamic behavior in a tapered-bed of upward flow

Affinity chromatography operated in a tapered-bed with the continuous up-flow was researched. Purification of Lysozyme by Blue Dextran Sepharose 4B was chosen as a model system. A series of experiments such as the batchwise adsorption, tapered columnwise adsorption, elution and pressure drop determination were carried out to find out characteristics such as adsorption equilibrium, hydrodynamic performance and adsorption efficiency for this kind of new affinity process. A mathematical model to describe this system was also established and verified. Research results showed that the affinity purification by the tapered-bed with up-flow could be operated in three regimes, i.e. the fixed-bed regime, fully fluidized-bed regime and partially fluidized-bed regime, which depended on the flow rate. The adsorption efficiencies were the highest in the fixed-bed regime, and poor adsorption efficiencies were shown in the fully fluidized-bed regime.

A Predictive Model for Salt Effects on the Dye-Ligand Affinity Adsorption Equilibrium of Protein

A predictive model is developed to describe the salt effects on the adsorption equilibrium of protein to Cibacron Blue-modified agarose gel (Sepharose CL-6B). This model assumes that, with the addition of salt, a fraction of dye-ligand molecules will lodge to the surface of the agarose gel resulting from the induced strong hydrophobic interaction between the dye ligands and agarose surface. This leads to a decrease in the dye ligands accessible to protein adsorption and consequently decreases the adsorption capacity of protein. The effect of salt on the dye-ligand lodging is presented by the equilibrium between salt and the dye ligands. Combined with the basic concept of steric−mass action theory, which considers both the multipoint nature and the macromolecule steric shielding of protein adsorption, an implicit isotherm of the protein adsorption equilibrium on Cibacron Blue 3GA-modified Sepharose CL-6B is formulated, involving salt concentration as a variable. Good agreement between experimental data and the predicted adsorption isotherms for single-component protein systems has been demonstrated, and fairly good matching between the predicted and experimental data for the binary adsorption of bovine serum albumin and bovine hemoglobin is obtained under most conditions. This model is expected to be useful in the design and optimization of the salt-gradient elution process of dye-ligand affinity chromatography.

Determination of kinetic parameters for the adsorption of a protein on porous beads using symbolic computation and numerical Laplace inversion

The work deals with the determination of the kinetic parameters for the adsorption of human serum albumin (HSA) onto spherical resin beads (Blue Sepharose CL-6B) in a batch stirred tank. By noting the practically linear equilibrium adsorption isotherm during experimental conditions and assuming that the adsorption rate is very rapid compared with the diffusion in the adsorbent particles, the differential equations describing the process with the appropriate boundary conditions are solved in the Laplace domain thus facilitating the expression of the bulk concentration of protein in the tank in terms of the unknown kinetic parameters and the Laplace variable. In order to cheaply and accurately invert the Laplace transform (which cannot be done analytically), we employ I MN approximants using mathematica. This gives an explicit expression of the bulk concentration as a function of time and the unknown parameters. Subsequent nonlinear regression facilitates rapid determination of the desired kinetic parameters, viz., the film transfer coefficient, k f; the effective diffusion coefficient, D e, and the modified diffusion coefficient D. The values obtained are in agreement with those in the literature.

Expression of Torpedo californica creatine kinase in Escherichia coli and purification from inclusion bodies

The pET17 expression vector was used to express creatine kinase from the electric organ of Torpedo californica as inclusion bodies in Escherichia coli BL21(DE3) cells. The insoluble aggregate was dissolved in 8 M urea and, following extraction with Triton X-100, the enzyme was refolded by dialysis against Tris buffer (pH 8.0) containing 0.2 M NaCl. After two buffer changes, chromatography on Blue Sepharose was used as a final step in the purification procedure. Approximately 54 mg active protein was recovered from a 1 L culture and the refolded enzyme had a specific activity of 75 U/mg. The molecular mass of the purified protein was consistent with that predicted from the amino acid sequence and the CD spectrum of the refolded enzyme was essentially identical to that of creatine kinase from human muscle (HMCK). The K m values of ATP and ADP were also similar to those of HMCK, while the K m values for both phosphocreatine and creatine were approximately 5–10-fold higher. The purification described here is in marked contrast with earlier attempts at purification of this isozyme where, in a process yielding less than 1 mg/L culture, enzyme with a specific activity of ca. 5 U/mg was obtained.

