Assay of glycoamidases and endo-beta-N-acetylglucosaminidases by lectin capture and dissociation-enhanced lanthanide fluorescence immunoassay

We have developed an assay system for endo-beta-N-acetylglucosaminidase and glycoamidase (PNGase), using Eu3+-labeled Man(9)GlcNAc(2) glycopeptides as substrates in combination with lectin capture. Two glycopeptides of differ ent peptide lengths, derived from soybean agglutinin, were labeled with Eu3 + via a diethylenetriaminepentaacetate (DTPA) chelating linker and served a s substrates for two types of enzymes: one with (Man(9)GlcNAc(2))Asn for en do-beta-N-acetylglucosaminidase and the other with Ala-Ser-Phe-(Man(9)GlcNA c(2))Asn-Phe-Thr for glycoamidase activities. Following enzymatic hydrolysi s, concanavalin A, immobilized or soluble, was added to the mixture to bind unreacted substrate and unlabeled hydrolysis product. The labeled peptide product could then be separated from the lectin-bound complexes by filtrati on for quantification by dissociation-enhanced lanthanide fluorescence immu noassay. Activities as low as 2 fmol min(-1) could be rapidly quantified fo r both types of enzymes, and enzymological parameters could be determined w ithin minutes. Applicability of the assay was tested for identification of a glycoamidase activity peak in the fractionation of sweet almond emulsin, a classic example. This assay offers sensitivity, ease of use, and high thr oughput. In addition, it is versatile and should be applicable to other gly cobiology enzyme systems, (C) 2000 Academic Press.