Chromatographic Method For Purification Research Articles

Membrane chromatographic method for the rapid purification of vitellogenin from fish plasma

A membrane chromatographic method was developed for the rapid purification of vitellogenin (Vtg) from the plasma of 17β-estradiol induced loach ( Misgurnus angaillicaud atus) and carp ( Cyprinus carpio). The time required for the proposed procedure is less then 10 min at a flow-rate of 5 ml/min of the mobile phase, and 0.5 ml of fish plasma could be separated in one cycle. Multistep gradient elution was more suitable for the separation than linear gradient elution. Under optimized conditions, a single Vtg peak can be obtained and its identity was confirmed by SDS–PAGE and gel-permeation chromatography assessment. This method is rapid and easy to operate compared to conventional HPLC and FPLC columns for Vtg separation.

Chromatographic purification and characterization of B-phycoerythrin from Porphyridium cruentum: Semipreparative high-performance liquid chromatographic separation and characterization of its subunits

A fast preparative two-step chromatographic method for purification of B-phycoerythrin from Porphyridium cruentum is described. This biliprotein was homogeneous as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis yielding three closely migrating bands corresponding to its three subunits. Baseline separation of its α-, β- and γ-subunits was achieved by a reversed-phase HPLC gradient semipreparative method with a C 4 large-pore column and a solvent system consisting of 0.05% trifluoroacetic acid (TFA) in water and 0.05% TFA in acetonitrile. B-Phycoerythrin in different aggregation states and its subunits have been spectroscopically characterized. Hexameric B-phycoerythrin has similar secondary and tertiary structure than dissociated B-phycoerythrin determined by circular dichroism.

Partial purification and characterization of a hemolysin (CAH1) from Hawaiian box jellyfish (Carybdea alata) venom

We have isolated and characterized a novel hemolytic protein from the venom of the Hawaiian box jellyfish (Carybdea alata). Hemolysis of sheep red blood cells was used to quantitate hemolytic potency of crude venom extracted from isolated nematocysts and venom after fractionation and purification procedures. Hemolytic activity of crude venom was reduced or lost after exposure to the proteolytic enzymes trypsin, collagenase and papain. The activity exhibited lectin-like properties in that hemolysis was inhibited by d-lactulose and certain other sugars. Activity was irreversibly lost after dialysis of crude venom against divalent-free, 20mM EDTA buffer; it was optimal in the presence of 10mM Ca2+ or Mg2+. Two chromatographic purification methods, size fractionation on Sephadex G-200 and anion exchange with quaternary ammonium, provided fractions in which hemolytic activity corresponded to the presence of a protein band with an apparent molecular weight of 42kDa by SDS–PAGE. We have designated this protein as CAH1. The N-terminal sequence of CAH1 was determined to be: XAADAXSTDIDD/GIIG.

Application of the high-performance liquid chromatographic method for separation, purification and characterisation of p-bromophenylacetylurea and its metabolites

This study presents a HPLC method for the separation and purification of p-bromophenylacetylurea (BPAU) and its metabolites. The method effectively separated and purified BPAU and its metabolites. Three metabolites of BPAU, M1, M2 and M3 were characterised by mass spectroscopy and nuclear magnetic resonance. They are named as N′-hydroxy- p-bromophenylacetylurea, 4-(4-bromophenyl)-3-oxapyrrolidine-2,5-dione and N′-methyl- p-bromophenylacetylurea, respectively. The major metabolic pathways of BPAU were proposed. The establishment of the HPLC method and characterisation of BPAU metabolites make it possible for further pharmacokinetic studies to explore the mechanism of BPAU-induced delayed neuropathy.

Epiregulin is a potent vascular smooth muscle cell-derived mitogen induced by angiotensin II, endothelin-1, and thrombin.

