A novel analytical procedure for assaying lysozyme activity using an end-blocked chitotetraose derivative as substrate
Abstract An end-modified β- D -galactosyl chitotetraose derivative [4 4 - O -β- D -galactosyl-β-tri- N -acetylchitotriosyl 2-acetamide-2,3-dideoxy-glucopyranose; Gal(GlcN) 3 D] was designed and synthesized from chitin tetrasaccharide. The derivative was chemically modified by dehydration of the reducing end GlcN and enzymatic addition of a Gal group to the non-reducing end GlcN. Hydrolysis of Gal(GlcN) 3 D and related compounds using hen egg-white lysozyme was then examined. Gal(GlcN) 3 D was specifically cleaved to Gal(GlcN) 2 and GlcND. Kinetic studies and docking simulations were further conducted to elucidate its mode of binding to lysozyme. These analyses revealed the binding of Gal(GlcN) 3 D to lysozyme is more favorable than that of (GlcN) 4 D. We conclude the 4- O -substituted Gal group at the non-reducing end of Gal(GlcN) 3 D does not prohibit the action of lysozyme, but gives some affinity to the subsite (i.e. equivalent to GlcN). From these results, a new assay method for quantifying lysozyme was established by utilizing the Morgan-Elson reaction based on the generation of product D (2-acetamide-2,3-dideoxy-glucopyranose), which serves as a chromophore, formed from Gal(GlcN) 3 D by lysozyme through a conjugated reaction involving β- N -acetylhexosaminidase. The assay system gave a linear dose-response curve in the range of 2–31 μg of lysozyme during a 15 min incubation. This novel assay method for the quantification of lysozyme is highly specific, sensitive, accurate and reproducible. Highlights We synthesized an end-modified β- D -galactosyl chitotetraose derivative [Gal(GlcN) 3 D]. The Gal(GlcN) 3 D was specifically hydrolyzed to Gal(GlcN) 2 and GlcN by action of lysozyme. We established lysozyme assay system by adding β-NAHase using Gal(GlcN) 3 D. This assay method for the quantification of lysozyme is highly specific, sensitive and accurate. Graphical abstract [DISPLAY OMISSION]
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