RESUMO
Chitosan partially functionalized with aldehyde groups was used for enzyme immobilization, favoring first the enzyme adsorption through its amino groups and then the covalent bonding of the adsorbed catalyst through the aldehyde groups of the support. Using this strategy, immobilized A. oryzae ß-galactosidase had a better performance than when only the aldehyde groups were used. The performance was further improved by modifying the support aldehyde group density to 200⯵molesâ g-1. The biocatalyst under optimized immobilization conditions had 2951â¯IUâ g-1 and half-life of 46.3â¯min at 60⯰C, while its agarose counterpart had 2294â¯IUâ g-1 and half-life of 59.5â¯min. Both biocatalysts were applied in galacto-oligosaccharide synthesis. After 10 sequential batches, the cumulative productivity (gGOSâ h-1Ëgprotein-1) obtained with the chitosan and the agarose biocatalysts were 4.7 and 4.0 times the value when soluble enzyme was used respectively. This methodology had not been reported previously with chitosan, showing the high versatility of this low cost carrier and its high potential for enzyme immobilization.
Assuntos
Quitosana/metabolismo , Enzimas Imobilizadas/metabolismo , beta-Galactosidase/metabolismo , Adsorção/efeitos dos fármacos , Aldeídos/metabolismo , Catálise/efeitos dos fármacos , Sefarose/metabolismoRESUMO
BACKGROUND: The histone-like Hlp protein is emerging as a key component in mycobacterial pathogenesis, being involved in the initial events of host colonization by interacting with laminin and glycosaminoglycans (GAGs). In the present study, nuclear magnetic resonance (NMR) was used to map the binding site(s) of Hlp to heparan sulfate and identify the nature of the amino acid residues directly involved in this interaction. RESULTS: The capacity of a panel of 30 mer synthetic peptides covering the full length of Hlp to bind to heparin/heparan sulfate was analyzed by solid phase assays, NMR, and affinity chromatography. An additional active region between the residues Gly46 and Ala60 was defined at the N-terminal domain of Hlp, expanding the previously defined heparin-binding site between Thr31 and Phe50. Additionally, the C-terminus, rich in Lys residues, was confirmed as another heparan sulfate binding region. The amino acids in Hlp identified as mediators in the interaction with heparan sulfate were Arg, Val, Ile, Lys, Phe, and Thr. CONCLUSION: Our data indicate that Hlp interacts with heparan sulfate through two distinct regions of the protein. Both heparan sulfate-binding regions here defined are preserved in all mycobacterial Hlp homologues that have been sequenced, suggesting important but possibly divergent roles for this surface-exposed protein in both pathogenic and saprophic species.
Assuntos
Adesinas Bacterianas/química , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Mycobacterium leprae/química , Sequência de Aminoácidos , Sítios de Ligação , Cromatografia de Afinidade , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Sefarose/análogos & derivados , Sefarose/metabolismo , Cloreto de Sódio/metabolismoRESUMO
The present paper reports the partial characterization of a subset of atypical cruzipain molecules which do not bind to Concanavalin A-Sepharose column. They are present in different strains of epimastigote forms of Trypanosoma cruzi and represent a 2-4% of total cruzipain. They were purified by affinity chromatography on Cystatin-Sepharose, recognized by the polyclonal anti-cruzipain serum, and their activity in gelatin-containing gels was completely abolished by E-64, TLCK, leupeptin, and aprotinin but not by PMSF, pepstatin A, EDTA or 1,10-phenantroline. These cysteine proteinases, as well as cruzipain showed to be endoproteinases able to hydrolize azocasein, hemoglobin, and bovine serum albumin at acidic pHs. However, evidences are presented indicating that this subset of cruzipain isoforms were also able to use the same blocked chromogenic peptidyl substrates than cruzipain at similar optimal alkaline pH values although with a different order of preference. Moreover, they showed a different oligosaccharide pattern after enzymatic treatment by high pH anion exchange chromatography, suggesting that this structural difference may account for the atypical behaviour in the lectin column.
