A FLUORESCENCE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY ASSAY FOR ENZYMES ACTING ON THE DI-N-ACETYLCHITOBIOSYL PART OF ASPARAGINE-LINKED GLYCANS

The glycoasparagine, Man(7)GlcNAc(2)Asn ('Man(7)') was labelled with r esorufin and used as a specific substrate for the detection and quanti fication of endo-beta-N-acetyl glucosaminidases (Endos) acting on the di-N-acetylchitobiosyl part of asparagine-linked glycans. Peptide-N-4- (N-acetyl-beta-glucosaminyl) asparagine amidases (PNGases) cannot tran sform this substrate but they can be detected by the procedure describ ed 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 mo nitor Endo and PNGase activities during isolation and purification pro cesses, or studies of the evolution of such activities during cultivat ion of the producing cells.