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PubMed1976

Use and abuse of sepharose-conjugated antibodies for the isolation of lymphocyte-surface immunoglobulins.

Immunoadsorbents of Sepharose-4B-conjugated antibodies were shown to be suitable for the characterization, by subsequent SDS-polyacrylamide gel electrophoresis, of splenocyte-membrane Immunoglobulin (Ig) solubilized by detergent lysis of surface 125I-labelled cells. However, high non-specific binding of 125I-labelled lymphocyte-membrane components to Sepharose-4B prevented accurate quantition of Ig in such lysates. 125I-labelled lymphocyte-membrane components solubilized by metabolic release also showed high non-specific binding to Sepharose-4B, and this interfered with both quantitative and qualitative analysis of Ig solubilized in this manner. Initial attempts to overcome the nonspecific binding of 125I-labelled lymphocyte-membrane components to Sepharose-4B were unsuccessful, and attention is drawn to the technical problems of using Sepharose-4B as a matrix for solid-phase immunoadsorbent studies of lymphocyte-membrane Ig.

Animals↗
PubMed1976

Immunoadsorbents: non-specific binding of proteins to albumin-sepharose.

Human serum albumin-Sepharose was prepared by coupling human serum albumin to cyanogen bromide activated Sepharose 4B. This immunoadsorbent showed considerable non-specific protein adsorption. The adsorbed proteins were mainly immunoglobulins which could not be separated from required antibody. It is suggested that basic groups formed in the preparation of the albumin-Sepharose are responsible for this non-specific protein adsorption. Non-specific protein adsorption could be completely eliminated by neutralizing the basic groups of the albumin-Sepharose with the anionic dye blue dextran. The resultant blue dextran-albumin-Sepharose allowed purification of anti-albumin-antibody with high yield. It is shown that the isolated antibody is pure and retains its native properties.

Adsorption↗
PubMed1983

Selective binding of heat- and antigen-aggregated IgG to Blue-Sepharose. Radioimmunoassay of circulating immune complexes.

In an attempt to find a direct method for the determination of circulating immune complexes in pathological sera we studied the ability of Cibacron Blue 3GA covalently bound to Sepharose 4B to bind heat- or antigen-aggregated human IgG. Blue-Sepharose was found to bind significant amounts of aggregated IgG (about 1.5 mg/ml packed gel) but very low amounts of monomeric IgG (about 0.1 mg/ml gel). The ability of Blue-Sepharose to bind immune complexes was tested on human pathological sera containing variable amounts of circulating immune complexes. Using both a turbidimetric measurement and Barkas' method we found that Blue-Sepharose is able to remove a very high percent of circulating immune complexes (greater than 80%) from sera. Considering the above observations a new method is described for the direct determination of circulating immune complexes using Blue-Sepharose as serum adsorbent and either 125I-labeled protein A or 125I-labeled rabbit IgG anti-human IgG to demonstrate the binding of immune complexes.

Animals↗
PubMed1982

Sepharose-insolubilization of the dihydrolipoyl transacetylase core component of the pyruvate dehydrogenase complex: preparation and characterization.

The dihydrolipoyl transacetylase core components of the bovine kidney and heart pyruvate dehydrogenase complexes were covalently attached through the lipoyl moiety to Sepharose by the thiol-crosslinking reagent, N,N'-p-phenylenedimaleimide. In one approach, the N,N-p-phenylenedimaleimide was allowed to react with glutathione which was in turn linked by its N-terminal to Sepharose CL-6B. In addition, we found that N,N-p-phenylenedimaleimide would react directly with Sepharose CL-6B (at undetermined sites) and could be used as the sole bridge in forming a stable linkage of the transacetylase core to Sepharose. With the latter approach the extent of multiple-linkage of the 60-subunit core could more easily be controlled. This should be a generally useful approach for linking proteins with reactive surface thiol residues. Insolubilization of the core of the pyruvate dehydrogenase complex by these methods did not appear to significantly alter the binding of other protein components of the complex, but the catalytic activities of the complex requiring the lipoyl moiety were appreciably altered. Procedures for coupling the transacetylase core to various derivatives of phenylenedimaleimide-Sepharose and techniques described for studying the protein products should be useful in preparation of specialized matrices for both protein purification and the study of protein-protein interactions.

