Concanavalin A-Sepharose Research Articles

Thermal Inactivation of Myrosinase from White Mustard Seeds

Myrosinases (thioglucosidases) catalyze the hydrolysis of a class of compounds called glucosinolates, of which the aglycones show various biological functions. It is often necessary to minimize the loss of myrosinase activity during thermal processing of cruciferous vegetables. Myrosinase was isolated from a popular spice, white mustard (Sinapis alba), and its thermal inactivation kinetics was investigated. The enzyme was extracted from white mustard seeds and purified by a sequential processes of ammonium sulfate fractionation, Concanavalin A-Sepharose column chromatography, and gel permeation chromatography. At least three isozymes were revealed by Concanavalin A-Sepharose column chromatography. The purity of the major myrosinase was examined by native polyacrylamide gel electrophoresis and on-gel activity staining with methyl red. The molecular weight of the major enzyme was estimated to be 171 kDa. When the consecutive step model was used for the thermal inactivation of the major myrosinase, its inactivation energy was 44.388 kJ/mol for the early stage of destruction and 32.019 kJ/mol for the late stage of destruction. When the distinct two enzymes model was used, the inactivation energy was 77.772 kJ/mol for the labile enzyme and 95.145 kJ/mol for the stable enzyme. The thermal inactivation energies lie within energy range causing nutrient destruction on heating.

Efficient hydrolysis of wine and grape juice anthocyanins by Malbranchea pulchella β-glucosidase immobilized on MANAE-agarose and ConA-Sepharose supports

β-glucosidases (BGLs) hydrolyze short-chain cellulooligosaccharides. Some BGLs can hydrolyze anthocyanins and be applied in the clarification process of food industries, especially grape juice and wine. Enzyme immobilization is a valuable tool to increase enzyme stabilization. In this work, Malbranchea pulchella BGL was immobilized on Monoaminoethyl-N-ethyl-agarose ionic support, MANAE-agarose, and Concanavalin A-Sepharose affinity support, Con-A-Sepharose. The formed biocatalysts, denominated BLG-MANAE and BLG-ConA, were applied in the grape juice and red wine clarification. BGL-MANAE and BGL-ConA hyperactivated M. pulchella BGL 10- and 3-fold, respectively. Both biocatalysts showed at least 70% activity at pH range 2–11, until 24 h incubation. BGL-MANAE and BGL-ConA showed activity of 60% and 100%, respectively, at 50 °C, up to 24 h. Both biocatalysts were efficiently reused 20-fold. They were stable in the presence of up to 0.1 M glucose for 24 h incubation, and with 5%, 10% and 15% ethanol kept up to 70% activity. BGL-MANAE biocatalyst was 11% and 25% more efficient than BGL-ConA in clarification of concentrate and diluted wines, respectively. Likewise, BGL-MANAE biocatalysts were 14% and 33% more efficient than the BGL-ConA in clarification of diluted and concentrated juices, respectively. Therefore, the BGL-MANAE biocatalyst was especially effective in red wine and grape juice clarification.

Proteomic Toolbox To Standardize the Separation of Extracellular Vesicles and Lipoprotein Particles.

Circulating in blood, extracellular vesicles (EVs) and lipoprotein particles (LPs) have diagnostic and prognostic value. To unambiguously define their functions, separation protocols need to be developed. However, because of their similar size and density, traditional approaches to separate EVs and LPs often fail to provide the required resolution. Further development and standardization of affinity-based protocols is necessary, and a quantitative method is needed to assess the efficiency of LP depletion from EV samples. In the present study, we propose the simultaneous quantification of three groups of proteins by mass spectrometry as a toolbox to evaluate prospective separation protocols. We generated 15N-labeled internal standards for quantification of (i) EV-specific proteins, (ii) all classes and subclasses of apolipoproteins constituting LPs, and (iii) several major serum proteins. These standards were then used in multiple reaction monitoring assay to evaluate the performance of size-exclusion chromatography, heparin-Sepharose, lipopolysaccharide-Sepharose, (2-hydroxypropyl)-β-cyclodextrin-Sepharose, and concanavalin A-Sepharose in separating serum EVs and LPs. The efficiency of a resin to separate EVs from non-EV substances could be jeopardized by simultaneous EV aggregation. Therefore, dynamic light scattering analysis was used in this study in addition to the proteomic toolbox when making a recommendation to use particular resin for EV isolation. On the basis of our measurements, we concluded that none of the individual separation protocols used in this study resulted in LP-free EVs, and the combination of two protocols may be complex due to low EV yield. Overall, this further points to the importance of proposed proteomic toolbox for the future evaluation of EV separation protocols.

