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At least 181 records · Page 10Linked to original sources
PubMed1993

Isolation of plasma proteins from the clotting cascade by heparin affinity chromatography.

The use of heparin affinity chromatography for the isolation of plasma proteins from the clotting cascade is described. The separation is carried out with heparin agarose and, in parallel operations, with different rigid gels on a polymer base. The quality of the separation and the reproducibility of the results were investigated and the stability of the materials at high pH was tested. The affinity supports were used for the isolation of antithrombin III from human plasma and for the separation of factor IX from factor X, after partial purification by anion-exchange chromatography. The isolation of antithrombin III from human plasma served as a model. The non-specific bindings were investigated, together with the resistance of the support when treated with 0.2 and 0.5 M sodium hydroxide. Heparin agarose has low non-specific bindings, but it cannot be exposed to high pH. The supports on a polymer base are resistant to high pH, up to 13.7. However, they may remain slightly hydrophobic, and the hydrophobicity of the matrix leads to an increase in non-specific bindings. When antithrombin III is isolated, the non-specific bindings result in contamination of the final product. The lack of resistance of the matrix at high pH causes a weaker binding of antithrombin III, and the product is eluted at lower and lower sodium chloride concentrations. The results can be indicative of the behaviour of the support in the separation of factor IX from factor X. High non-specific bindings will lead to contamination of the factor IX product and consequently to low specific activity. Insufficient resistance of the support at high pH will result in failure to separate the two clotting factors satisfactorily. The separation can be monitored by heparin high-performance membrane affinity chromatography (HPMAC). Contamination of the sample, which occur in sodium dodecyl sulphate-polyacrylamide gel electrophoresis are detected within minutes by fast heparin HPMAC.

Antithrombin III↗
PubMed2000

[Study on protein separation using immobilized metal ion affinity chromatography].

Immobilized metal ion affinity chromatography (IMAC) has shown promise of isolating desired proteins from a mixture based on their difference of affinity for chelated metal ions. With its technological superiority, such as large adsorption capacity, mild separation condition, simple ligands and wide applications, IMAC has become powerful tool for biotechnological products separation, such as proteins, amino acids and gene products. In spite of many sophisticated applications for IMAC, the theoretical analysis of Immobilized metal chromatography has remained insufficient. In this paper, the eluted efficiencies of bovine serum albumin (BSA) in a single-component system under different elution conditions are studied. The effects of several elution factors, such as pH value, ammonium concentration and anion species on protein separation are studied. Comparing the elution data of BSA in IDA-Cu and IDA-Zn columns, the different ability of affinity between metal ions and proteins is found. In addition, the elution behaviors of different proteins are investigated. This work facilitates the further research in separation of real systems.

Chromatography, Affinity↗
PubMed1996

Purification of Mycobacterium bovis BCG Tokyo antigens by chromatofocusing, lectin-affinity chromatography, and hydrophobic interaction chromatography.

A combination of chromatofocusing, lectin-affinity chromatography, and hydrophobic interaction chromatography resulted in a simple purification of protein antigens of Mycobacterium bovis BCG Tokyo culture filtrate. Identification was established on the basis of chromatographic separation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis determination of molecular weights, and N-terminal amino acid determination. Chromatofocusing on PBE 94 accomplished the separation of BCG85B from other BCG85 complex antigens and partial separation of MPB64 and MPB70 antigens. Subsequently, MPB64 and MPB70 were completely separated on a high-performance liquid chromatography TSK Phenyl 5PW hydrophobic interaction chromatography column. This column also separated BCG85B from a 17-kDa protein with an N-terminal amino acid sequence of A-V-P-I-T-G-K-L-G-S-E-L-T-M-T-D-( )-V-G-Q, which is similar to the sequence of MPT63. Concanavalin A-Sepharose-affinity chromatography separated MPB64 from a 43- and 47-kDa doublet with an amino acid sequence of D-P-E-P-A-P-P-V-P-P-V-P-A-( )-A-A-S-P, which is similar to the sequence of MPT32 and which appears to be glycosylated.

Amino Acid Sequence↗
PubMed1978

Purification of the hexokinases by affinity chromatography on sepharose-N-aminoacylglucosamine derivates. Design of affinity matrices from free solution kinetics.

