Lectin affinity chromatography has been applied to the separation of the sialyloligosaccharides of human milk. A human milk sialyloligosaccharide fraction was tritium labeled and applied to a highly substituted WGA-agarose column (20 mg/ml). Only a single component from the complete sialyloligosaccharide fraction was retarded in the WGA-agarose column. The WGA-bound fraction when applied to paper chromatography migrated with identical mobility as the sialylhexasaccharide fraction (S-5) of human milk, previously isolated and described by Kobata and Ginsburg in 1972 [Arch. Biochem. Biophys., 150:273-281]. A purified sialylhexasaccharide fraction (S-5), isolated according to the method of Kobata and Ginsburg, was radiolabeled and applied to the WGA-agarose column. The WGA-bound (60%) and WGA-unbound (40%) sialylhexasaccharide fractions were isolated. The WGA-bound sialylhexasaccharide fraction was subjected to neuraminidase digestion to remove sialic acid, and the resulting neutral oligosaccharide had more affinity for the WGA-agarose column. Sequential exoglycosidase digestion of the asialo derivative of the WGA-bound fraction with jack bean β -galactosidase and β -hexosaminidase demonstrated the presence of a lacto-N-neohexaose core. The position of sialic acid in the sialyllacto-N-neohexaose was determined by simultaneous digestion of the sialylhexaose with jack bean β -galactosidase and β -hexosaminidase, which removed the non-sialylated branch from the sialylhexaose and produced a sialyltetraose. The sialyltetraose was found to be sialyltetrasaccharide c as demonstrated by its elution time on HPLC and direct binding to monospecific anti-sialyltetrasccharide c serum. The structural data indicated that the WGA-bound sialylhexaose is a sialyl derivative of lacto-N-neohexaose with sialic acid linked to the 3 branch of this core structure which represents a previously undescribed sialyloligosaccharide in human milk. The structure of the WGA-bound sialylhexaose is, / M.S.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/94481 |
| Date | January 1986 |
| Creators | Tarrago-Trani, Maria T. |
| Contributors | Biochemistry |
| Publisher | Virginia Polytechnic Institute and State University |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | en_US |
| Detected Language | English |
| Type | Thesis, Text |
| Format | x, 58 leaves, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | OCLC# 16655879 |
Page generated in 0.0021 seconds