Purification and characterisation of a pronase-inducible lectin isolated from human serum

A new lectin was purified to electrophoretic homogeneity from pronase treated human serum by a single-step of affinity chromatography on concanavalin A-Sepharose 4B. The isolated lectin agglutinated five types of vertebrate RBC, with highest titer against hen RBC. This activity was independent of divalent cations, insensitive to EDTA and specific to mannosamine, glucosamine as well as galactosamine. Purified lectin gave a single symmetrical peak in its native form with a molecular mass estimate of 6kDa in FPLC analysis and 6.5kDa by MALDI-TOF MS. SDS-PAGE analysis of the lectin revealed that it is a homo-oligomer of a 3kDa subunit protein. Isolated lectin did possess both, hemagglutinating and phenoloxidase activities, but did not exhibit any antibacterial or antifungal activities. In addition, this lectin could oxidize all nine different phenolic substrates tested, with hydroquinone proving to be the best among them. Phenoloxidase inhibitors namely, phenylthiourea and tropolone inhibited this oxidation activity.

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.

Isolation and Chemical Analysis of Potent Anti-Complementary Polysaccharides from Fruiting Bodies of the Fomes fomentarius

The five anti-complementary polysaccharides (MFKF-NP, MFKF-AP1, , and MFKF-AP2, ) were separated from hot water extracts of fruiting bodies of Fomes fomentarius by two subsequent column chromatography using DEAE-sepharose FF and Concanavalin A-sepharose 4B. The order of anti-complementary activity was MFKF-AP1 > MFKF-AP1 > MFKF-AP2 > MFKF-AP2 > MFKF-NP > Polysaccharide Krestine (PSK). Especially, MFKF-AP1 among those showed the most excellent anti-complementary activity (70% of ITCH50 value at ). The monosaccharide composition analysis by gas chromatography indicates that MFKF-AP1 and are a kind of homoxylan consisted mainly of xylose above 97%. Molecular weight of MFKF-AP1, major anti-complementary polysaccharide, was estimated to be about 12,000 by high performance liquid chromatography (HPLC). After the incubation of the serum with MFKF-AP1 in the presence or absence of and ions, its anti-complementary activity was investigated. This result indicated that MFKF-AP1 seems to be activator both on the classical and the alternative pathway of complement activation.

Immunomodulatory Response of Mice Splenocytes Induced by RcaL, a Lectin Isolated from Cobia Fish (Rachycentron canadum) Serum

This work reports the isolation of a serum lectin from cobia fish (Rachycentron canadum) named RcaL. Immunomodulatory activity on mice splenocyte experimental cultures through cytotoxic assays and cytokine production were also performed. RcaL was obtained through precipitation with ammonium sulphate and affinity chromatography on a Concanavalin A-Sepharose 4B column. The ammonium sulphate fraction F3 showed the highest specific hemagglutinating activity and was applied to affinity chromatography. The lectin was eluted with methyl-α-D-mannopyranoside. RcaL showed highest affinity for methyl-α-D-mannopyranoside and D-mannose; eluted fractions of RcaL agglutinated rabbit erythrocytes (titre, 128(-1)) retained 66% of chromatographed lectin activity, and the obtained purification factor was 1.14. Under reducing conditions, a polypeptide band of 19.2kDa was revealed in sodium dodecyl sulphate polyacrylamide gel electrophoresis (PAGE). PAGE confirmed RcaL as an acidic protein revealed in a single band. Cytotoxic and immunomodulatory assays with RcaL in mice splenocyte cultures showed that the lectin was not cytotoxic and induced higher interferon gamma and nitric oxide production in splenocyte cultures. Purified RcaL induced preferential Th1 response, suggesting that it acts as an immunomodulatory compound.