Vasoactive GTP-binding protein-coupled receptor agonists such as angiotensin II (AII), endothelin-1 (ET-1), and alpha-thrombin (alpha-Thr) have been reported to indirectly stimulate vascular smooth muscle cell (VSMC) proliferation by regulating the expression of one or more autocrine growth factors. Using ion-exchange, gel-filtration, and reverse-phase chromatographic purification methods, we isolated a major mitogenic protein present in AII-stimulated rat aortic smooth muscle (RASM) cell conditioned medium. Twenty N-terminal amino acids of the purified peptide were identified, and they had 75% amino acid sequence identity with mouse epiregulin, an epidermal growth factor (EGF)-related growth factor. We cloned the cDNA for rat epiregulin to determine its pattern of expression in G-protein-coupled receptor agonist-stimulated cells and confirm its activity as a mitogen. After treatment of RASM cells with AII, ET-1, or alpha-Thr for 1 h, induction of two epiregulin transcripts was observed, including a 4.8-kb transcript and a novel transcript of approximately 1.2 kb. Recombinant rat epiregulin was strongly mitogenic for RASM cells, stimulating DNA synthesis to levels similar to those induced by serum or platelet-derived growth factor and approximately 3-fold above that observed with saturating concentrations of EGF. In addition, epiregulin caused rapid EGF receptor activation in RASM cells. However, relative levels of EGF receptor tyrosine phosphorylation stimulated by epiregulin were less than those induced by EGF or betacellulin. Taken together, these results indicate that epiregulin is a potent VSMC-secreted mitogen, induced in common by AII, ET-1, and alpha-Thr, that may contribute to VSMC proliferation and vascular remodeling stimulated by vasoactive agonists.

Chromatographic Separation and Purification of trans-Astaxanthin from the Extracts of Haematococcus pluvialis

A gradient reversed-phase HPLC method was developed for the separation of astaxanthin esters and the isomers of astaxanthin from the unsaponified and saponified pigment extracts of the microalga Haematococcus pluvialis. Four kinds of isomers of astaxanthin and astaxanthin esters, (3S,3‘S)-trans-astaxanthin, (3S,3‘S)-9-cis-astaxanthin, (3S,3‘S)-13-cis-astaxanthin, (3R,3‘R)-trans-astaxanthin, and their esters, were separated and identified. A small amount of (3S,3‘S)-15-cis-astaxanthin was also detected from the saponified extract. In addition, a chromatographic purification method was established for the preparation of natural trans-astaxanthin from the saponified extract of H. pluvialis. With this method, 3.7 mg of astaxanthin was isolated from 1 g of dry biomass of H. pluvialis. The purified astaxanthin contained approximately 97.7% trans-astaxanthin, 1.1% cis-astaxanthin, and 1.2% impurity. Keywords: Astaxanthin; astaxanthin esters; purification; carotenoids; Haematococcus pluvialis; HPLC

Efficient purification of mouse anti-FGF receptor IgM monoclonal antibody by magnetic beads.

Affinity chromatography has been widely used for the purification of monoclonal antibodies (MAb). Traditionally, activated agarose beads conjugated with specific antisera have been used as a solid support in chromatographic protein purification. Magnetic beads conjugated with various antibodies have recently become an alternative method for the isolation of diverse proteins, nucleic acids, and cell types. In this study, murine anti-fibroblast growth factor receptor 1 (FGFR1) immunoglobulin M (IgM) was isolated from protein solutions to compare immunoaffinity column chromatography and magnetic bead IgM purification methods. Using immobilized rat anti-mouse IgM MAb, an UltraLink 1-ethyl-3-(3-dimethylaminopropyl)carboiimide (EDC)/diaminodipropylamine (DADPA) immunoaffinity column and polystyrene-coated magnetic beads were used for the purification of mouse IgM from bovine serum albumin/phosphate-buffered saline (BSA/PBS) as well as from crude ascites. Protein quantitation and percent IgM yield were determined by reducing SDS-PAGE electrophoresis followed by silver staining, then IgM and protein contaminants were quantitated using densitometry analysis. IgM anti-FGFR1 binding specificity and immunologic activity were determined by enzyme-linked immunosorbant assay (ELISA). This study demonstrates that magnetic bead isolation of IgM from ascites is more effective than traditional affinity chromatography purification as determined by greater IgM yield, purity, and immunologic activity.

