THE GLYCANS OF HORSERADISH-PEROXIDASE

Horseradish peroxidase (E.C. 1.11.1.7) isozyme c (HRPc) is a glycoprot ein found to contain 21.8% carbohydrate with the average composition: 2 mol GlcNAc, 2.6 mol Man, and 0.8 mol each of Fuc and Xyl. The oligos accharides of HRPc were investigated by a combination of High pH Anion -Exchange Chromatography with Pulsed Amperometric Detection, methylati on analysis and Matrix-Assisted Laser Desorption/Ionization Time-of-Fl ight Mass Spectrometry. The structure of the major oligosaccharide rel eased by digestion with glycopeptidase A, accounting for between 75 an d 80% of the total, was confirmed to be alpha-Man-(1 --> 6)[alpha-Man- (1 --> 3)][beta-Xyl-(1 --> 2)]-beta-Man-(1 --> 4)-beta-GlcNAc-(1 --> 4 )[alpha-Fuc-(1 --> 3)]-GlcNAc. Most of the remaining oligosaccharides were found to belong to the (Xyl)(x)Man(m)(Fuc)(f)GlcNAc(2) (m = 2, 4, 5, 6; f = 0 or 1, x = 0 or 1) family. Less than 5% of the oligosaccha rides were of the Man(m)GlcNAc(2) (m = 4 to 7) type. Methylation analy sis of holo- and apo-HRPc and its tryptic glycopeptides support the st ructures proposed for the oligosaccharides. Furthermore, methylation a nalysis of the tryptic glycopeptides provides evidence for the heterog eneity of the oligosaccharides occurring at each of the N-linked sites . (C) 1996 Elsevier Science Ltd.