The purification is described of rat hepatic hexokinase type III and kidney hexokinase type I on a large scale by using a combination of conventional and affinity techniques similar to those previously used for the purification of rat hepatic glucokinase [Holroyde, Allen, Storer, Warsy, Chesher, Trayer, Cornish-Bowden & Walker (1976) Biochem. J. 153, 363-373] and muscle hexokinase type II [Holroyde & Trayer (1976) FEBS Lett. 62, 215-219]. The key to each purification was the use of a Sepharose-N-aminoacylglucosamine affinity matrix in which a high degree of specificity for a particular hexokinase isoenzyme could be introduced by either varying the length of the aminoacyl spacer and/or varying the ligand concentration coupled to the gel. This was predicted from a study of the free solution kinetic properties of the various N-aminoacylglucosamine derivatives used (N-aminopropionyl, N-aminobutyryl, N-aminohexanoyl and N-aminooctanoyl), synthesized as described by Holroyde, Chesher, Trayer & Walker [(1976) Biochem. J. 153, 351-361]. All derivatives were competitive inhibitors, with respect to glucose, of the hexokinase reaction, and there was a direct correlation between the Ki for a particular derivative and its ability to act as an affinity matrix when immobilized to CNBr-activated Sepharose 4B. Muscle hexokinase type II could be chromatographed on the Sepharose conjugates of all four N-aminoacylglucosamine derivatives, although the N-aminohexanoylglucosamine derivative proved best. This same derivative was readily able to bind hepatic glucokinase and hexokinase type III, but Sepharose-N-amino-octanoyl-glucosamine was better for these enzymes and was the only derivative capable of binding kidney hexokinase type I efficiently. Separate studies with yeast hexokinase showed that again only the Sepharose-N-amino-octanoylglucosamine was capable of acting as an efficient affinity matrix for this enzyme. Implications of these studies in our understanding of affinity-chromatography operation are discussed.

Animals↗
PubMed1999

Purification of human chorionic gonadotrophin from urine by membrane filtration affinity chromatography with a positively charged membrane.

UNLABELLED: A new method of affinity chromatography, termed membrane filtration affinity chromatography (MFAC), has been developed and applied to purify HCG from urine. By filtrating urine through ZBM (HCG in urine would bind to the antibody on ZBM) and by dissociating the HCG from the antibody on ZBM in purified form, we developed the MFAC and purified HCG from urine of pregnant women by MFAC. The purified HCG showed a single band in polyacrylamide gel electrophoresis. ZBM (1 cm(2)) could harvest 90.3 microg HCG, which showed immunoactivity of 8554 IU/mg. The rate of recovery was 87%. CONCLUSION: MFAC with ZBM is an effective method, which is much easier and cheaper than conventional affinity chromatography for purification of proteins from solution, especially from highly diluted solution.

Chorionic Gonadotropin↗
PubMed1978

Affinity chromatography of H+-translocating adenosine triphosphatase isolated by chloroform extraction of Rhodospirillum rubrum chromatophores. Modification of binding affinity by divalent cations and activating anions.

1. ATPase isolated from Rhodospirillum rubrum by chloroform extraction and purified by gel filtration or affinity chromatography shows three bands (alpha, beta and gamma) upon electrophoresis in sodium dodecyl sulphate. 2. Ca2+-ATPase activity of the preparation is inhibited by aurovertin and efrapeptin but not by oligomycin. Activity may be inhibited by treatment with 4-chloro-7-nitrobenzofurazan and subsequently restored by dithiothreitol. 3. The enzyme fails to reconstitute photophosphorylation in chromatophores depleted of ATPase by sonic irradiation. 4. Most of the active protein from the crude chloroform extract binds to an affinity chromatography column bearing an immobilised ADP analogue but not to a column bearing immobilised pyrophosphate. 5. In the absence of divalent cations, a component with a very high specific activity for Ca2+-ATPase is eluted from the column by 1.6 mM ATP. This protein migrates asa single band on 5% polyacrylamide gel electrophoresis and only possesses three subunits. At 12 mM ATP an inactive protein is eluted which does not run on acid or alkali polyacrylamide gels and shows a complex subunit structure. 6. ATPase preparations prepared by acetone extraction or by sonic irradiation of chromatophores may also be purified 10-fold by affinity chromatography. 7. The inclusion of 5 mM MgCl2 or CaCl2 during affinity chromatography of chloroform ATPase increases the capacity of the column for the enzyme and demands a higher eluting concentration of ATP. 8. When the enzyme is more than 90% inhibited by efrapeptin or 4-chloro-7-nitrobenzofurazan, the binding characteristics of the enzyme are not affected. 9. 10 mM Na2SO3, which greatly stimulates the Ca2+- and Mg2+-dependent ATPase activity of the enzyme and increases Ki (ADP) for Ca2+-ATPase from 50 to 850 micron, prevents binding to the affinity column. Binding may be restored by the addition of divalent cations. 10. Na2SO3 increases the rate of ATP hydrolysis, ATP-driven H+ translocation and ATP-driven transhydrogenase in chromatophores. 11. It is proposed that anions such as sulphite convert the chromatophore ATPase into a form which is a more efficient energy transducer.

