A New Approach to Assay Endo-Type Carbohydrases: Bifluorescent-Labeled Substrates for Glycoamidases and Ceramide Glycanases

Abstract

Glycoamidases and ceramide glycanases are important "endo-type" enzymes for structural elucidations of glycoconjugates as well as for construction of neoglycoconjugates. The assay methods currently available for these enzymes are tedious and do not permit continual assay of the enzyme activities. We modified a desialylated biantennary glycopeptide with 2-naphthylacetic acid at the N-terminus and at the nonreducing terminal galactosyl residues with mono-N-dansylethylenediamine, via a specific oxidation of the C-6 hydroxyl group with galactose oxidase. In such a substrate, the naphthyl fluorescence (λem = 335 nm) is quenched due to absorption of its emitted light by the dansyl group, which in turn results in emission of fluorescence (λex = 520 nm) by the latter. However, when the link between the two fluorophores is severed by glycoamidase (PNGase), the energy transfer ceases to occur. Consequently the emission of the dansyl fluorescence and the quenching of naphthyl fluorescence diminish or disappear. Likewise, the energy transfer between the fluorophores in an alkyl lactoside containing a dansyl group at the terminal position of aglycon and a 2-naphthylmethyl group on the galactosyl residue is also eliminated by the glycosidic cleavage by a ceramide glycanase from American leech, Macrobdella decora, resulting in enhancement of the naphthyl emission and decrease in the dansyl emission. The substrates presented here permit continuous fluorescent monitoring of the enzymatic reaction. This allows precise analyses of enzyme kinetics not possible with the conventional assay methods for the endo-type enzymes which usually require separation of reaction products.