A new approach to assay endo-type carbohydrases: bifluorescent-labeled substrates for glycoamidases and ceramide glycanases

Analytical Biochemistry
K B LeeY C Lee

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 non-reducing 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 (lambda em = 335 nm) is quenched due to absorption of its emitted light by the dansyl group, which in turn results in emission of fluorescence (lambda ex = 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 th...Continue Reading

Citations

Nov 13, 2013·Experimental Psychology·Patrick Lemaire, Thomas Hinault
Jul 19, 2011·Developmental Neuropsychology·Korbinian MoellerHans-Christoph Nuerk
Dec 25, 2012·Journal of Clinical and Experimental Neuropsychology·Kim Uittenhove, Patrick Lemaire
Oct 27, 1997·The Journal of Biological Chemistry·J Q Fan, Y C Lee
Aug 10, 2000·Analytical Biochemistry·K G Rice
Feb 23, 2010·Bioorganic & Medicinal Chemistry Letters·Hiroyuki OkaKoji Matsuoka

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