Acetyltransferases↗
PubMed1983

Interaction of purified brush-border membrane aminopeptidase N and dipeptidyl peptidase IV with lectin-sepharose derivatives.

The glycoprotein nature of two peptidases purified from the rat intestinal brush-border membrane was examined by their interaction with several lectin-Sepharose derivatives. Aminopeptidase N (EC 3.4.11.2), which contains 20% carbohydrate by weight, was bound minimally (less than 30%) by columns of Con A-, RCAI- and WGA-Sepharose. Alternatively, a greater proportion of dipeptidyl peptidase IV (EC 3.4.14.-) was bound by these immobilized lectins with 50% of the enzyme binding to Con A-Sepharose. Treatment of both enzymes with neuraminidase enhanced the binding of aminopeptidase to RCAI-Sepharose by 4-fold but did not alter the binding patterns of dipeptidyl peptidase IV. A sequential fractionation of the two peptidases with columns of Con A- and RCAI-Sepharose gave four fractions of each enzyme with differing lectin-binding specificities. Approximately 60% of the dipeptidyl peptidase IV interacted with either one or both of the lectins while only 30% of the aminopeptidase N did so. Kinetic analysis of the four isolated fractions revealed some differences, possibly related to variations in the carbohydrate moiety. The findings confirm that these two purified rat intestinal brush-border membrane peptidases are glycoproteins and, while they share a common physiologic function and source, they apparently have very different and possibly unique asparagine-linked oligosaccharide side-chains. In addition, a considerable degree of microheterogeneity exists in the carbohydrate structure of these two enzymes.

Aminopeptidases↗
PubMed1975

Purification of SV-40 messenger RNA by hybridization to SV-40 DNA covalently bound to Sepharose.

SV-40 DNA sheared form was coupled in a stable covalent bond to cyanogen bromide activated Sepharose. Under the conditions used at least 80% of the DNA was bound to Sepharose. The T 1/2 of hybridization of 0.5 mug/ml of SV-40 cRNA to SV-40 DNA-Sepharose was 1 hr. This rate of hybridization is sufficiently rapid to purify SV-40 sequences from solutions containing as little as 0.05-0.1 mug/ml. Nonspecific hybridization of RNA is in the range of 0.1-0.2% of the total input RNA. The DNA-Sepharose is fairly stable and can be reused several times to purify RNA. The SV-40 DNA-Sepharose was used to select large quantities of virus specific RNA from SV-40 infected BS-C-1 cells. The virus specific RNA when added to cell-free extracts from wheat germ was shown to direct the synthesis of the major viral structural protein VP-1.

Cells, Cultured↗
PubMed1997

Characteristics of the adsorption of immunoglobulin M onto Q Sepharose Fast Flow ion-exchangers.

Measurement of adsorption breakthrough curves in packed beds has shown that the amounts and rates of uptake of immunoglobulin M (IgM) onto the commonly used anionic ion-exchanger Q Sepharose Fast Flow (based on 6% agarose) are severely limited as a result of the large molecular size of this adsorbate (RMM 950,000). A similar ion-exchanger based on a more porous 4% agarose, Q Sepharose 4 Fast Flow was evaluated as an alternative adsorbent for the purification of IgM. Equilibrium adsorption isotherms and the effective diffusivities of IgM within these two adsorbents were measured. Q-Sepharose 4 Fast Flow was found to have a maximum capacity for IgM 2.5 times greater than that of Q Sepharose 6 Fast Flow and the effective diffusivity of IgM was found to be between 6 and 7 times greater than with the latter material. Comparison of the breakthrough curves obtained for these adsorbents at a variety of flow velocities confirm that Q Sepharose 4 Fast Flow is a superior adsorbent for the capture and purification of large proteins.

Adsorption↗
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↗
PubMed1996

Binding of anti-band 3 autoantibody to sialylated poly-N-acetyllactosaminyl sugar chains of band 3 glycoprotein on polyvinylidene difluoride membrane and sepharose gel: further evidence for anti-band 3 autoantibody binding to the sugar chains of oxidized and senescent erythrocytes.