A Single Rainbow Trout Cobalamin-binding Protein Stands in for Three Human Binders

Cobalamin uptake and transport in mammals are mediated by three cobalamin-binding proteins: haptocorrin, intrinsic factor, and transcobalamin. The nature of cobalamin-binding proteins in lower vertebrates remains to be elucidated. The aim of this study was to characterize the cobalamin-binding proteins of the rainbow trout (Oncorhynchus mykiss) and to compare their properties with those of the three human cobalamin-binding proteins. High cobalamin-binding capacity was found in trout stomach (210 pmol/g), roe (400 pmol/g), roe fluid (390 nmol/liter), and plasma (2500 nmol/liter). In all cases, it appeared to be the same protein based on analysis of partial sequences and immunological responses. The trout cobalamin-binding protein was purified from roe fluid, sequenced, and further characterized. Like haptocorrin, the trout cobalamin-binding protein was stable at low pH and had a high binding affinity for the cobalamin analog cobinamide. Like haptocorrin and transcobalamin, the trout cobalamin-binding protein was present in plasma and recognized ligands with altered nucleotide moiety. Like intrinsic factors, the trout cobalamin-binding protein was present in the stomach and resisted degradation by trypsin and chymotrypsin. It also resembled intrinsic factor in the composition of conserved residues in the primary cobalamin-binding site in the C terminus. The trout cobalamin-binding protein was glycosylated and displayed spectral properties comparable with those of haptocorrin and intrinsic factor. In conclusion, only one soluble cobalamin-binding protein was identified in the rainbow trout, a protein that structurally behaves like an intermediate between the three human cobalamin-binding proteins.

Purification of an elicitor from Magnaporthe oryzae inducing defense resistance in rice

Inducible defenses that contribute to overall resistance in plant can be triggered by elicitors. A novel elicitor, derived from the mycelia of the blast fungus Magnaporthe oryzae , was purified to homogeneity by HiPrep 16/20 DEAE-Sepharose FF, Concanavalin A-Sepharose 4B and HiPrep 16/60 Sephacryl S-100 column chromatography. The purified elicitor appeared as single band corresponding to a molecular weight of 48.53 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophresis (SDS-PAGE) and a pI of 6.01 on isoeletric focusing (IEF) gel. Treatment with the purified elicitor increased the activities of phenylalanine ammonium-lyase (PAL ) and peroxidase ( POD) in rice susceptible cultivar CO39. Timecourse analysis showed peak accumulation of PAL appeared at 24 h after treatment, and it was higher in challenge-inoculated plants than non-challenge plants. POD accumulation showed similar kinetics with PAL, but the largest peak appeared at 36 h after treatment. Compared to the untreated control plants, pretreatment of rice leaves with the purified elicitor provided an enhanced level of protection against M. oryzae . N-terminal blocked elicitor was identified as hypothetical protein MG 05155.4 with 26.28% mass fingerprint coverage by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The results suggest that the purified elicitor is involved in inducing resistance against blast fungus.