Chromatographic purification of biliproteins from Spirulina platensis high-performance liquid chromatographic separation of their α and β subunits

A fast preparative two-step chromatographic method for purification of C-phycocyanin and allophycocyanin from Spirulina platensis is described. Both biliproteins were homogeneous as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielding two closely migrating bands. Separation of α and β subunits from C-phycocyanin and allophycocyanin was also accomplished by a chromatographic procedure under denaturing conditions. The phycocyanobilin chromophore from both biliproteins has been isolated, tested for purity by reversed-phase high performance liquid chromatography and spectroscopically characterized.

Purification of Commercial Coomassie Brilliant Blue R-250 and Characterization of the Chromogenic Fractions

Coomassie brilliant blue R-250 (CBB) is a popular and widely used dye for detection of proteins by gel electrophoresis. However, commercially available CBBs are complex mixtures of numerous chromogenic compounds that vary from lot to lot, thereby giving an undesirable level of variation in reproducibility, precision, and specificity in staining gels. We have developed a silica gel column chromatographic method for purification of commercial CBBs in high yield and have standardized each lot to perform equivalently in staining proteins as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and quantitative scanning densitometry. This is a major improvement in protein purity determinations by quantitative scanning densitometry. A thin-layer chromatographic method for quality control testing of the purified CBB lots was also developed. Plasma desorption mass spectrometry was used to identify components of silica gel column fractions. Scanning densitometry was the technology used to establish performance equivalency between different CBB preparations. The less polar chromogenic compounds are nonblue and/or fluorescent in color, contain mono- or unsulfonated structures, and lack significant protein binding capacity. The more polar chromogenic compounds are green and blue–green in color, contain tri- and tetrasulfonated moieties, compared to the disulfonated structure of CBB, and bind to protein at least 40 times more effectively than pure CBB. The concentrations of these highly polar chromogens differ from lot to lot and act as “inhibitors” in protein staining, thereby causing variability in protein staining.

Biosynthesis of labdenediol and sclareol in cell-free extracts from trichomes of Nicotiana glutinosa

Biosynthesis of the diterpenes labdenediol and sclareol from all trans-geranylgeranyl pyrophosphate was observed in cell-free extracts prepared from leaf midvein epidermal peels of Nicotiana glutinosa 24A. The bioactivities were shown to be exclusively localized in trichomes, to be conferred by a soluble enzyme(s), and to resemble other plant terpene cyclase activities in basic characteristics. Chromatographic methods for protein purification including gel filtration, anion exchange, hydroxyapatite chromatography, and free-flow isoelectric focusing. Thermal inactivation and end-product inhibition experiments did not afford separation of the biosynthetic activities. Results indicate that these labdane diterpenes are direct products of cyclization reactions catalysed by a soluble cyclase(s). To the best of our knowledge, this report represents the first case for direct biosynthesis of diterpene di-alcohols by cyclization of the acyclic precursor. A unified scheme for formation of labdane mono- and di-alcohols in Nicotiana species is presented.

Low-level endoglucanase contamination in a Trichoderma reesei cellobiohydrolase II preparation affects its enzymatic activity on β-glucan

A routinely employed and specific affinity chromatography purification method was used to isolate cellobiohydrolase II (CBHII) from a Trichoderma reesei culture supernatant. Two different identically purified batches of CBHII were found to have distinctly different properties in barley β-glucan hydrolysis. The abilities of the two preparations to produce small oligosaccharides and reduce the viscosity of β-glucan were substantially different, but no contamination or heterogeneity was detected in sodium dodecyl sulfate-electrophoresis used to assess the protein purity. Furthermore, the specific activities of the preparations on cellotetraose substrate were similar. Careful analysis with specific chromogenic oligosaccharide substrates showed that the considerable variation in β-glucan hydrolysis was caused by a minor endoglucanase contamination consisting of only 0.4% of the total protein. The contamination is possibly caused by nonspecific interactions between the proteins and chromatographic column material or by unfavorable protein-protein interactions during the chromatographic separation. The data demonstrate clearly the caution needed in the interpretation of hydrolysis studies on polymeric substrates with cellulolytic enzyme preparations.

Chromatography in plasma fractionation: benefits and future trends.

