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Analytical biochemistry v.538, 2017년, pp.64 - 70   SCI SCIE
본 등재정보는 저널의 등재정보를 참고하여 보여주는 베타서비스로 정확한 논문의 등재여부는 등재기관에 확인하시기 바랍니다.

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

Ogata, Makoto (Department of Applied Chemistry and Biochemistry, National Institute of Technology, Fukushima College, 30 Nagao, Iwaki, Fukushima 970-8034, Japan ) ; Matsui, Megumi (Department of Applied Chemistry and Biochemistry, National Institute of Technology, Fukushima College, 30 Nagao, Iwaki, Fukushima 970-8034, Japan ) ; Kono, Haruka (Department of Applied Chemistry and Biochemistry, National Institute of Technology, Fukushima College, 30 Nagao, Iwaki, Fukushima 970-8034, Japan ) ; Matsuzaki, Yuka (Department of Applied Chemistry and Biochemistry, National Institute of Technology, Fukushima College, 30 Nagao, Iwaki, Fukushima 970-8034, Japan ) ; Kato, Yuna (Department of Applied Chemistry and Biochemistry, National Institute of Technology, Fukushima College, 30 Nagao, Iwaki, Fukushima 970-8034, Japan ) ; Usui, Taichi (Integrated Bioscience Research Division, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan ) ;
  • 초록  

    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]


  • 주제어

    Lysozyme .   Hexosaminidase .   Enzyme assay .   Kinetics .   Chitin oligosaccharide derivatives.  

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