Carbohydrates in protein. 3. The preparation and some of the properties of a glycopeptide from hen's-egg albumin

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Abstract
The preparation of a glycopeptide from egg albumin has been described, in which enzymic digestion of the protein is followed by chromatography on charcoal-Celite columns and electrophoresis on Porath cellulose columns. Evidence is presented showing that the poly-saccharide is present in the whole protein as a single unit. The main constituents of this glycopeptide have been quantitatively analysed and found to be mannose, 5 residues; glucosamine, 3; acetyl, 3; aspartic acid, 1; leucine, 0.78; serine, 0.32; threonine, 0.35. The possibility has been discussed that these values may not represent accurately the composition of the isolated glycopeptide. Experiments with fluorodinitro-benzene have shown that aspartic acid is N-terminal in the peptide chain (with traces of N-terminal tyrosine in some samples). These results, together with those obtained with carboxypeptidase and by hydrazinolysis, are in agreement with the sequence Tyr. Asp (carbohydrate). (Leu. Ser. Thr). Val. The carbohydrate seems to be linked to aspartic acid through one of its carboxyl residues, probably the [beta]-group. Titration-curve data indicate the presence of a group with pK 3.5 and another with pK 6-7. These represent the ionization of the C-terminal and N-terminal groups respectively. The possible presence of a third group with pK about 9.5 cannot be exluded with certainty. Acid hydrolysis of the glycopeptide gives rise to the formation of ammonia approximately 1 residue is produced by hydrolysis in N-sulphuric acid at 100[degree] for 4 hr., increasing to about 1.7 equiv. after hydrolysis in 2 N-hydrochloric acid at 100[degree] for 3 hr. The possible sources of this ammonia have been discussed. The nature of the linkage between the polypeptide and the carbohydrate in the whole protein has been considered. The most likely structure from our evidence is that of a [beta]-aspartylglycosylamine, but this suggestion must be regarded as tentative at present.