Industrial-scale chromatographic fractionation and purification methods have been used increasingly in the last few years for plasma fractionation. This has resulted in the development of a new generation of therapeutic plasma derivatives, especially coagulation factors, protease inhibitors and anticoagulants. Implementation and combination of ion-exchange, affinity and size-exclusion chromatography have allowed the development of new therapeutic products with improved purity and safety for treating congenital or acquired plasma protein deficiencies in patients. More recently, the benefit of chromatographic purification of plasma proteins in the removal of plasma-borne viruses has been revealed. Development of packing materials with improved characteristics for industrial applications, including higher capacity and rigidity, should further promote the use of chromatography as an essential plasma fractionation tool and confine more and more the traditional ethanol precipitation methods to the final processing stages used to recover albumin.

3-Butyl-5,6-dihydro-4H-isobenzofuran-1-one, a sensorial active phthalide in parsley roots

The analysis of parsley root revealed the presence of a novel phthalide derivative with low odor threshold. The structure 3-butyl-5,6-dihydro-4H-isobenzofuran-1-one followed from interpretation of MS, GC-FTIR, and NMR spectroscopic data. It can be derived from sedanenolide. Due to its restricted stability, a high-speed countercurrent chromatography isolation and purification method, which allows separation from other phthalides, had to be elaborated

Selective high-performance liquid chromatographic purification of bispecific monoclonal antibodies

The recent development of improved production techniques for bispecific monoclonal antibodies (biMAbs) has significantly increased interest in specific purification procedures. In this investigation, a general high-performance liquid chromatographic (HPLC) purification method is proposed that allows highly purified biMAbs to be obtained from mouse ascites fluid containing a mixture of different antibodies, i.e., parental MAbs, active biMAb and a mixture of randomly assembled heavy and light chains. Proteins from ascites fluid were precipitated with ammonium sulphate and applied to a high-performance protein A column to separate the total immunoglobulin fraction. BiMAbs were isolated from other immunoglobulins by two subsequent passages through a high-performance hydroxyapatite (HPHT) column. This purification protocol combines specificity of protein A for immunoglobulin G (IgG) and high selectivity of hydroxyapatite for different IgG idiotypes. All purification steps were performed rapidly and reliably by HPLC. This method was applied to the purification of six different biMAbs with consistently high yields, purity and homogeneity. This general purification method may prove extremely valuable when highly pure preparation of biMAbs is required, as for in vivo use.

High-performance liquid chromatographic method for the simultaneous purification of cathepsins B, H and L from human liver

A high-performance liquid chromatographic procedure for the isolation of the three cysteine proteinases, namely cathepsins B, H and L, is described. The method is based on the following four steps. (1) A classical AcA 44 gel permeation separation with a 30–70% ammonium sulphate fraction from the human liver homogenate is used to remove the non-enzymic high-molecular-mass components. (2) Preparative cation-exchange chromatography on a CM-SW TSK column can separate the three proteinases. (3) An anion-exchange step on a semi-preparative DEAE-SW TSK column for the cathepsin H fraction is used to remove a small amount of cathepsins B and L activities. (4) The three separated enzymes are purified on an analytical TSK gel 2000 SW column. The purity of each enzyme is assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and electrofocusing on polyacrylamide gels. To check the activities of the purified proteinases, the kinetic constants [Michaelis constant ( K M) and catalytic constant ( K cat)] and the ratio K cat/ K M against the fluorigenic substrates Arg-NH-Mec, Z-Arg-Arg-NH-Mec and Z-Phe-Arg-NH-Mec after active-site titration using E-64, were determined. Z-Phe-Phe-CNH 2 was also used as a specific inhibitor of cathepsin L. This method requires only 6 g of human liver, and gives a high yield of the three lysosomal cysteine-proteinases: thus, about 150 μg of cathepsin B and 50 μg each of cathepsins L and H are obtained in a single run.

