RESUMO
A glycoconjugate constituted by the Streptococcus pneumoniae serotype 14 capsular polysaccharide (CPS14) and bovine serum albumin (BSA) was prepared, and the unique properties of Sephadex LH-20 were used to separate the conjugate from the unconjugated material. The strength of this approach consists in its capacity to produce pure polysaccharide-protein conjugate in good yield and free from unconjugated material, a common residual contaminant of this type of immunobiologicals. The CPS14-BSA conjugate prepared via an improved 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP)-activation technique was characterized chemically and its immunogenicity was evaluated in mice. The purified conjugate, unlike the corresponding polysaccharide, produced a T-cell-dependent response in this species.
Assuntos
Cápsulas Bacterianas/imunologia , Cápsulas Bacterianas/isolamento & purificação , Vacinas Bacterianas/isolamento & purificação , Glicoconjugados/síntese química , Glicoconjugados/isolamento & purificação , Streptococcus pneumoniae/imunologia , Animais , Feminino , Camundongos , Soroalbumina BovinaRESUMO
This work exploits the combination of the lectin affinity chromatography (LAC) with an ultra-sensitive immunochromatographic assay to differentiate several types of erythropoietin (EPO). The chromatographic behaviours of different commercial types of recombinant human EPO (rhEPO), EPO analogues (Aranesp) and urine human EPO (uhEPO) from healthy individuals on eight lectin-Sepharose columns, have been worked out. Results show that when using wheat germ agglutinin (WGA)-Sepharose columns, a careful desorption regime starting with very low concentration (2mM) of the competitive sugar N-acetylglucosamine (GlcNAc) makes it possible to efficiently distinguish endogenous EPO from recombinant EPO and EPO analogues.
Assuntos
Cromatografia de Afinidade/métodos , Eritropoetina/análogos & derivados , Eritropoetina/química , Eritropoetina/urina , Lectinas/química , Acetilglucosamina/química , Adsorção , Humanos , Imunoensaio/métodos , Proteínas Recombinantes , Aglutininas do Germe de Trigo/químicaRESUMO
The presence of carbohydrate-binding proteins, namely lectins, -galactosidases and amylases, was determined in aqueous extracts of plants collected in Uruguay. Twenty-six extracts were prepared from 15 Uruguayan plants belonging to 12 Phanerogam families. Among them, 18 extracts caused hemagglutination (HAG) that was inhibited by mono- and disaccharides in 13 cases, indicating the presence of lectins. The other 8 extracts did not cause any HAG with the four systems used to detect HAG activity (rabbit and mouse red cells, trypsin-treated rabbit and mouse red cells). For the extracts prepared from Solanum commersonii, HAG activity and HAG inhibition were similar for those prepared from tubers, leaves and fruits, with the chitocompounds being responsible for all the inhibitions. Purification of the S. commersonii tuber lectin was carried out by affinity chromatography on asialofetuin-Sepharose, and SDS-PAGE under reducing conditions gave a single band of Mr of approximately 80 kDa. The monomer N-acetylglucosamine did not inhibit HAG induced by the purified lectin, but chitobiose inhibited HAG at 24 mM and chitotriose inhibited it at 1 mM. -Galactosidase activity was detected in leaves and stems of Cayaponia martiana, and in seeds from Datura ferox. Only traces of amylase activity were detected in some of the extracts analyzed. The present screening increases knowledge about the occurrence of carbohydrate-binding proteins present in regional plants.
