Enzymatic methods, determining oligosaccharide

Whilst the first and second questions can be satisfactorily answered by NMR, they are perhaps better approached in tandem with chemical or enzymatic methods, such as hydrolysis, followed by reduction and analysis of alditol acetates by GC-MS, or digestion with specific glycosidases. Such a concerted approach has been used to successfully determine the composition and configuration of many oligosaccharides and glycans. 

The structures of oligosaccharides and polysaccharides are usually determined by a combination of methods specific enzymatic hydrolysis to determine stereochemistry and produce smaller fragments for further analysis methylation analysis to locate glycosidic bonds and stepwise degradation to determine sequence and configuration of anomeric carbons. 

The full structural determination of the structure of an oligosaccharide by this method includes complete sequencing by successive enzymatic digests using specific exoglycosi-dases and analysis of the products of each hydrolysis by mass spectrometry to observe the effectiveness of the hydrolysis by the decrease of molecular weight. [249]... 

The first structure of human renin was obtained from prorenin produced by expression of its cDNA in transfected mammalian cells. Prorenin was cleaved in the laboratory to renin using the protease trypsin. Because the carbohydrates in renin are not required for bioactivity, oligosaccharides were removed enzymatically. This process facilitates crystallization in some cases and also removes the contribution of the heterogeneous sugar chains to the diffraction pattern. The structure was determined without the use of heavy-atom derivatives, by application of molecular replacement techniques based on the atomic coordinates of porcine pepsinogen as the model. The molecular dynamic method of refinement was used extensively to arrive at a 2.5 A resolution structure. However, some of the loop regions were not well resolved in this structure (Sielecki et al, 1989 Sail et al, 1990). 

For this reason, glycoproteins must first be isolated from the biological matrix by dialysis, preparative chromatography, isoelectric focusing, and so forth or by a combination of several methods. For a structural determination, degradation steps such as a site-specific proteolysis (e.g., with trypsin), removal of oligosaccharides from the polypeptide (by an enzymatic hydrolysis or hydrazine... 

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