A fluorescence high-performance liquid chromatography assay for enzymes acting on the di-N-acetylchitobiosyl part of asparagine-linked glycans.
The glycoasparagine, Man7GlcNAc2Asn ('Man7') was labelled with resorufin and used as a specific substrate for the detection and quantification of endo-beta-N-acetyl glucosaminidases (Endos) acting on the di-N-acetylchitobiosyl part of asparagine-linked glycans. Peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidases (PNGases) cannot transform this substrate but they can be detected by the procedure described earlier using the resorufin-labelled N-glycopeptide [Glycoconjugate J., 9 (1992) 162-167]. These two substrates can be used in a simple, reproducible and very sensitive fluorescence HPLC assay in order to monitor Endo and PNGase activities during isolation and purification processes, or studies of the evolution of such activities during cultivation of the producing cells.
Bourgerie
S
S
Institut de Biotechnologie, Université de Limoges, France.
Berger
S
S
Strecker
G
G
Julien
R
R
Karamanos
Y
Y
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D005453:Fluorescence; D006020:Glycopeptides / Q000737:chemistry*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000032:analysis*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry*; D010078:Oxazines; D011134:Polysaccharides / Q000737:chemistry*; D013379:Substrate Specificity
D006020:Glycopeptides; D009844:Oligosaccharides; D010078:Oxazines; D011134:Polysaccharides; C007872:glycoasparagines; C014180:resorufin; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase