Isolation and Separation of the Glycan Strands from Murein of Escherichia Coli by Reversed-phase High-performance Liquid Chromatography

The length distribution of the glycan strands in the murein (peptidoglycan) sacculus of Escherichia coli has been analyzed after solubilization of the murein by complete digestion with human serum amidase. The glycan strands released were separated according to length by reversed-phase HPLC on wide-pore Nucleosil 300 C18 material at 50 degrees C, employing a convex gradient from 5 to 11% acetonitrile. The length of the fractionated glycan strands, which carry a nonreducing 1,6-anhydromuramic acid as a natural end group, was calculated from the ratio of total to nonreducing terminal muramic acid residues. This was possible after complete hydrolysis of the isolated glycan strands by muramidase followed by separation of the released nonreducing and reducing di- and tetrasaccharides by reversed-phase HPLC on Hypersil C18. The method established allows the separation of the glycan strands of murein, a poly-GlcNAc(beta 1-4)MurNAc-polysaccharide, up to a degree of polymerization of approximately 60. The predominant lengths of the glycan strands were 5 to 10 GlcNAc(beta 1-4)MurNAc disaccharide units.