Assuntos
Carboidratos/isolamento & purificação , Lectinas de Plantas/isolamento & purificação , Amilases/análise , Animais , Metabolismo dos Carboidratos , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Eritrócitos/metabolismo , Hemaglutinação/efeitos dos fármacos , Camundongos , Lectinas de Plantas/metabolismo , Ligação Proteica , Coelhos , Uruguai , beta-Galactosidase/análiseRESUMO
The presence of carbohydrate-binding proteins, namely lectins, ß-galactosidases and amylases, was determined in aqueous extracts of plants collected in Uruguay. Twenty-six extracts were prepared from 15 Uruguayan plants belonging to 12 Phanerogam families. Among them, 18 extracts caused hemagglutination (HAG) that was inhibited by mono- and disaccharides in 13 cases, indicating the presence of lectins. The other 8 extracts did not cause any HAG with the four systems used to detect HAG activity (rabbit and mouse red cells, trypsin-treated rabbit and mouse red cells). For the extracts prepared from Solanum commersonii, HAG activity and HAG inhibition were similar for those prepared from tubers, leaves and fruits, with the chitocompounds being responsible for all the inhibitions. Purification of the S. commersonii tuber lectin was carried out by affinity chromatography on asialofetuin-Sepharose, and SDS-PAGE under reducing conditions gave a single band of Mr of approximately 80 kDa. The monomer N-acetylglucosamine did not inhibit HAG induced by the purified lectin, but chitobiose inhibited HAG at 24 mM and chitotriose inhibited it at 1 mM. ß-Galactosidase activity was detected in leaves and stems of Cayaponia martiana, and in seeds from Datura ferox. Only traces of amylase activity were detected in some of the extracts analyzed. The present screening increases knowledge about the occurrence of carbohydrate-binding proteins present in regional plants
Assuntos
Animais , Camundongos , Coelhos , Carboidratos , Plantas , Carboidratos , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Eritrócitos , Hemaglutinação , Ligação Proteica , UruguaiRESUMO
A lectin from cat liver has been identified and purified by affinity chromatography on asialofetuin-Sepharose. One hundred micrograms of lectin was obtained from one cat liver with a purification factor of 1561. The lectin agglutinates trypsin-treated rabbit and cow erythrocytes. Hemagglutination was inhibited only by saccharides containing -galactosyl residues, of which the 1-amine-1-deoxy- -D-galactose was the most potent one by inhibiting hemagglutination at a concentration of 12.5 mM, followed by melibiose, trehalose and galactose. The lectin has a subunit molecular mass of 14.4 kDa determined by SDS-PAGE under reducing conditions and a pI of 4.85. Compared with the composition of lectins from calf heart and porcine heart, cat liver lectin contains approximately the same amount of cysteine, half the amount of glycine, twice as much arginine and threonine, and three times the amounts of tyrosine and methionine. Cat liver lectin contains four cysteine residues per subunit, all of them in the reduced form. Their lack of reactivity towards thiol-reactive supports suggests they are not exposed on the lectin surface. The protein apparently has a blocked N-terminus. The purified lectin was stable for up to 20 months stored at +4 C in buffer supplemented with 4 mM -mercaptoethanol. Results indicated that this lectin belongs to the family of soluble -galactoside-binding lectins, also known as galectins, which are expressed in a wide range of vertebrate tissues.
Assuntos
Galectinas/isolamento & purificação , Fígado/química , Animais , Gatos , Bovinos , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Eritrócitos/metabolismo , Galectinas/química , Galectinas/efeitos dos fármacos , Testes de Inibição da Hemaglutinação , Peso Molecular , Coelhos , Reagentes de Sulfidrila/farmacologiaRESUMO
A lectin from cat liver has been identified and purified by affinity chromatography on asialofetuin-Sepharose. One hundred micrograms of lectin was obtained from one cat liver with a purification factor of 1561. The lectin agglutinates trypsin-treated rabbit and cow erythrocytes. Hemagglutination was inhibited only by saccharides containing á-galactosyl residues, of which the 1-amine-1-deoxy-á-D-galactose was the most potent one by inhibiting hemagglutination at a concentration of 12.5 mM, followed by melibiose, trehalose and galactose. The lectin has a subunit molecular mass of 14.4 kDa determined by SDS-PAGE under reducing conditions and a pI of 4.85. Compared with the composition of lectins from calf heart and porcine heart, cat liver lectin contains approximately the same amount of cysteine, half the amount of glycine, twice as much arginine and threonine, and three times the amounts of tyrosine and methionine. Cat liver lectin contains four cysteine residues per subunit, all of them in the reduced form. Their lack of reactivity towards thiol-reactive supports suggests they are not exposed on the lectin surface. The protein apparently has a blocked N-terminus. The purified lectin was stable for up to 20 months stored at +4ºC in buffer supplemented with 4 mM á-mercaptoethanol. Results indicated that this lectin belongs to the family of soluble á-galactoside-binding lectins, also known as galectins, which are expressed in a wide range of vertebrate tissues
Assuntos
Animais , Coelhos , beta-Galactosidase , Fígado , beta-Galactosidase , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Eritrócitos , Testes de Inibição da Hemaglutinação , Peso MolecularRESUMO
This review surveys recent developments in chromatographic methods for the separation of amylases from complex extracts, including the separation of isozymes. It contains two tables with the properties and molecular characteristics of alpha-and beta-amylases from different sources as well as an updated review of methods for the determination of amylase activity. The main subject of this review is a detailed evaluation of the application of newly developed chromatographic methods for the purification of amylases.