title: Mild chemical deglycosylation of horseradish peroxidase yields a fully active, homogeneous enzyme.
abstract: Horseradish peroxidase isoenzyme C (HRP) contains eight N-linked glycans composed of Man, Xyl, Fuc, and GlcNAc. These glycans were resistant to enzymatic hydrolysis by endoglycosidases peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F, endo-beta-N-acetyl-glucosaminidase H, and endo-beta-N-acetylglucosaminidase F under conditions where ovalbumin was deglycosylated. However, using anhydrous trifluoromethanesulfonic acid (TFMS) in the presence of 90 mM phenol for 5 min at--10 degrees C, all carbohydrate except GlcNAc was removed. Sixty percent of deglycosylated HRP was active after this TFMS treatment. Benzhydroxamic acid affinity chromatography separated active and inactive deglycosylated HRP. TFMS treatment, however, introduced negative charges in all inactive HRP and in about 90% of the active deglycosylated HRP. The nature of this modification has not been identified. After ion-exchange chromatography, homogeneous and fully active deglycosylated HRP, showing the original pI of 9, electronic absorption spectrum, and enzyme kinetics, was obtained, In this purified product no amino acid modifications were detected by amino acid analysis, partial sequencing, and mass spectrometry of tryptic peptides. The deglycosylated product showed greatly reduced solubility in salt solution compared to that of authentic HRP.
authors.affiliation: Department of Protein Chemistry, University of Copenhagen, Denmark.
authors.forename: J W
authors.initials: JW
authors.lastname: Tams
authors.forename: K G
authors.initials: KG
authors.lastname: Welinder
mesh_terms: D004789:Enzyme Activation; D006031:Glycosylation; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000302:isolation & purification; D010944:Plants / Q000201:enzymology
chemical_list: D006735:Horseradish Peroxidase