Detection of serum proteins by native polyacrylamide gel electrophoresis using Blue Sepharose CL-6B-containing stacking gels

Analysis of serum proteins by native polyacrylamide gel electrophoresis is difficult because albumin is abundant in serum and interferes with the resolution of other proteins, especially α-antitrypsin which has mobility that is very similar to that of albumin. We present here a method in which serum proteins are separated by polyacrylamide gel electrophoresis using stacking gels containing Blue Sepharose CL-6B, which has a high affinity for albumin, lipoproteins, kinases, and pyridine-nucleotide-dependent oxidoreductases. During electrophoresis, proteins that bind to Blue Sepharose CL-6B stay in the stacking gel and do not migrate into the separating gel. As a consequence, certain proteins, including α 1-antitrypsin, can be detected as clear bands. This method overcomes the requirement for fractionation of serum samples prior to electrophoresis to remove albumin and allows the simultaneous analysis of many samples.

A protein kinase activated by darkness phosphorylates nitrate reductase in Komatsuna (Brassica campestris) leaves.

Although it has been shown that leaf nitrate reductase (NR: EC 1.6.6.1) is phosphorylated by subjecting plants to darkness, there is no evidence for the existence of dark-activated or dark-induced NR kinase. This study was undertaken to investigate the occurrence of a protein kinase phosphorylating NR in response to dark treatments. Immediately after transferring Komatsuna (Brassica campestris L.) plants to darkness, we observed rapid increases in the phosphorylating activity of the synthetic peptide, which is designed for the amino acid sequence surrounding the regulatory serine residue of the hinge 1 region of Komatsuna NR, in crude extracts from leaves. The activity reached a maximum after 10 min of darkness. Inactivation states of NR estimated from relative activities with or without Mg2+ were correlated to activities of the putative dark-activated protein kinase. Using the synthetic peptide as a substrate, we purified a protein kinase from dark-treated leaves by means of successive chromatographies on Q-Sepharose, Blue Sepharose, FPLC Q-Sepharose, and ATP-gamma-Sepharose columns. The purified kinase had an apparent molecular mass of 150 kDa with a catalytic subunit of 55 kDa, and it was Ca2+-independent. The purified kinase phosphorylated a recombinant cytochrome c reductase protein, a partial protein of NR, and holo NR, and inactivated NR in the presence of both 14-3-3 protein and Mg2+. The kinase also phosphorylated synthetic peptide substrates designed for sucrose phosphate synthase and 3-hydroxy-3-methylglutaryl-Coenzyme A reductase. Among inhibitors tested, only K252a, a potent and specific serine/threonine kinase inhibitor, completely inhibited the activity of the dark-activated kinase. The activity of the purified kinase was also specifically inhibited by K252a. Taken together with these findings, results obtained suggest that the putative dark-activated protein kinase may be the purified kinase itself, and may be responsible for in vivo phosphorylation of NR and its inactivation during darkness.

Isolation of lysophosphatidic acid phosphatase from developing peanut cotyledons.