Purification and Characterization of a Mannose Recognition Lectin from Oreochromis niloticus (Tilapia Fish): Cytokine Production in Mice Splenocytes

The aim of this work was to purify and partially characterize a mannose recognition lectin from Nile tilapia (Oreochromis niloticus) serum, named OniL. OniL was isolated through precipitation with ammonium sulfate and affinity chromatography (Concanavalin A-Sepharose 4B). In addition, we evaluated carbohydrate specificity, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) profiles, and in vitro immunomodulatory activity on mice splenocyte experimental cultures through cytotoxic assays and cytokine production. The ammonium sulfate fraction F2 showed the highest specific hemagglutinating activity (331) and was applied to affinity matrix. Adsorbed proteins (OniL) were eluted with methyl-α-D: -mannopyranoside. OniL, a 17-kDa protein by SDS-PAGE constituted by subunits of 11 and 6.6kDa, showed highest affinity for methyl-α-D: -mannopyranoside and D: -mannose. Immunological assays, in vitro, showed that OniL did not show cytotoxicity against splenocytes, induced higher IFN-γ production and lower IL-10 as well as nitrite release. In conclusion, OniL lectin was successfully purified and showed a preferential Th1 response in mice splenocytes.

Novel 1-O-indole-3-acetyl-β-d-glucose-dependent acyltransferase transferring indoleacetyl moiety to some mono- di- and oligosaccharides

1-O-(indole-3-acetyl)-β-d-glucose: sugar indoleacetyl transferase (1-O-IAGlc-SugAc) is a novel enzyme catalyzing the transfer of the indoleacetyl (IA) moiety from 1-O-(indole-3-acetyl)-β-d-glucose to several saccharides to form ester-linked IAA conjugates. 1-O-IAGlc-SugAc was purified from liquid endosperm of Zea mays by fractionation with ammonium sulphate, anion-exchange, Blue Sepharose chromatography, affinity chromatography on Concanavalin A-Sepharose, adsorption on hydroxylapatite and preparative PAGE. The obtained enzyme preparation indicates only one band of Rf 0.67 on 8% non-denaturing PAGE consisting of two polypeptides of 42 and 17 kDa in SDS/PAGE. Highly purified 1-O-IAGlc-SugAc shows maximum transferase activity with monosaccharides (mannose, glucose, and galactose), lower activity with disaccharides (melibiose, gentobiose) and trisaccharide (raffinose) and minimal enzymatic activity with oligosaccharides from the raffinose family as well. The novel acyltransferase exhibits, besides its primary indoleacetylation of sugar, minor hydrolytic and disproportionation activities producing free IAA and supposedly 1,2-di-O-(indole-3-acetyl)-β-glucose, respectively. Presumably, 1-O-IAGlc-SugAc, like 1-O-indole-3-acetyl-β-d-glucose-dependent myo-inositol acyltransferase (1-O-IAGlc-InsAc), is another member of the serine carboxypeptidase-like (SCPL) acyltransferase family.

Purification of Soluble Acetylcholinesterase from Sheep Liver by Affinity Chromatography

The purpose of this study was to develop a protocol for the purification of acetylcholinesterase (AChE, acetylcholine acetylhydrolase, E.C.3.1.1.7) enzyme and to extend a purification method for further enzyme characterization. A further aim was to study whether the edrophonium's pharmacologic action is due primarily to the inhibition or inactivation of AChE at sites of cholinergic transmission. The purification of a soluble AChE from sheep liver using affinity chromatography on Concanavalin A-Sepharose 4B and edrophonium-Sepharose 6B is studied. The affinity matrix was synthesized by coupling an inhibitor edrophonium to epoxy-activated Sepharose at flow rate of 0.5ml/min. AChE is a pivotal enzyme in the cholinergic nervous system. Its primary function is to catalyze hydrolysis of released acetylcholine (ACh) and thus maintain homeostasis of this neurotransmitter in the central and peripheral nervous systems. Hence, AChE is important in both pharmacological and toxicological mechanisms. It was purified 842-fold with a specific activity of 21U/mg protein. Sodium dodecyl sulfate (SDS) electrophoresis resulted in a monomeric molecular weight of 67.04kDa, while on gel chromatography using Sephacryl S-200 under nondenaturing conditions to be 201.5kDa. Based on the molecular weight obtained by gel filtration, the purified AChE was assumed to be a tetrameric form.