Assuntos
Concanavalina A/metabolismo , Cisteína Endopeptidases/química , Sefarose/metabolismo , Trypanosoma cruzi/enzimologia , Adsorção , Animais , Western Blotting , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Cistatinas/metabolismo , Cisteína Endopeptidases/isolamento & purificação , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Eletroforese em Gel de Poliacrilamida , Peptídeos/metabolismo , Proteínas de Protozoários , Especificidade por SubstratoRESUMO
Procedures for IgG depletion in visceral leishmaniasis (VL) and schistosomiasis sera using Sepharose-protein G beads also deplete IgE. In this study, the presence of IgG anti-IgE autoantibodies in sera from patients with VL (n = 10), and hepatic-intestinal schistosomiasis (n = 10) and from healthy individuals (n = 10) was investigated. A sandwich ELISA using goat IgG anti-human IgE to capture serum IgE and goat anti-human IgG peroxidase conjugate to demonstrate the binding of IgG to the IgE captured was performed. VL sera had higher titers (p < 0.05) of IgG anti-IgE autoantibodies (OD = 2.01 +/- 0.43) than sera from healthy individuals (OD = 1.35 +/- 0.16) or persons infected with Schistosoma mansoni (OD = 1.34 +/- 0.18). The immunoblotting carried out with eluates from Sepharose-protein G beads used to deplete IgG from these sera and goat anti-human IgE peroxidase conjugate, showed a similar pattern of bands, predominating the 75 kDa epsilon-heavy chain and also polypeptides resulting from physiological enzymatic digestion of IgE. A frequent additional band immediately above 75 kDa was observed only in VL sera.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Autoanticorpos/imunologia , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Leishmaniose Visceral/imunologia , Anticorpos Anti-Idiotípicos/sangue , Autoanticorpos/sangue , Proteínas de Ligação ao GTP/metabolismo , Humanos , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Leishmaniose Visceral/sangue , Ligação Proteica , Esquistossomose mansoni/sangue , Esquistossomose mansoni/imunologia , Sefarose/metabolismoRESUMO
Granulocyte-macrophage colony-stimulating factor (GM-CSF) controls growth and differentiation of hematopoietic cells. Previous reports have indicated that the mitogenic activity of GM-CSF may be modulated by the glycosidic moiety of proteoglycans associated with the membrane of stromal cells. In this work, we have performed in vitro studies of the interaction between GM-CSF and glycosaminoglycans. The addition of heparin promoted a marked blue shift in the fluorescence emission spectrum of GM-CSF as well as a 30-fold increase in the intensity of light scattering, which indicates formation of large molecular weight complexes between the two molecules. Interestingly, heparin-induced changes in the spectral properties of GM-CSF were only observed at acidic pH. The dependence on acidic pH, together with a strict dependence on glycosaminoglycan sulfation and the fact that high ionic strength destabilized the interaction, indicates that the association between GM-CSF and glycosaminoglycans is mediated by electrostatic interactions. These interactions probably involve sulfate groups in the glycosaminoglycans and positively charged histidine residues in GM-CSF. We propose that negatively charged glycolipids present on the plasma membrane of the hematopoietic and/or the stromal cell could promote an acidic microenvironment capable of triggering interaction between GM-CSF and membrane-bound proteoglycans in vivo.
Assuntos
Glicosaminoglicanos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Sítios de Ligação , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Heparina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Lipossomos/metabolismo , Modelos Moleculares , Naftalenossulfonatos/metabolismo , Fosfolipídeos , Ligação Proteica , Proteínas Recombinantes/metabolismo , Sefarose/análogos & derivados , Sefarose/metabolismo , Espectrometria de FluorescênciaRESUMO
A system of immunoadsorption was developed for in vitro depletion of xenoreactive natural antibodies of classes IgG and IgM from monkey and human plasma. Porcine endothelial cell membrane proteins, platelet membrane proteins, and endothelial cells were used as affinity ligands, and cyanogen bromide-activated Sepharose 6 Fast Flow and Sepharose CL-4B gels were used for chromatography. Adsorption capacity was evaluated by means of ELISA, immunonephelometry, and cytotoxicity testing. Several consecutive adsorption-desorption cycles were performed. Different parameters influencing immunoadsorption were examined: ligand density on the column gel, adsorbent-plasma contact time, ratio of plasma volume to immunoadsorbent volume, desorption conditions, and temperature. After 2 adsorption-desorption cycles, 99% and 82 to 85% of IgG and IgM antipig antibodies were adsorbed, respectively. Furthermore, there was a 74 to 77% decrease in cytotoxicity. In vivo, we observed that after one adsorption-desorption cycle, 97% of antipig IgG antibodies and 96% of antipig IgM antibodies were adsorbed, and there was an 85% decrease in cytotoxicity. The immunoadsorption method studied and optimized in vitro and in vivo therefore efficiently depleted xenoantibodies and reduced the cytotoxicity. Thus, it can be used in xenotransplantation experiments without eliminating non-specific antibodies.
Assuntos
Imunoglobulina G/sangue , Imunoglobulina M/sangue , Imunoadsorventes/metabolismo , Proteínas de Membrana/metabolismo , Transplante Heterólogo/imunologia , Adsorção , Animais , Plaquetas/citologia , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Membrana Celular/metabolismo , Separação Celular , Células Cultivadas , Brometo de Cianogênio/química , Testes Imunológicos de Citotoxicidade , Eletroforese em Gel de Poliacrilamida , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Endotélio Vascular/ultraestrutura , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Imunoglobulina M/imunologia , Imunoglobulina M/metabolismo , Técnicas de Imunoadsorção , Imunoadsorventes/imunologia , Macaca mulatta , Masculino , Nefelometria e Turbidimetria , Ensaio Radioligante , Sefarose/metabolismo , SuínosRESUMO
Surface antigen profiles of Leishmania donovani promastigote isolates have been studied. Surface patterns of Brazilian and African isolates display remarkable similarities and are extremely simple, consisting of three major peptides of 65,000, 25,000, and 23,000 mol wt. Surface iodination and biosynthetic labeling coupled to immunoprecipitation techniques revealed that a single major determinant of 65,000 mol wt is recognized in all strains by sera from kala-azar patients from both Brazil and Africa. This major determinant is not brought down by sera from normal individuals and shows no significant cross-reactivity with sera from Chagas' disease, leprosy, or syphilis patients. Binding to concanavalin A suggests a glycoprotein nature for this antigen. Sera from patients with cutaneous leishmaniasis (L. braziliensis) also recognized the same 65,000-mol wt determinant, although to a lesser extent. The possibility that this major surface antigen is shared, with minor differences, not only by L. donovani strains but between Leishmania species in general is suggested.