Binding specifically of naturally occurring anti-band 3 IgG antibody isolated from human plasma was investigated in a cell-free binding system. 125I-labeled human anti-band 3 IgG specifically bound to band 3 glycoprotein and lactoferrin, a glycoprotein that has poly-N-acetyllactosamine-type sugar chains like band 3, on the polyvinylidene difluoride blotting membrane. Binding was decreased by 50-70% when band 3 and lactoferrin were pretreated with N-glycosidase F, endo-beta-galactosidase, or neuraminidase. Binding of 125I-anti-band 3 IgG to band 3-Sepharose gel was partially inhibited by band 3 oligosaccharides or lactoferrin, but was less inhibited by them after they had been treated with N-glycosidase F or endo-beta-galactosidase. A significant part of 125I-anti-band 3 IgG that bound to the band 3-Sepharose gel was released upon treatment of the gel with N-glycosidase F or endo-beta-galactosidase. IgG that binds to lactoferrin (anti-lactoferrin IgG) was isolated from normal human plasma. 125I-Anti-lactoferrin IgG bound to the band 3-Sepharose gel as effectively as to the lactoferrin-Sepharose. The antibody specifically bound to the band 3- and lactoferrin-blotted membrane depending on the poly-N-acetyllactosaminyl sugar chains of the blotted glycoproteins. The results indicate that a major part (about 70%) of anti-band 3 IgG recognizes the sialylated poly-N-acetyllactosaminyl sugar chains of band 3 and lactoferrin, and the remaining part (about 30%) of the antibody may recognize the polypeptide portion of band 3. This was supported by the observation that anti-band 3 IgG effectively bound to lactoferrin-Sepharose but 33% of the antibody did not. Anti-band 3 IgG with the carbohydrate-binding property was equally obtained whether fully denatured or barely denatured band 3 was used for isolation of anti-band 3 IgG by affinity chromatography. These results provide further evidence for our proposal that the binding sites of anti-band 3 IgG to oxidized and senescent erythrocytes reside on the locally condensed sialylated poly-N-acetyllactosaminyl sugar chains of band 3 on the cell surface.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1982

A simple and sensitive proteinase assay using Sepharose 4B-coupled fluorescamine-labeled casein as a substrate.

A simple and sensitive method for proteinase assay was developed, which uses fluorescamine-labeled casein-Sepharose 4B as a substrate. Casein-Sepharose 4B was prepared most effectively by coupling casein to cyanogen bromide-activated Sepharose 4B at pH 10.0. Fluorescamine-labeled casein-Sepharose 4B was then prepared by mixing fluorescamine and casein-Sepharose 4B suspension at pH 8.0 and used for the assay as a substrate after removal of the excess reagent and/or its hydrolysis products. The assay can be done by simple measuring the fluorescence (excitation at 390 nm and emission at 475 nm) of the filtrate of the assay mixture after incubation of the substrate with enzyme solution. This method is suited for the assay of proteinases active at neutral to slightly alkaline pH values, and the activity of 3 ng of trypsin or 10 ng of alpha-chymotrypsin can be determined with reasonable accuracy. This method is therefore almost as sensitive as those using radioisotope-labeled proteins as substrates.

Animals↗
PubMedDate not supplied

The application of the sepharose bead immunofluorescence test for the detection of allergen-specific IgE and IgG antibodies in pollinosis.

A new application of the Sepharose bead immunofluorescence test for detection of allergen-specific IgE and IgG antibodies is described. Allergen extracts of four different grass pollens were coupled to CNBr-activated Sepharose 4B. Twenty normal and allergic sera were incubated with the allergen-coupled beads, washed and incubated with fluorescence-conjugated anti-gamma E and G globulins. After washing and staining with 0.5% trypan blue, the percentage of fluorescent beads was detected by fluorescence microscopy. In the IgG/anti IgG system, the smallest amount of IgG demonstrated was 20ng/ml; in the IgE/anti-IgE system it was 40ng/ml. Independent examination of tests gave a mean difference between observations of 4.1%. Reproducibility was also very good (var. coeff = 18%). The number of beads stained with IgE correlated well with intensity of skin reactions to the same extracts (r = 0.64; p = 5 x 10-7), the percentage of IgG stained beads being independent of skin reactivity. The Sepharose-IgE test allowed clear distinction between allergic and normal sera (p = 4 x 10-7), while the Sepharose IgG test did not distinguish between them at the time of diagnosis. However, the number of beads stained with IgG significantly increased in patients undergoing immunotherapy (p = 3.8 x 10-3). The Sepharose bead immunofluorescence test requires a very small amount of materials, is highly sensitive and easy to handle. It may be valuable in the "in vitro" diagnosis of grass pollen allergy and useful in evaluating immunotherapy.

