Concanavalin A-Sepharose 4B Affinity Chromatography Research Articles

An easy two-step purification method for human leucocyte myeloperoxidase / İnsan lökosit miyeloperoksidazı saflaştırılması için kolay 2 basamaklı yöntem

Abstract Objective: The object of this study is to describe a simple, rapid and cost effective method for purification of human leucocyte myeloperoxidase from a single donor. Myelopeoxidase (MPO) was purified by a two step procedure consisting of concanavalin-A Sepharose 4B affinity chromatography followed by CM-Sephadex cation exchange chromatography. Methods: Leucocytes from a single donor collected by leucopheresis were used in purification studies. MPO was solubilized and extracted from leucocytes by homogenization in phosphate buffer containing 1% HETAB (hexadecyltrimethylammonium bromide). MPO containing soluble material was applied onto concanavalin-A Sepharose 4B affinity gel, and was eluted with methyl-a- D-manno-piranoside. Fractions with MPO activity were pooled, dialyzed and applied onto CM-sephadex cation exchange gel, and was eluted from the column at weak cationic pH with linear NaCl gradient. Results: By the use of two chromatographic procedures, MPO was purified from human leucocytes with 70% yield. Purity of MPO was checked by determining the Reinheit Zahl (RZ) value (A430/A280). The RZ value of 0.86 indicated that the purified enzyme was highly homogenous as compared to reported experimental values (ranging from 0.82 to 0.88) and pure commercial enzyme with the RZ value of 0.84. Conclusion: In comparison with earlier purification methods, the purification method reported here has higher recovery rate and high purity together. Use of leucocytes with leucopheresis origin help us to omit the leucocyte isolation step and omitting of ammonium sulphate precipitation steps also help us to reduce the cost and is shortened the time of purification.

Antifungal and antibacterial activities of lectin from the seeds of Archidendron jiringa Nielsen

A Archidendron jiringa Nielsen lectin was purified by aqueous extraction, 90% ammonium sulphate precipitation and concanavalinA-Sepharose 4B affinity chromatography. Its specific activity was of 88.3×102 hemagglutination unit/mg protein for a yield of 51.6% total protein. The molecular weight is of 35.7kDa. It has hemagglutinating activity against human blood group, rabbit, mouse, rat, guinea pig, geese and sheep erythrocytes. The hemagglutination activity of lectin was relatively insensitive to acidic pH above 2, had an optimal activity at pH 8, and stable below 45°C for 30min. The activity was stimulated by Ca2+, Mg2+ and Mn2+. The internal sequence indicated similarity with legume lectin family. Moreover, even at low concentrations antifungal activity was observed against Exserohilum turcicum, Fusarium oxysporum and Colletotrichum cassiicola. The minimal inhibitory concentrations were 0.227, 0.0567 and 0.0567mg/ml for Bacillus subtilis, Staphylococcus aureus, and Candida albicans, respectively.

ELISA FOR QUANTITATION OF SERUM C-erbB-2 ONCOPROTEIN IN BREAST CANCER PATIENTS

ABSTRACT C-erbB-2 is a protooncogene that is overexpressed in various cancers, either due to its amplification and/or increased transcription, and has been associated with more aggressive disease and a poor clinical prognosis in 20–30% of patients with breast cancer. Besides the prognostic factors such as tumor size, tumor grade, lymph node status, etc., which are significant in the management of breast cancer, C-erbB-2 level might also serve as an additional factor. Immunohistochemistry is the most frequently used method to study the expression of C-erbB-2 in breast cancer. We have generated a panel of monoclonal antibodies against C-erbB-2 oncoprotein with a view to evaluate their application for the diagnosis and therapy of breast cancer. In the present study, a simple, quantitative sandwich ELISA has been developed that uses two monoclonal antibodies directed against the extracellular domain of C-erbB-2 product, designated as CIBCgp185 as the capture antibody and CIBCHER-2 as the detector antibody. C-erbB-2 protein, isolated from BT474 cells, a human breast carcinoma cell line with high expression of C-erbB-2 and purified by Concanavalin A-Sepharose 4B affinity chromatography and HPLC has been used to develop the ELISA procedure. Sera samples of 150 healthy women donors and of 300 breast cancer patients with different histological types of malignancies have been analysed. The control women had serum C-erbB-2 in the range of 4.0–13.2 ng/mL, whereas the 300 breast cancer patients studied had a range of 4.8–75.2 ng/mL with a cut off value of 13.8 ng/mL. Our study showed that 18.6% of breast cancer patients had elevated levels of circulating C-erbB-2. These results might suggest that the serum C-erbB-2 level can be used as a potential tumor marker for breast cancer and that the Sandwich ELISA procedure might serve as an excellent alternative to immunohistochemistry in the near future.

