The isolation and characterization of the glycopeptides from horseradish peroxidase isoenzyme C.
The purity of horseradish peroxidase isoenzyme C was demonstrated using isoelectric focusing, polyacrylamide gel electrophoresis at two pH values and cellulose acetate electrophoresis at two pH values. The glycopeptides obtained upon trypsin digestion were isolated using the plant lectin, concanavalin A, and were resolved using paper electrophoresis. The carbohydrate content of the native peroxidase was 86% accounted for by the carbohydrate content of the glycopeptides thus suggesting little loss of carbohydrate during glycopeptide isolation and purification. In each of the seven glycopeptides isolated glucosamine was associated with asparagine, thus suggesting the carbohydrate chains are covalently bound to the peptide chain through N-glycosidic linkages. The purity of each glycopeptide was demonstrated by the sequential release of single amino acid residues by Edman degradation. As six glycopeptides had unique amino acid sequences, it was concluded that the carbohydrate prosthetic group was distributed in at least six units along the protein backbone. Five glycopeptides possessed the amino acid sequence about the point of carbohydrate attachment of Asn-X-(Ser, Thr) where X is any amino acid. The size of the carbohydrate units ranged from 1600 to 3000 daltons. The predominant carbohydrate residues in each glycopeptide were mannose and glucosamine with lesser and varying amounts of fucose, xylose, and arabinose. There was no apparent correlation of the carbohydrate composition with the amino acid sequence.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001089:Arabinose / Q000032:analysis; D002849:Chromatography, Gas; D004588:Electrophoresis, Cellulose Acetate; D004591:Electrophoresis, Polyacrylamide Gel; D005643:Fucose / Q000032:analysis; D005944:Glucosamine / Q000032:analysis; D005947:Glucose / Q000032:analysis; D006020:Glycopeptides / Q000302:isolation & purification; D006735:Horseradish Peroxidase; D007527:Isoenzymes; D008358:Mannose / Q000032:analysis; D010446:Peptide Fragments / Q000032:analysis; D010544:Peroxidases; D014357:Trypsin; D014994:Xylose / Q000032:analysis
D000596:Amino Acids; D006020:Glycopeptides; D007527:Isoenzymes; D010446:Peptide Fragments; D005643:Fucose; D014994:Xylose; D001089:Arabinose; D006735:Horseradish Peroxidase; D010544:Peroxidases; D014357:Trypsin; D005947:Glucose; D005944:Glucosamine; D008358:Mannose