The Glycopeptide Linkage of Ribonuclease B

A micro method, employing the fluorescent 1-dimethyl-aminonaphthalene-5-sulfonyl (dansyl) group, was developed for the detection of the 1-l-β-aspartamido[-2-acetamido]-1, 2-dideoxy-β-d-glucose linkage in glycoproteins. With this procedure, dansyl 1-l-β-aspartamido[-2-acetamido]-1,2-dideoxy-β-d-glucose was isolated from an acid hydrolysis of the dansylated ribonuclease B glycopeptide. Similar results were obtained with the glycopeptide from ovalbumin. Confirmation for the N-acetylglucosamine-asparagine linkage was obtained with a glycopeptide amidohydrolase isolated from hen oviduct, an enzyme that hydrolyzes 1-l-β-aspartamido[-2-acetamido]-1,2-dideoxy-β-d-glucose to N-acetyl-glucosamine, aspartic acid, and ammonia. With the use of this enzyme it was shown that N-acetylglucosamine was on the reducing end of the polysaccharides enzymically released from the ribonuclease B and ovalbumin glycopeptides. Treatment of the ribonuclease B glycopeptide with periodate or β-N-acetylglucosaminidase did not affect the glucosamine content of the glycopeptide. These results indicate that the second glucosamine moiety of the ribonuclease glycopeptide is located internally in the polysaccharide and is glycosidically linked at either position 3 or 4.