Isolation, purification and properties of the peroxidase from the hull ofGlycine max var HH2

Soybean hull peroxidase (EC 1.11.1.7), an acidic peroxidase isolated from soybean (Glycine max var HH2) hulls was purified to electrophoretic homogeneity by a combination of ammonium sulphate fractionation, DEAE-Sephadex A-50 chromatography, concanavalin A-Sepharose 4B affinity chromatography and Bio-Gel P-60 gel filtration. The specific activity of purified peroxidase was about 57-fold higher than that of crude extract. The yield was about 16.4%. The molecular weight of the enzyme was estimated to be 38 000 by SDS-polyacrylamide gel electrophoresis. The peroxidase was a glycoprotein containing about 18.7% carbohydrate, approximately one-quarter of which was shown to be glucosamine residues. It was found to have an isoelectric point of 3.9. The enzyme was most active at pH 4.6 and 45°C, and was stable in the pH range 2.5–11.5. The enzyme could tolerate heating for 10 min at 75°C without being inactivated, and at 85°C, it took 40 min to inactivate the enzyme 50%, confirming that the peroxidase was a novel thermostable enzyme. Fe 2+, Fe3+, Sn2+, CN − and N3− inhibited enzyme activity, while Hg2+, Ag+, Pb 2+, Cr3+, EDTA and SDS were not significantly inhibitory. © 1999 Society of Chemical Industry

Antitumor components ofAgrocybe cylindracea

To find pharmacologically active components ofAgrocybe cylindracea, its basidiocarps were extracted with water. The extracts were separated by DEAE cellulose column chromatography, Sepharose CL-4B gel filtration, and Concanavalin A-Sepharose 4B affinity chromatography. Among the obtained fractions fromA. cylindracea, fraction IN which was the neutral proteinbound-polysaccharide fraction exhibited a marked antitumor activity and it was tentatively named “Cylindan”. It showed about 70% of tumor inhibition against the solid form of sarcoma 180 when a dose of 30 mg/kg/day was intraperitoneally injected into ICR mice. When each fraction was examined by chemical analysis, Cylindan consisted of 85% polysaccharide, 3% protein and 1% hexosamine. Its polysaccharide moiety contained glucose, mannose, fucose and galactose and its protein moiety contained the comparatively large amounts of aspartic acid and glycine, and other 11 amino acids.

Purification and ultrastructural localization of surface glycoproteins of Taenia solium (Cestoda) cysticerci

Purification and ultrastructural localization of surface glycoproteins of Taenia solium (Cestoda) cysticerci. International Journal for Parasitology24: 265–269. A glycoprotein-enriched fraction was obtained by Concanavalin A-Sepharose 4B affinity chromatography from a crude extract of T. solium cysticerci. The six most prominent glycoproteins with molecular sizes of 180, 103, 96, 68, 55 and 45 kDa were purified by electro-elution from polyacrylamide gel slices. Ultrastructural localization assays using hyperimmune rabbit sera to each glycoprotein, demonstrated their presence on the tegumentary surface of the bladder wall of T. solium cysticerci. Similar studies showed that the 180 kDa glycoprotein is also present on the surface of the T. solium and T. saginata adult worms, as well as in T. saginata, T. pisiformis and T. crassiceps cysticerci. The 55 kDa glycoprotein, which is one of the most abundant on the cyst surface, was found to correspond to the heavy chain of pig IgG by Western blotting.

Multiple forms of rat prolactin and growth hormone in pituitary cell subpopulations separated using a Percoll gradient system: disulphide-bridged dimers and glycosylated variants.