287 Development and validation of a real-time multiplex PCR assay for the simultaneous quantification of CK-19, MAGE-A3, HER-2 and PBGD in circulating tumor cells of breast cancer patients

Synaptosomal plasma membrane glycoproteins were solubilized in 0.08% sodium dodecyl sulfate (SDS) and separated by affinity chromatography on concanavalin A-Sepharose. Three fractions were obtained.Fraction CO (unadsorbed) contained 63% of the protein, but only 23% of the sugar and was rich in fucose, galactose and N-acetyl-neuraminic acid (NANA) relative to the other sugars. Many proteins were detected in this fraction by polyacrylamide gel electrophoresis, but only one band stained well for carbohydrate.Fraction CR (retarded) contained glycoproteins which reacted weakly with concanavalin A and stained poorly with periodic acid-Schiff reagent (PAS). There was no enrichment in total sugar/mg protein relative to the original fraction, but there was a marked enrichment in N-acetyl-glucosamine and NANA relative to the other sugars. The protein profile of this fraction was complex, but only one major PAS-positive band was detected. However, most, if not all, the proteins seemed to be weakly PAS-positive.Fraction C1 (adsorbed) was markedly enriched (5-fold) in sugar/mg protein, particularly in mannose and N-acetyl-glucosamine. It had a relatively simple protein profile, and most of the protein bands stained well with the PAS reaction.Glucose was detected in the initial fraction, and in all the subfractions, but it could not be shown definitely to be either a contaminant or an intrinsic constituent of synaptosomal plasma membrane (SPM) glycoproteins. Minor sugars, if present, could, at most, account for less than 0.25% of the carbohydrate.

Stimulation of Platelet-derived Growth Factor Receptor β (PDGFRβ) Activates ADAM17 and Promotes Metalloproteinase-dependent Cross-talk between the PDGFRβ and Epidermal Growth Factor Receptor (EGFR) Signaling Pathways

Binding of the platelet-derived growth factor (PDGF)-B to its receptor PDGFRbeta promotes proliferation, migration, and recruitment of pericytes and smooth muscle cells to endothelial cells, serving to stabilize developing blood vessels. The main goals of this study were to determine whether the extracellular domain of the PDGFRbeta can be proteolytically released from cell membranes and, if so, to identify the responsible sheddase and determine whether activation of the PDGFRbeta stimulates its shedding and potentially that of other membrane proteins. We found that the PDGFRbeta is shed from cells by a metalloproteinase and used loss-of-function experiments to identify ADAM10 as the sheddase responsible for constitutive and ionomycin-stimulated processing of the PDGFRbeta. Moreover, we showed that ligand-dependent activation of the PDGFRbeta does not trigger its own shedding by ADAM10, but instead it stimulates ADAM17 and shedding of substrates of ADAM17, including tumor necrosis factor alpha and transforming growth factor alpha. Finally, we demonstrated that treatment of mouse embryonic fibroblasts with PDGF-B triggers a metalloproteinase-dependent cross-talk between the PDGFRbeta and the epidermal growth factor receptor (EGFR)/ERK1/2 signaling axis that is also critical for PDGF-B-stimulated cell migration, most likely via ADAM17-dependent release and activation of ligands of the EGFR. This study identifies the principal sheddase for the PDGFRbeta and provides new insights into the mechanism of PDGFRbeta-dependent signal transduction and cross-talk with the EGFR.

Measurement‐specific bioavailable testosterone using concanavalin A precipitation: Comparison of calculated and assayed bioavailable testosterone