The soluble fraction of immature peanut (Arachis hypogaea) was capable of dephosphorylating [(3)H]lysophosphatidic acid (LPA) to generate monoacylglycerol (MAG). The enzyme responsible for the generation of MAG, LPA phosphatase, has been identified in plants and purified by successive chromatography separations on octyl-Sepharose, Blue Sepharose, Superdex-75, and heparin-agarose to apparent homogeneity from developing peanuts. This enzyme was purified 5,048-fold to a final specific activity of 858 nmol min(-1) mg(-1). The enzyme has a native molecular mass of approximately 39 kD determined by gel filtration and migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a subunit molecular mass of 39 +/- 1.5 kD. The K(m) values for oleoyl-, stearoyl-, and palmitoyl-sn-glycerol-3-phosphate were determined to be 28.6, 39.3, and 47.9 microM, respectively. The LPA phosphatase was specific to LPA and did not utilize any other substrate such as glycerol-3-phosphate, phosphatidic acid, or p-nitrophenylphosphate. The enzyme activity was stimulated by the low concentrations of detergents such as Triton X-100 and octylglucoside. Cations had no effect on the enzyme activity. Fatty acids, sphingosine, and sphingomyelin at low concentrations stimulated the enzyme activity. The identification of LPA phosphatase in plants demonstrates the existence of MAG biosynthetic machinery in plants.

Purification and characterization of a Galactomyces reessii hydratase that converts 3-methylcrotonic acid to 3-hydroxy-3-methylbutyric acid.

Cell free extracts of Galactomyces reessii contain a hydratase as the key enzyme for the transformation of 3-methylcrotonic acid to 3-hydroxy-3-methylbutyric acid. Highest levels of hydratase activity were obtained during growth on isovaleric acid. The enzyme, an enoyl CoA hydratase, was purified 147-fold by precipitation with ammonium sulphate and successive chromatography over columns of DE-52, Blue Sepharose CL-6B and Sephacryl S-200. During purification, hydratase activity was measured spectrophotometrically (OD change at 263 nm) for 3-methylcrotonyl CoA and crotonyl CoA as substrates. The enzyme displayed highest activity with crotonyl CoA with a Kcat of 1,050,000 min(-1). The ratio of crotonyl CoA to 3-methylcrotonyl CoA activities was constant (20:1) during all steps of purification. The Kcat for crotonyl CoA was also about 20 times greater than the Kcat for 3-methylcrotonyl CoA (51,700 min(-1). The enzyme had pH and temperature optima at 7.0 and 35 degrees C, a native Mr of 260 +/- 4.5 kDa and a subunit Mr of 65 kDa, suggesting that the enzyme was a homotetramer. The pI of the purified hydratase was 5.5, and the N-terminal amino acid sequence was VPEGYAEDLLKGKMMRFFDS. Hydratase activity for 3-methylcrotonyl CoA was competitively inhibited by acetyl CoA, propionyl CoA and acetoacetyl CoA.

Glyceraldehyde-3-phosphate dehydrogenase from the newt Pleurodeles waltl. Protein purification and characterization of a GapC gene

The NAD +-dependent cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) has been purified to homogeneity from skeletal muscle of the newt Pleurodeles waltl (Amphibia, Urodela). The purification procedure including ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography resulted in a 24-fold increase in specific activity and a final yield of approximately 46%. The native protein exhibited an apparent molecular weight of approximately 146 kDa with absolute specificity for NAD +. Only one GAPDH isoform (p I 7.57) was obtained by chromatofocusing. The enzyme is an homotetrameric protein composed of identical subunits with an apparent molecular weight of approximately 37 kDa. Monospecific polyclonal antibodies raised in rabbits against the purified newt GAPDH immunostained a single 37-kDa GAPDH band in extracts from different tissues blotted onto nitrocellulose. A 510-bp cDNA fragment that corresponds to an internal region of a GapC gene was obtained by RT-PCR amplification using degenerate primers. The deduced amino acid sequence has been used to establish the phylogenetic relationships of the Pleurodeles enzyme — the first GAPDH from an amphibian of the Caudata group studied so far — with other GAPDHs of major vertebrate phyla.

Hepatocyte proliferation factors from neonatal pig liver: purification and characterization.

