A novel analytical procedure for assaying lysozyme activity using an end-blocked chitotetraose derivative as...

An end-modified β-d-galactosyl chitotetraose derivative [4⁴-O-β-d-galactosyl-β-tri-N-acetylchitotriosyl 2-acetamide-2,3-dideoxy-glucopyranose; Gal(GlcN)₃D] 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)₃D and related compounds using hen egg-white lysozyme was then examined. Gal(GlcN)₃D 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)₃D to lysozyme is more favorable than that of (GlcN)₄D. We conclude the 4-O-substituted Gal group at the non-reducing end of Gal(GlcN)₃D 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)₃D 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.