To assess the value of calculated bioavailable testosterone (cBT) and assayed BT (aBT) for the diagnosis of late-onset hypogonadism (LOH) in middle-aged and elderly subjects. In order to assay serum BT, sex hormone-binding globulin was precipitated with concanavalin-A and then testosterone was measured using liquid chromatography-tandem mass spectrometry. To validate the non-sex-hormone-binding-globulin-bound testosterone, gel filtration chromatography and concanavalin-A sepharose were used. Following this validation, the usefulness between aBT and cBT was evaluated in clinical samples. Eighty-eight healthy male volunteers (mean age 65.6 years, range: 50-86) were recruited for this study. A significant correlation was found between cBT and aBT (R(2) = 0.53, P < 0.01). Mean value ratio (cBT/aBT) was 2.48. Both cBT (R(2) = 0.122) and aBT (R(2) = 0.251) decreased with age. Variations in aBT were less marked than those for cBT, suggesting that aBT can be used to determine age-related reduced testosterone levels. aBT levels are more reliable than cBT levels for the diagnosis of LOH in middle-aged and elderly subjects.

Properties of a purified thermostable glucoamylase from Aspergillus niveus

A glucoamylase from Aspergillus niveus was produced by submerged fermentation in Khanna medium, initial pH 6.5 for 72 h, at 40 degrees C. The enzyme was purified by DEAE-Fractogel and Concanavalin A-Sepharose chromatography. The enzyme showed 11% carbohydrate content, an isoelectric point of 3.8 and a molecular mass of 77 and 76 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or Bio-Sil-Sec-400 gel filtration, respectively. The pH optimum was 5.0-5.5, and the enzyme remained stable for at least 2 h in the pH range of 4.0-9.5. The temperature optimum was 65 degrees C and retained 100% activity after 240 min at 60 degrees C. The glucoamylase remained completely active in the presence of 10% methanol and acetone. After 120 min hydrolysis of starch, glucose was the unique product formed, confirming that the enzyme was a glucoamylase (1,4-alpha-D-glucan glucohydrolase). The K(m) was calculated as 0.32 mg ml(-1). Circular dichroism spectroscopy estimated a secondary structure content of 33% alpha-helix, 17% beta-sheet and 50% random structure, which is similar to that observed in the crystal structures of glucoamylases from other Aspergillus species. The tryptic peptide sequence analysis showed similarity with glucoamylases from A. niger, A. kawachi, A. ficcum, A. terreus, A. awamori and A. shirousami. We conclude that the reported properties, such as solvent, pH and temperature stabilities, make A. niveus glucoamylase a potentially attractive enzyme for biotechnological applications.

Experimental "chronic" African trypanosomiasis: endocrine dysfunctions generated by parasitic components released during the tryptanolytic phase in rats.

The disorders of the gonadotropic axis have been studied during the course of a "chronic" african trypanosomiasis induced experimentally in rats inoculated by the variant Trypanosoma brucei brucei AnTat 1.1.E. The levels of serum and pituitary LH as well as serum testosterone and corticosterone have been determined, during the infestation, at a particular period of the circadian cycle, in regard to the parasitemia variations. In addition, the inoculation of trypanosomal component fractions [obtained by concanavalin-A sepharose chromatography (conA-components)], has been performed in an attempt to define more exactly the nature of factor(s) producing the hypotestosteronemia in rats. This work evidenced that the hormonal parameter levels were predominantly decreased at the trypanolytic phase during the evolution of the disease. The action towards the hypothalamo-pituitary gonadal axis was attributed not only to peculiar trypanosomal enzyme(s) [a serine, thiol-dependent, cation sensitive endoprotease with a post-proline cleaving activity (purified from unretained conA fraction)], but also to protein and/or glycoprotein factor(s) released by the trypanosomes (components with affinity to the lectin).

Kinetic and redox properties of MnP II, a major manganese peroxidase isoenzyme from Panus tigrinus CBS 577.79