Prolactin and GH cells from rat pituitary glands were separated into three main fractions on discontinuous Percoll gradient layers. SDS-PAGE and subsequent immunoblotting of these fractions revealed that: (1) multiple rat prolactin (rPRL) molecular variants were present in total culture, Percoll layer 1 and 2; four variants were clear-cut: Mr approximately 23,000, Mr doublet approximately 25,000-26,000, Mr approximately 40,000 and Mr approximately 42,000; (2) cell cytosol from Percoll gradient layer 1 was particularly enriched in prolactin; (3) cells from gradient layer 1 secreted into the culture medium only prolactin in detectable amounts; (4) three distinct molecular forms of rat growth hormone (rGH) were recorded in layer 3: Mr approximately 36,000, 24,000 and 20,000; the 20,000 variant was paramount; and (5) cells from layer 3 secreted both rPRL and rGH into the culture medium. Reduction experiments showed that, on the one hand, 42,000 and 40,000 rPRL variants and, on the other hand, 36,000 rGH variants are disulphide-bridged dimers. An important finding was the presence of glycosylated rPRL and rGH: indeed Concanavalin A-Sepharose 4B affinity chromatography indicated that 26,000 rPRL and 24,000 rGH display a very strong affinity for lectin. Competitive inhibition tests showed that this affinity is specific and not due to hydrophobic binding. When rPRL was submitted to deglycosylation in conditions specific for O-linked glycoproteins, the 26,000 rPRL variant disappeared. The biological role of glycosylated rPRL is as yet unknown.

Composition and properties of very low density lipoproteins secreted by the perfused rat liver and subfractionated by affinity chromatography

Very low density lipoproteins (VLDL) were isolated from the perfusate of rat livers infused with a complex of oleic acid bound to bovine serum albumin. Very low density lipoprotein (VLDL) secretion, bile flow, histopathology, and transmission electron microscopy indicated that secretory functions but not morphologic integrity of the livers were maintained during the procedure. Plasma VLDL and liver perfusate VLDL did not have similar size distribution. VLDL isolated from recycling perfusate and single pass perfusate were also subfractionated with concanavalin A-Sepharose 4B affinity chromatography. Three subfractions were eluted sequentially from the perfusate VLDL: a non-adherent fraction A and two adherent fractions B and C. The size of these VLDL, determined after negative staining and examination by transmission electron microscopy, was significantly decreased by affinity chromatography. VLDL in fractions A, B and C were spherical and had diameters of 935 ± 17, 881 ± 34 and 415 ± 30 A ̊ respectively. Fraction A, which did not adhere to the column, contained 65% of the lipid applied to the column. The carbohydrate composition of fraction A VLDL was 11.2 ± 0.6% fucose, 14.7 ± 1.2% galactose, 43.7 ± 2.3% N-acetylglucosamine, and 30.5 ± 1.9% sialic acid. Sugars such as glucose and mannose, which bind to concanavalin A, were not detected. In contrast, VLDL fractions B and C, which adhered to the column, contained both glucose (17.7 and 2.5%) and mannose (5.8 and 8.3%) as well as the other sugars present in VLDL fraction A. Sodium dodecyl sulfate gradient gel electrophoresis revealed that the affinity column procedure clearly altered the apolipoprotein patterns of the applied VLDL, thereby producing abnormal fractions B and C. Fractions B and C also differed from unfractionated VLDL and fraction A VLDL in lipid composition, in surface/interior core lipid ratio, and in fatty acid composition of the interior core lipids, primarily triacylglycerols. The steady-state anisotropy, the limiting anisotropy and the lipid order parameter of fluorescence probe molecules 1,6-diphenyl-1,3,5-hexatriene and trans-parinaric acid incorporated into the VLDL were of the following order: fraction B > fraction A > fraction C. These results are consistent with the interpretation that concanavalin A-Sepharose 4B affinity chromatography may artificially produce a series of VLDL subfractions whose composition and structural properties do not resemble those of native VLDL.

Granulocyte-macrophage colony-stimulating factor in the sera of Schistosoma japonicum-infected mice

Infection of mice with Schistosoma japonicum engendered high levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) in serum. The rise in GM-CSF levels in serum was closely associated with the acute phase of the infection and seemed to be dependent on the dose of infection. GM-CSF activity was detected as a sharp single peak in DE-52 anion-exchange chromatography and Sephacryl S-200 and Sephadex G-200 gel chromatography and was almost entirely adsorbed to concanavalin A-Sepharose 4B affinity chromatography. The possible immunological and immunopathological importance of GM-CSF in S. japonicum infection is discussed.

Mouse uterine alkaline phosphatase: improved purification by affinity chromatography and further characterization of the enzyme.

