Separation of Biosynthetic Oligosaccharide Branch Isomers Using High-Performance Liquid Chromatography on a Porous Two-Dimensional Graphite Stationary Phase
✍ Scribed by Peter H. Lipniunas; David C.A. Neville; Robert B. Trimble; R.Reid Townsend
- Publisher
- Elsevier Science
- Year
- 1996
- Tongue
- English
- Weight
- 196 KB
- Volume
- 243
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
fying enzymes such as phosphotransferases (3) and sul-Oligomannosidic branch isomers (structures diffotransferases (4). In most cases, these biosynthetic fering only in the branch location of a single residue) enzymes remove or add residues to specific branches, which are biosynthetic intermediates in yeast and presumably by recognizing multiple, interresidue feahigher eukaryotics have been separated using hightures of their oligosaccharide substrates (5,6). The performance liquid chromatography (HPLC) on poyeast a-mannosidase removes only the terminal a1 r rous graphatized carbon (PGC) columns, a stationary 2-linked Man residue from the Mana(1 r 2)Mana(1 r phase of two-dimensional crystalline carbon. A mix-3)r unit attached to Man residue a (Scheme 1) of the ture of two Man 6 GlcNAc isomers from IgM, which was archetypal Man 9 GlcNAc 2 oligosaccharide (7). GlcNAc determined from 1 H NMR analysis, was completely transferase I attaches GlcNAc only to Man residue b separated by PGC-HPLC. Mixtures of larger yeast oliof the pentasaccharide core (Scheme 1) (8). The (a2 r gomannosidic branch isomers were also chromato-6) sialyl transferase links Neu5Ac preferentially to spegraphically resolved using PGC-HPLC. Man 10 GlcNAc cific branches of bi-, tri-, and tetraantennary structures and Man 11 GlcNAc species from invertase expressed in and the transferase also recognizes the intact chitobi-Pichia pastoris showed three and five peak fractions, ose core (9). On the same amino acid residue in a glycorespectively, by PGC-HPLC in agreement with the protein, the biosynthetic apparatus produces an array of number of isomeric forms from one-and two-dimenoligosaccharides, which can differ in size, monosacchasional 1 H NMR analyses of the individual sized fracride composition, sequence, linkages between residues tions (Trimble, R.