Carbohydrate chain structure enzymic analysis

Over the years, several strategies have been developed for determination of the structure of carbohydrate chains for concise reviews, see Refs. 3 and 4. In particular, the refinements of methylation analysis,15,53 and chemical6-8 and enzymic degradation methods9-10 have permitted... 

Chemical structure elucidation of the carbohydrate moiety of a glycoprotein is a difficult problem because there is often chain branching and because the individual carbohydrate chains are not always identical. Specific exoglycosidases which release monosaccharide units only from the terminal nonreducing end of the carbohydrate chain are most useful in structural analysis. Among the enzymes available for sequence analysis... 

The uses of enzymic methods in the structural analysis of polysaccharides and the carbohydrate chain(s) of glycoproteins have been discussed. 

The side chains of proteins can undergo post-translational modification in the course of thermal processes. The reaction can also result in the formation of protein cross-links. A known reaction which mainly proceeds in the absence of carbohydrates, for example, is the formation of dehydroalanine from serine, cysteine or serine phosphate by the elimination of H2O, H2S or phosphate. The dehydroalanine can then lead to protein cross-links with the nucleophilic side chains of lysine or cysteine (cf. 1.4.4.11). In the presence of carbohydrates or their degradation products, especially the side chains of lysine and arginine are subject to modification, which is accompanied by a reduction in the nutritional value of the proteins. The structures of important lysine modifications are summarized in Formula 4.95. The best known compounds are the Amadori product -fructoselysine and furosine, which can be formed from the former compound via the intermediate 4-deoxyosone (Formula 4.96). To detect of the extent of heat treatment, e. g., in the case of heat treated milk products, furosine is released by acid hydrolysis of the proteins and quantitatively determined by amino acid analysis. In this process, all the intermediates which lead to furosine are degraded and an unknown portion of already existing furosine is destroyed. Therefore, the hydrolysis must occur under standardized conditions or preferably by using enzymes. Examples showing the concentrations of furosine in food are presented in Table 4.13. 

An early analysis of the spleen enzyme showed the presence of hexose and hexo-samine A more recent study has demonstrated an enzyme from human hairy leukemic cells which appears identical to the purple spleen phosphatase, and which binds to ConA-Sepharose and is eluted with methylmannoside Very likely, therefore, the spleen enzyme, Uke uteroferrin, is a high mannose glycoprotein, but the structure of its carbohydrate has yet to be determined. In each protein the oligosaccharide chain appears to be attached to an asparagine residue (at position 97 in uteroferrin), since this residue was observable only after treatment with N-glycanase which removes N-asparagine-linked oUgosaccharides. ... 

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