Two factors were found in the condition medium of neonatal pig liver fragments, which were capable of stimulating DNA synthesis in primary hepatocytes. They were named hepatocyte proliferation factor (HPF)-1 and HPF-2 and purified 1,025- and 2,580-fold, respectively. Both HPF-1 and HPF-2 seem to be anionic at pH 8.0 judged from the elution pattern of DEAE (DE52) column chromatography. HPF-1 was recovered as a non-adsorbed fraction in blue Sepharose and heparin Sepharose columns, and had a molecular weight of 26-31 kDa as estimated by gel filtration in high salt condition. Purified HPF-1 stimulated DNA synthesis of primary rat hepatocytes, but suppressed that of HepG2 cells. HPF-2 strongly bound to blue Sepharose and heparin Sepharose columns, and had a molecular weight of 71-90 kDa as estimated by SDS-PAGE under non-reduced condition. Purified HPF-2 stimulated DNA synthesis of primary rat hepatocytes dose dependently but did not suppress that of HepG2 cells. From further biological and chemical characteristics studied in this paper, HPF-1 and HPF-2 may be novel stimulating proteins for hepatocyte proliferation, although the possibility that they are already known growth factors can not be excluded without complete purification and its cloning.

An associated process for the purification of immuno globulin G, catalase, superoxide dismutase and albumin from haemolysed human placenta blood.

The human placenta is a rich raw material for production of many biopharmaceutical products. Here we describe a co-purification process for the production of four different proteins from haemolysed human placenta blood: IgG, catalase (Cat), superoxide dismutase (Sod) and albumin (Alb). The process can be divided in two parts: the common steps and the specific separation techniques for each protein. The common steps are: extraction, haemoglobin precipitation, concentration/diafiltration and the first Q-Sepharose chromatography step. At this chromatography step the process is branched: while IgG and Cat were recovered in the flow-through, Sod and Alb were eluted separately. IgG and Cat were separated in a second Q-Sepharose chromatography step during which IgG was recovered in the flow-through, whereas Cat bound to the resin. IgG was purified by S-Sepharose chromatography, followed by selective precipitation with n-octanoic acid, yielding about 0.4 g of IgG per kg of placenta. Cat was eluted at the second Q-Sepharose chromatography step and was purified by Blue Sepharose chromatography. A total of 1.8 x 10(6) units of Cat were recovered/kg of placenta, with a specific activity of 45000 units/mg of protein. Sod was further purified by S-Sepharose and Phenyl-Sepharose chromatography steps and recovered in the non-adsorbed fractions. The yield of Sod was 2.1 x 10(5) units/kg of placenta, with a specific activity of 1194 units/mg of protein. Alb purification was followed by a combined process including thermocoagulation and treatment with activated charcoal. The final step was Phenyl-Sepharose chromatography. The process yielded 3.1 g of Alb/kg of placenta. The described methodology was designed to be easily scaled-up for industrial production.

Oscillatory electroosmosis-enhanced intra/inter-particle liquid transport and its primary applications in the preparative electrochromatography of proteins

The concept of generating an oscillatory electroosmotic flux inside the porous particle to enhance the intra-particle mass transport was presented and a new kind of electrochromatography carried out in a five-compartment electrolyzer were developed. The adsorbent was packed in the central compartment, while the neighboring compartments were used as the elution compartments and the electrode compartments, respectively. Chromatographic separations of human serum albumin on Blue Sepharose Fast Flow, bovine serum albumin (BSA) on DEAE–Sepharose Fast Flow, and BSA on hydroxyapatite were carried out, respectively. The adsorption isotherms were shown to be independent of electric field, while the increase in the electric field strength resulted in a linear increase in the magnitude of electroosmotic flux and the improvement of the breakthrough behavior in all cases. The experiment results have demonstrated the effectiveness of the oscillatory electroosmosis in enhancing intra- and inter-particle mass transport and its high potential to large-scale chromatography.