An improved procedure for the purification of alkaline phosphatase from about 10 g of day 7 pregnant mouse uterine tissue is described. Following homogenization, the procedure involved solubilization and extraction with 0.8% (v/v) Triton X-100 and 20% (v/v) n-butanol, ammonium sulfate precipitation, concanavalin A-Sepharose 4B affinity chromatography, DEAE-cellulose anion-exchange chromatography and Sephacryl S200 gel filtration. On subjecting 2162-fold purified enzyme preparations to polyacrylamide-gel electrophoresis, a single band of protein coincident with the zone of enzyme activity and having an apparent molecular weight of 205 000 +/- 10 000 was identified. Affinity chromatography yielded the largest increase in purity of any step in the procedure and established the glycoprotein nature of the uterine enzyme. Some metalloenzyme properties of the phosphatase were also studied and it was demonstrated that both Mg2+ and Zn2+ ions are necessary for hydrolytic activity. Treatment with neuraminidases retarded the anodal migration of the enzyme during electrophoresis on cellulose acetate membranes but did not influence its activity or catalytic properties. These results suggest that the uterine alkaline phosphatase is a sialoglycoprotein. The sialic acid residues, however, do not appear to constitute part of the active centre of the enzyme. In addition, the optimum pH for activity depended on substrate concentration and decreased with decreasing substrate concentration. Apparent Km values also depended on variations in pH and decreased with decreasing pH. Plots of pKm versus pH revealed a functional group with a pK value of 9.45. The enzyme also hydrolysed a variety of compounds having either phosphomonoester or pyrophosphate linkages and was inactivated after heating at 60 degrees C for 15 min. The activation energy, determined from a linear Arrhenius plot, was 50.1 kJ mol-1.

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°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.

Congenital Abnormalities Induced by Heterologous Antisera Directed Against Rat Kidney Glycoproteins Isolated by Concanavalin A Affinity Chromatography

It has been well established that heterologous antisera against whole rat kidney homogenate when injected into pregnant rats during the embryonic organogenetic period may induce abnormal embryonic development. Attempts were made to isolate the active components from soluble rat kidney extract by ammonium sulfate precipitation, anion-exchange chromatography, and concanavalin A-Sepharose 4B affinity chromatography. The glycoproteins isolated were capable of stimulating the production of potent rabbit antisera. When injected ip into the 9th day pregnant rats, these antisera induced embryonic death, congenital abnormalities, and fetal growth retardation. Eighty-four surviving fetuses were examined, all of them were malformed. The most frequently observed congenital defects were anophthalmia and microphthalmia. Attempts were made to analyze the glycoprotein fraction by discontinuous and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The results indicate that the glycoproteins were of high molecular weight and could be dissociated by SDS into a multitude of molecules or subunits. Although double immunodiffusion indicated that there were one major and two minor antigens in the glycoprotein fraction, attempts to identify the antigens as to their size by analytical gel electrophoresis have not been successful. Electron microscopic study seemed to suggest that the glycoproteins might tend to aggregate to form particulates. The underlying mechanism whereby the antisera to these glycoproteins induce abnormal embryonic development is not understood. The hypotheses to explain the possible sites of teratogenic antibody interaction are discussed.

Studies on the appearance and location of hemagglutinins from a common lentil during the life cycle of the plant

A Archidendron jiringa Nielsen lectin was purified by aqueous extraction, 90% ammonium sulphate precipitation and concanavalinA-Sepharose 4B affinity chromatography. Its specific activity was of 88.3 × 102 hemagglutination unit/mg protein for a yield of 51.6% total protein. The molecular weight is of 35.7 kDa. It has hemagglutinating activity against human blood group, rabbit, mouse, rat, guinea pig, geese and sheep erythrocytes. The hemagglutination activity of lectin was relatively insensitive to acidic pH above 2, had an optimal activity at pH 8, and stable below 45 °C for 30 min. The activity was stimulated by Ca2+, Mg2+ and Mn2+. The internal sequence indicated similarity with legume lectin family. Moreover, even at low concentrations antifungal activity was observed against Exserohilum turcicum, Fusarium oxysporum and Colletotrichum cassiicola. The minimal inhibitory concentrations were 0.227, 0.0567 and 0.0567 mg/ml for Bacillus subtilis, Staphylococcus aureus, and Candida albicans, respectively.