Removal of Glycosides from Glycoproteins for Structure Determination

11 Removal of Glycosides from Glycoproteins for Structure Determination 

To achieve -elimination, various strengths of alkali (0.05-0.5 N NaOH), temperatures of 0-45°C, and lengths of 15-216 hr are used. The sodium borohydride is 0.15-1.0 M. A standard procedure uses 0,1 N NaOH and 0.3 M NaBH at 37°C for 48 hr. The conditions, however, should be established for each glycoprotein or glycopeptide [61], The 3-elimination reaction does not proceed satisfactorily if the glycosylated amino acid is the C- or N-terminal residue. In such cases, the amino or carboxyl group has to be derivatized to eliminate the charge [62]. 

The asparagine AMinked glycosides can be cleaved by hydrazinolysis [63]. The glycoprotein is heated at 100°C with anhydrous hydrazine for 8-12 hr. A series of reactions take place (Fig. 12.7) The first and second reactions proceed relatively quickly. The third reaction is much slower. The procedure is carried out by suspending 0,2-1 mg of glycoprotein in 0.5-1.0 mL of freshly distilled anhydrous hydrazine. The solution is heated in a sealed tube at 100°C for 8-12 hr. The glycoprotein sample usually dissolved after 1 hr. 

Various endoglycosidases such as endo-p-V-acetylglucosaminidase can be used to liberate asparagine V-linked oligosaccharide chains [64]. 

Chemical methods that liberate both O- and V-linked oligosaccharides from glycoproteins involve anhydrous trifluoromethane sulfonic acid (TFMS) or anhy- 

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