Allergens↗
PubMed1978

[Biospecific chromatography of poly(A)-containing RNA on poly(U)-Sepharose].

It is shown that in addition to specific binding of polyadenylic sequence with poly(U), the chromatography of poly(A)-containing RNAs on poly(U)-Sepharose is accompanied by nonspecific irreversible adsorption of polynucleotides on Sepharose gel. This disadvantage may be overcome by establishing optimal BrCN/Sepharose rations during Sepharose activation and by many-fold treatment of poly(U)-Sepharose with ethanolamine immediately before chromatography of RNAs. It was also found that the efficient separation of poly(A+)-RNA preparations from poly(A-)-RNAs is achieved only after double chromatography of RNA on poly(U)-Sepharose. The amount of poly(A+)-RNA in total RNA preparations isolated from bound polyribosomes of 10-day-old chick embryos is equal to 1%. Data from PAAG gel electrophoresis are indicative of the lack of degradation and high heterogeneity of the preparations under study.

Chemical Phenomena↗
PubMedDate not supplied

Affinity purification of bacterial luciferase and NAD(P)H:FMN oxidoreductases by FMN-sepharose for analytical applications.

A modified purification method for bacterial luciferases and NAD(P)H:FMN oxidoreductases is described which uses FMN-Sepharose alone or coupled to DEAE ion exchange chromatography for the simultaneous purification of luciferase and the various oxidoreductases from Vibrio harveyi, a bright mutant of Vibrio harveyi, Vibrio fischeri, and Photobacterium phosphoreum. This purification method is compared with DEAE-Sepharose Cl 6B fractionations from these organisms. Both methods allow the separation of oxidoreductases specific for either NADH or NADPH. The use of FMN-Sepharose coupled to DEAE-Sepharose fractionation allows the isolation of highly purified enzymes. Lacking interfering factors, these are very suitable for various analytical applications based on bacterial bioluminescence enzymes. The partially purified enzymes from the affinity column have higher specific activities than those obtained using DEAE-Sepharose.

Chromatography, Affinity↗
PubMed2000

Arsanilic acid-Sepharose chromatography of pyruvate kinase from KB cells.

In the present study, arsanical-based affinity chromatography for pyruvate kinase (PK) isolation was explored. p-Arsanilic acid (4-aminophenyl arsonic acid), which contains an arsonic acid moiety structurally similar to inorganic pentavalent arsenate, was conjugated to Sepharose 4B via its para-amino group to form an As(V)-Sepharose matrix. The cellular proteins from KB cells bound to arsonic acid moieties were eluted by 50 mM sodium arsenate in Tris-HCl buffer (50 mM, pH 7.6). A single protein band with a molecular mass of 58 kDa was shown on a sodium dodecyl sulfate-polyacrylamide gel. By immunoblotting, amino acid sequencing and enzymatic analysis, the sodium arsenate-eluted 58-kDa protein was demonstrated to be a human PK (type M2). By using this one-step As(V)-Sepharose chromatography, PK from KB cells was purified 35.4-fold with a specific activity of 153.15 U/mg protein in the presence of 6 mM fructose-1,6-biphosphate. Although PK was eluted from an As(V)-Sepharose column with sodium arsenate, PK activity was apparently inhibited by the used eluent system, but not by p-arsanilic acid, indicating a specific interaction of As(V) to PK. In summary, our results indicate that As(V)-Sepharose can serve as a simple and efficient chromatographic support for PK purification from KB cells.

Arsanilic Acid↗
PubMed1978

Blue Sepharose chromatography of human alcohol dehydrogenase: evidence for interlocus and interallelic differences in affinity characteristics.