Quantitation and Confirmation of Sulfamethazine Residues in Swine Muscle and Liver by LC and GC/MS

The present paper describes a liquid chromatographic (LC) method for purification of crude swine tissue extracts before gas chromatographic/mass spectrometric (GC/MS) quantitation and confirmation of sulfamethazine at low ppb levels. Fractions corresponding to sulfamethazine were collected, evaporated to dryness, N-methylated with diazomethane, concentrated, and analyzed by GC/MS. A mass spectrometer was set to selected ion monitoring (SIM) mode. Ions 233, 227, 228, and 92 m/z were detected. Ratio 227/233 m/z (sulfamethazine/internal standard, [phenyl 13C6] sulfamethazine) was used for quantitation, while ratios 228/227 and 92/227 m/z, respectively, were used for confirmation. Quantitation in spiked blank muscle tissue was tested from 100 to 1 ppb and found acceptable at all concentrations studied; coefficients of variations ranged from 4.9 to 14.4%. Similar results were obtained for liver tissue from 5 to 20 ppb; coefficients of variation ranged from 1.2 to 9.1%.

Preparative purification of tetrabromotetrachlorofluorescein and phloxine B by centrifugal counter-current chromatography

A centrifugal counter-current chromatographic method for preparative purification of commercial tetrabromotetrachlorofluorescein and Phloxine B (D&C Red Nos. 27 and 28, respectively) was developed. Ethyl acetate— n-butanol–0.01 M ammonium acetate (1:1:2) was used as the two-phase solvent systems. Each purification trial involved 50 mg of sample and yielded 22 mg (±2 mg) of pure dye. The purity of the product was measured by hihg-performance liquid and thin-layer chromatography and was found to be 99.9%. The partition coefficients of these compounds were found to be highly concentration-dependent in the two-phase solvent system used. If this problem can be circumvented, then the counter-current chromatographic method can be extended for use with gram quantities of dye.

Purification of human thrombin by affinity chromatography for its use in preparations for biological coagulation

Biological glue is obtained by mixing different specific plasma proteins including a serine protease, thrombin. Surprisingly at present the thrombin used in such a mixture is from equine or bovine origin while all other components are from human. In this paper we described a particular efficient and specific chromatographic method for the purification of human thrombin usable as a serine protease in the preparation of biological glue. A pure and active thrombin is obtained from a plasma fraction after adsorption on benzamidine-Spherodex followed by an elution with non specific (sodium chloride gradient) or biospecific competitors (arginine methylester or benzamidine). The obtained thrombin with a yield close to 80% and a purification factor close to 160, showed good properties in the replacement of animal thrombin in the condition of biological glue.

63] Purification of rod outer segment GTP-Binding protein subunits and cgmp phosphodiesterase by single-step column chromatography

This chapter describes a novel and simple single-step chromatographic method for the extensive purification of the subunits of the GTP-binding protein and the phosphodiesterase (PDE). There is a well-established homology between the hormone-sensitive adenylate cyclase and the light-activated cGMP phosphodiesterase in rod outer segments (ROS). The light-activated PDE of ROS has three functional segments. The photopigment rhodopsin, the GTP-binding protein, and the PDE catalytic complex. Signal transduction from rhodopsin to PDE is dependent on the binding of GTP to the α subunit of the GTP-binding protein. Photon capture is necessary for the binding of GTP to the Gα subunit. The GTP/Gα complex is rapidly released from the disk membrane surface. The activation of PDE in ROS prepared from the amphibian retina occurs as a consequence of the presentation of photons and the addition of GTP. Moreover, this activation depends upon the physical release of an inhibitory moiety from the PDE that remains bound to the disk membrane throughout the activation cycle. The major components of the ROS light-sensitive PDE cascade have been isolated and purified. The purification of rhodopsin has been well characterized in a number of laboratories.

Purification of cellobiohydrolase I fromTrichoderma reesei using monoclonal antibodies in an immunomatrix

The several components of the fungal cellulase system present practical problems in devising facile and efficient schemes for their purification. We report on a new single-step affinity chromatographic method for purification of cellobiohydrolase I ofTrichoderma reesei based on its selective absorption and elution using an immunomatrix constructed with CnBr-activated Sepharose 4B and monoclonal antibody specific for the enzyme. Isoenzymes of cellobiohydrolase I were purified directly from crude culture filtrate. The method is fast, simple, and of high resolution.