Adenosine Triphosphatases↗
PubMed1984

Preparation of eight ovalbumin subfractions by combined lectin affinity chromatography.

Ovalbumin was fractionated by successive lectin affinity chromatography using concanavalin A/Sepharose and wheat germ agglutinin/Sepharose. Eight glycoprotein fractions, all behaving as ovalbumin on polyacrylamide gel electrophoresis, were obtained. To characterize the carbohydrate chains, the asparaginyl-carbohydrates were prepared from the Pronase digests of the ovalbumin fractions and their dansyl derivatives were analyzed by high-performance liquid chromatography. The elution profile of the dansylated asparaginyl-carbohydrates from each subfraction was compared with that from the unfractionated ovalbumin. The results indicated that the above eight subfractions could be separated from each other according to their carbohydrate chains and that three of the subfractions were homogeneous with respect to their carbohydrate chains.

Carbohydrate Sequence↗
PubMed2003

Characterization of p-aminobenzamidine-based sorbent and its use for high-performance affinity chromatography of trypsin-like proteases.

An affinity sorbent, hydrophilic polymer-based carrier of different pore size (Toyopearl) with immobilized p-aminobenzamidine (ABA), has been prepared. Its basic properties and some applications for protein purification were studied. ABA, which is a synthetic inhibitor for trypsin-like proteases, was covalently immobilized to Toyopearl by reductive amination. The ligand density and binding capacity for porcine trypsin varied depending on the pore size of Toyopearl. The maximum binding capacity of the immobilized p-aminobenzamidine Toyopearl (ABA-Toyopearl) for trypsin was more than 40 mg/ml gel. ABA-Toyopearl thus obtained was very stable below pH 8 and was successfully used for high-performance affinity chromatography of trypsin-like proteases such as trypsin, thrombin, tissue-type plasminogen activator or urokinase in a single step at 25 degrees C.

Benzamidines↗
PubMed1999

Characterisation of the binding of digitoxin and acetyldigitoxin to human serum albumin by high-performance affinity chromatography.

Zonal elution and high-performance affinity chromatography were used to examine interactions of the drugs digitoxin and acetyldigitoxin with the protein human serum albumin (HSA). This was done by injecting small amounts of digitoxin and acetyldigitoxin onto an immobilized HSA column in the presence of mobile phases that contained various concentrations of digitoxin, acetyldigitoxin or other solutes as competing agents. A fixed concentration of beta-cyclodextrin was also present in the mobile phase as a solubilising agent. It was found that digitoxin and acetyldigitoxin each had strong interactions at a single common binding site on HSA, but with slightly different equilibrium constants for this region. Neither compound showed any competition with warfarin or L-tryptophan, which were used as probes for binding at the warfarin-azapropazone and indole-benzodiazepine sites of HSA. These results confirmed the presence of a separate binding region on HSA for digitoxin-related compounds.

Acetyldigitoxins↗
PubMed1999

Characterization of minor site probes for human serum albumin by high-performance affinity chromatography.

This study used high-performance affinity chromatography (HPAC) and immobilized human serum albumin (HSA) columns to examine the specificity and cross-reactivity of various compounds that have been proposed as markers for the minor binding sites of HSA. These agents included acetyldigitoxin and digitoxin as probes for the digitoxin site, phenol red as a probe for the bilirubin site, and cisor trans-clomiphene as markers for the tamoxifen site. None of these probes showed any significant binding at HSA's indole-benzodiazepine site. However, phenol red did bind at the warfarin-azapropazone site of HSA, and cis/trans-clomiphene gave positive allosteric effects caused by the binding of warfarin to HSA. Digitoxin and acetyldigitoxin were found to bind to a common, unique region on HSA; cis- and trans-clomiphene also appeared to interact at a unique site, although trans-clomiphene displayed additional direct competition with phenol red. From these results it was possible to develop a model that described the general relationship between these binding regions on HSA. This information should be useful in future studies that employ HPAC for characterizing the binding of HSA to other drugs or clinical agents.

Binding Sites↗
PubMed1980

Affinity chromatography of cells and cell membranes.

This article attempts to introduce the concept of cellular affinity chromatography as it departs from molecular affinity chromatography. Special emphasis is placed on the selectio of a solid support as well as on the role of lectins as affinity ligands. Our major goal was to bring to light the basic principles involved, multiple options of ligands and matrices, and different techniques, which may be applied to separate the complex cell population as well as cell membranes. It is hoped that further developments in the field, especially in the selection of proper experimental conditions, ligands and matrix material may provide better results. We have tried to identify some of the potential problems which should be considered before these approaches can be used on a routine basis. Although the review deals primarily with the affinity chromatography of cells and cell membranes, examples are presented for diverse systems such as cell organelles, viruses and phages. A table summarizing the use of cellular affinity chromatography is included. It lists more than 80 examples covering the literature up to December, 1979.

