Assay of Glycoamidases and Endo-β-N-acetylglucosaminidases by Lectin Capture and Dissociation-Enhanced Lanthanide Fluorescence Immunoassay

Abstract

We have developed an assay system for endo-β-N-acetylglucosaminidase and glycoamidase (PNGase), using Eu3+-labeled Man9GlcNAc2 glycopeptides as substrates in combination with lectin capture. Two glycopeptides of different peptide lengths, derived from soybean agglutinin, were labeled with Eu3+ via a diethylenetriaminepentaacetate (DTPA) chelating linker and served as substrates for two types of enzymes: one with (Man9GlcNAc2)Asn for endo-β-N-acetylglucosaminidase and the other with Ala-Ser-Phe-(Man9GlcNAc2)Asn-Phe-Thr for glycoamidase activities. Following enzymatic hydrolysis, 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 filtration for quantification by dissociation-enhanced lanthanide fluorescence immunoassay. Activities as low as 2 fmol min−1 could be rapidly quantified for both types of enzymes, and enzymological parameters could be determined within 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 throughput. In addition, it is versatile and should be applicable to other glycobiology enzyme systems.