A manganese peroxidase (MnP) isoenzyme from Panus tigrinus CBS 577.79 was produced in a benchtop stirred-tank reactor and purified to apparent homogeneity. The purification scheme involving ultrafiltration, affinity chromatography on concanavalin-A Sepharose, and gel filtration led to a purified MnP, termed "MnP II," with a specific activity of 288 IU mg(-1) protein and a final yield of 22%. The enzyme turned out to be a monomeric protein with molecular mass of 50.5 kDa, pI of 4.07, and an extent of N-glycosylation of about 5.3% of the high-mannose type. The temperature and pH optima for the formation of malonate manganic chelates were 45 degrees C and 5.5, respectively. MnP II proved to be poorly thermostable at 50 and 60 degrees C, with half-lives of 11 min and 105 s, respectively. K (m) values for H(2)O(2) and Mn(2+) were 16 and 124 microM, respectively. Although MnP II was able to oxidize veratryl alcohol and to catalyze the Mn(2+)-independent oxidation of several phenols, it cannot be assigned to the versatile peroxidase family. As opposed to versatile peroxidase oxidation, veratryl alcohol oxidation required the simultaneous presence of H(2)O(2) and Mn(2+); in addition, low turnover numbers and K (m) values higher than 300 microM characterized the Mn(2+)-independent oxidation of substituted phenols. Kinetic properties and the substrate specificity of the enzyme markedly differed from those reported for MnP isoenzymes produced by the reference strain P. tigrinus 8/18. To our knowledge, this study reports for the first time a thorough electrochemical characterization of a MnP from this fungus.

Purification of three major forms of β-hCG from urine and production of polyclonal antibodies against them

Objectives This study has been conducted to develop a proper and relatively simple method for purification of three major forms of β-hCG from urine to use as clinical calibrators and also to produce anti-β-hCG polyclonal antibodies against these purified forms. Design and methods Major β-hCG forms from urine of pregnant women were purified using an ultrafiltration method followed by three successive steps: 1) affinity chromatography on a Concanavalin A-Sepharose column, 2) ion exchange chromatography on a DEAE-Sephacel column and 3) preparative gel electrophoresis. Specific polyclonal antibodies against the purified forms were produced by immunizing rats. We also extracted β-hCG from urine using trypsin affinity chromatography. Results Three β-hCG forms with the molecular mass (MW) of 28, 32 and 35 kDa were purified and identified by ELISA method using monoclonal antibodies against two distinct epitopes (β2 and β11) on β-hCG. The titer of prepared antibodies was comparable with common standard anti-β-hCG antibodies. Also we could purify a 37 kDa form of β-hCG by trypsin affinity chromatography. Conclusions Here we proposed two different proper methods for purification of major forms of β-hCG from urine. We also found that three prepared β-hCG forms expose the β11 epitope and have an hCGβcf epitope expressed. Specific antibodies against purified β-hCG forms had comparable titers and could be used in diagnostic kits.

Purification and immunological characterization of a 12-kDa allergen fromEpicoccum purpurascens

Exposure to Epicoccum purpurascens is implicated in respiratory allergies and asthma. Several allergens of clinical importance were identified in Epicoccum extract (EE), but only one allergen has been isolated and characterized. In the present study, a 12-kDa allergen was isolated from an Epicoccum spore-mycelial extract by concanavalin-A sepharose, reverse-phase hydrophobic and gel filtration chromatography. The purified protein was recognized as a single 12-kDa allergen on immunoblot with a serum pool of Epicoccum-sensitive patients. Of the 94 respiratory allergy patients tested intradermally, 17 showed marked positive skin reactions to EE and 12 of them reacted with the 12-kDa protein, indicating a diagnostic sensitivity of 70%. More than 80% patients' sera showed immunoglobulin E (IgE) reactivity to the purified protein in enzyme-linked immunosorbent assay and immunoblot, identifying it as a major allergen. Preincubation of pooled serum with the protein led to inhibition of IgE binding to solid-phase-bound EE (effective concentration 50%=180 ng). Twelve of the 17 serum samples showed significant basophil histamine release upon stimulation with purified protein. The protein induced significant proliferation of peripheral blood mononuclear cells of 13 patients. A high level of interleukin-4 in the culture supernatant of these cells indicated induction of a T-helper type 2 response. The purified 12-kDa protein is a clinically relevant allergen and has potential for the diagnosis and therapy of Epicoccum allergies.