RESUMEN
Using LiBH4/ButOH treatment, oligosaccharides were cleaved off the hen egg white riboflavin-binding glycoprotein. HPLC led to the isolation of four fucose-containing oligosaccharide alditols, whose structure was elucidated by means of 1H NMR 500 MHz spectroscopy. The main fucosylated oligosaccharide, also present in hen ovomucoid, was found to be a biantennary carbohydrate chain of N-acetyllactosamine type.
Asunto(s)
Proteínas Portadoras/química , Proteínas del Huevo/química , Fucosa/química , Glicoproteínas/química , Proteínas de Transporte de Membrana , Riboflavina/química , Animales , Secuencia de Carbohidratos , Pollos , Espectroscopía de Resonancia Magnética , Datos de Secuencia MolecularRESUMEN
Reductive cleavage of riboflavin-binding glycoprotein from hen egg white (RF-GPw) with LiBH4/tert-BuOH followed by NaBH4/NaOH treatment gave rise to oligosaccharide alditols, fractionated by a successive HPLC on muBondapak C18 and Zorbax NH2 columns. Seven main individual oligosaccharide alditols were isolated and their structure was investigated by 1H NMR 500-MHz spectroscopy. The structure and relative content of the main oligosaccharide chains were proved to be identical in RF-GPw and ovomucoid. Structure of polypeptide chains and their molecular weight, number of glycosylation sites and their structure had little or no effect on the glycosylation pattern in both glycoproteins. HPLC of the oligosaccharide alditols from another egg white glycoprotein, ovotransferrin, also revealed its high microheterogeneity and close resemblance to those of ovomucoid and RF-GPw.
Asunto(s)
Proteínas Portadoras , Conalbúmina , Proteínas del Huevo , Clara de Huevo , Proteínas de Transporte de Membrana , Oligosacáridos , Ovomucina , Animales , Secuencia de Carbohidratos , Pollos , Cromatografía Líquida de Alta Presión , Conalbúmina/aislamiento & purificación , Proteínas del Huevo/aislamiento & purificación , Femenino , Glicosilación , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Ovomucina/aislamiento & purificación , Oxidación-Reducción , Riboflavina/metabolismoRESUMEN
A new method of fractionation of hen egg glycoproteins has been developed. The procedure involves high-speed mass ion-exchange chromatography on ZetaPrep cartridges, differential precipitation, and ultrafiltration on "Minitan" tangential-flow system. Six fractions were obtained from egg white (ovomucin, avidin, riboflavin-binding glycoprotein RF-GPw, ovoinhibitor, ovalbumin-ovotransferrin, and ovomucoid fractions), and two fractions from egg yolk (riboflavin-binding glycoprotein RF-GPy and phosvitin fractions). Using ion-exchange HPLC on columns (150 X 21.5 mm) Protein PAK DEAE-5PW and SP-5PW, six homogenous glycoproteins (avidin, RF-GPw, ovalbumin, ovotransferrin, ovomucoid, and RF-GPy) were isolated in preparative quantities (0.1-1 g). Ion-exchange HPLC also resolves some glycoproteins' isoforms with different pI values.
Asunto(s)
Proteínas del Huevo/aislamiento & purificación , Glicoproteínas/aislamiento & purificación , Animales , Pollos , Cromatografía Líquida de Alta Presión , UltracentrifugaciónRESUMEN
Isolation and characterization of oligosaccharides of riboflavin binding glycoprotein from hen white is described. Reductive cleavage of the N-glycosylamide carbohydrate-peptide bond with LiBH4/tert-BuOH followed by NaBH4-NaOH treatment gave rise to alditols, which were fractionated by means of HPLC. Twelve alditols were isolated in quantities sufficient for the monosaccharide analysis. Possibility of an ovomucoid-type oligosaccharide structure for all the alditols is discussed.