Animals↗
PubMedDate not supplied

High-performance liquid chromatography of proteins on deformed nonporous agarose beads: immuno-affinity chromatography, exemplified with human growth hormone as ligand and a combination of ethylene glycol and salt for desorption of the antibodies.

An affinity chromatography column packed with nonporous agarose beads derivatized with human growth hormone via carbonyldiimidazol was used for the purification of antibodies against human growth hormone from antiserum. Desorption with 1 M sodium chloride in 60% ethylene glycol at pH 9.8 gave 100% total recovery of the antibodies, as measured by radioimmunoassay. The adsorption/desorption process is discussed in terms of hydrophobic and electrostatic interaction (these interactions may be involved in the bond between antibody and antigen in a cooperative fashion). The binding capacity of the column was estimated at about 50 micrograms of antibodies per gram sedimented agarose beads.

Antibodies↗
PubMed1986

Purification of cathepsin B by a new form of affinity chromatography.

Human cathepsin B was purified by affinity chromatography on the semicarbazone of Gly-Phe-glycinal linked to Sepharose 4B, with elution by 2,2'-dipyridyl disulphide at pH 4.0. The product obtained in high yield by the single step from crude starting material was 80-100% active cathepsin B. The possibility that this new form of affinity chromatography may be of general usefulness in the purification of cysteine proteinases is discussed.

2,2'-Dipyridyl↗
PubMedDate not supplied

Affinity chromatography of yeast alcohol dehydrogenase using immobilized monochlorotriazine colourless compounds.

Four new symmetrical colourless biomimetics bearing two monochlorotriazine rings have been rationally designed and synthesized. These immobilized colourless ligands and one analogue of Cibacron Blue F3GA on Sepharose CL-4B were used to purify alcohol dehydrogenase from baker's yeast extract. Twenty-two-fold purification with 78% enzyme recovery was achieved in a single step with specific elution of NAD+ (5 mM) from the colourless absorbent comprised of two ortho-aminobenzene sulphonates as terminal rings, which is comparable to the result obtained from the analogue of Cibacron Blue F3GA. Differential spectra between ligand-enzyme complexes and free ligands were studied.

Alcohol Dehydrogenase↗
PubMed1984

Synthesis and evaluation of immobilized androgens for affinity chromatography in the purification of nuclear androgen receptor.

Seven biospecific adsorbents containing immobilized androgens were synthesized: dihydrotestosterone-17 beta-succinyl agarose, and both the unsubstituted and the 17 beta-acetyl derivatives of dihydrotestosterone-7 alpha-undecanoyl agarose, testosterone-7 alpha-undecanoyl agarose, and 19-nortestosterone-7 alpha-undecanoyl agarose. The retention capacities for nuclear androgen receptor were generally between 40-80% with little variation in reproducibility; the amount of binding was greatest with dihydrotestosterone-17 beta-succinyl agarose and dihydrotestosterone-17 beta-acetoxy-7 alpha-undecanoyl agarose. Rapid flow rates were obtained with all gels, and no tendency for decomposition was observed over a period of 1 year. Factors that affected retention included the concentration of immobilized androgen, length of the linker arm, occupation of receptor sites, interval of contact with the gel, and temperature of incubation. Chemical dissociation of androgens from androgen receptor complexes with 0.2 mM mersalyl increased the retention of receptor by dihydrotestosterone-17 beta-succinyl agarose. Two elutants showed promise for the dissociation of gel-bound receptor: 1) 0.2 mM mersalyl in the presence of 1.5 mg/ml of ovalbumin; 2) 10% (v/v) dimethylformamide:water containing 30 microM [1,2-3H] dihydrotestosterone and 0.5 M sodium thiocyanate.

Androgens↗
PubMed1993

Characterization of porcine intestinal cytochrome c oxidase subunit VIIc, purified by affinity chromatography.

Cytochrome c oxidase subunit VIIc was isolated from porcine intestine. Its amino acid sequence, starting at position 17 of the predicted precursor peptide, differs at eight positions from the human form, two positions from the bovine and at one from the mouse form. Although the peptide does not contain cysteine, it was bound specifically to thiopropyl-Sepharose 6B. The developed method makes it possible to obtain this protein in high purity and large quantities for studies of its putative modulatory role in the catalytic actions of the whole enzyme complex.

Amino Acid Sequence↗