1. The various isozymes of human alcohol dehydrogenase have been examined by Blue Sepharose column chromatography. 2. The products (alpha, beta1 and gamma1) of the common alleles at the three ADH loci (ADH1 ADH2 and ADH3 respectively) were found to show slight, but significant differences in their affinities for Blue Sepharose. The order of affinity of the homodimeric isozymes was: alphaalpha less than gamma1gamma1 less than beta1beta1. The heterodimeric isozymes showed intermediate affinities. 3. The products (gamma1 and gamma2) of the common alleles (ADH31 and ADH32 respectively) at the ADH3 locus showed a pronounced difference in their affinities: the gamma1gamma1 isozyme was firmly adsorbed by Blue Sepharose, whereas the gamma2gamma2 isozyme was not adsorbed. The heterodimeric gamma1gamma2 isozyme was intermediate in its behaviour. 4. The 'usual' and 'atypical' forms of ADH were indistinguishable by Blue Sepharose column chromatography. 6. The 'anodal' form of ADH showed no affinity for Blue Sepharose.

Adult↗
PubMed1975

Demonstration of two molecular variants of carcinoembryonic antigen by concanavalin A sepharose affinity chromatography.

The carcinoembryonic antigen (CEA) active glycoproteins from perchloric acid extract of liver-metastasized primary colon tumor have been separated by concanavalin A Sepharose (Con A Sepharose) chromatography. The CEA activities separated by Con A Sepharose chromatography were designated as loosely bound and tightly bound which, respectively, eluted on the Con A Sepharose column between 0.12 and 0.15 M and 0.3 M alpha-methylmannose in a linear gradient of alpha-methylmannose. Further purification of these activities by Sephadex G-200, Bio-Gels A-1.5m and P-300 yielded two variants of glycoproteins (B1 and C2) with CEA activity. Both purified preparations of CEA had similar immunochemical properties. Their A280/A260 ratios were 1.30 and 1.56, respectively. The purified loosely bound CEA (B1) had immunological, chromatographic, and electrophoretic properties similar to those of 125I-CEA, whereas the tightly bound CEA (C2) had a lower molecular weight (120,000 to 140,000). Further, specificity to these two CEA's was established by their reactions in immunoelectrophoresis with preparations of specific goat anti-CEA anti-serum obtained from other investigators. The results indicate the practical use of Con A Sepharose affinity chromatography for the separation and characterization of glycoprotein tumor antigens.

Carcinoembryonic Antigen↗
PubMed1984

Representative applications of heparin-sepharose in the removal of polyamines from biological materials.

Many experimental systems would greatly benefit from the availability of a simple and effective technique to remove polyamines from biological materials. We have examined the possibility of utilizing heparin-sepharose in the removal of polyamines from rat heart mitochondria, DNA-spermine complex, and fetal calf serum. Heparin-sepharose removes 90% of spermine adsorbed to the cytoplasmic surface of rat heart mitochondria. Heparin-sepharose almost totally removes spermine from DNA-spermine complex, leaving less than 0.003 mol spermine/mol DNA phosphorus. Heparin-sepharose is highly effective in removing spermine and spermidine (99.5 and 95% adsorbed, respectively) from fetal calf serum. Under the same experimental conditions only 50% of putrescine is adsorbed. A higher amount of resin corresponding to an increased capacity for putrescine must be used to achieve a satisfactory removal of putrescine.

Animals↗
PubMed1980

Bovine plasma amine oxidase interactions with concanavalin A in solution and with concanavalin A-Sepharose.

The reaction of bovine plasma amine oxidase, a glycoprotein, with Concanavalin A in 0.1 M potassium phosphate buffer, pH 7.0 at 25 degrees C were investigated by equilibrium and kinetic methods. A tentative mechanism for the reaction was derived. The Concanavalin A-enzyme interaction was used to show that the carbohydrate is not essential for activity and that the carbohydrate is covalently attached to the protein at a site distant from the active site. Concanavalin A-Sepharose 4B affinity chromatography of the enzyme was found to be useful for obtaining the pure enzyme. Chromatographic conditions which elute the enzyme are reported. The superiority of the Concanavalin A-Sepharose 4B over that of the substrate-affinity Sepharose affinity support is discussed. The phenylhydrazone derivative of the enzyme, which was inactive, was shown to bind to the concanavalin A-Sepharose column.

Animals↗