p-nitrophenyl penta-N-acetyl-beta-chitopentaoside as a novel synthetic substrate for the colorimetric assay of lysozyme.
p-Nitrophenyl beta-glycosides of N-acetylchitooligosaccharides (PNP-(GlcNAc)n n = 3-5) were examined as substrates for lysozyme [EC 3.2.1.17]. The enzyme released predominantly p-nitrophenyl N-acetyl-beta-D-glucosaminide (PNP-GlcNAc) from each substrate. Furthermore, the initial rate of PNP-GlcNAc formation in lysozyme-catalyzed hydrolysis of p-nitrophenyl penta-N-acetyl-beta-chitopentaoside (PNP-(GlcNAc)5) was about 350 and 25 times faster than those of p-nitrophenyl tri-N-acetyl-beta-chitotrioside (PNP-(GlcNAc)3) and p-nitrophenyl tetra-N-acetyl-beta-chitotetraoside (PNP-(GlcNAc)4), respectively. From these results, a new colorimetric assay method of lysozyme using PNP-(GlcNAc)5 as a substrate was developed on the basis of the determination of p-nitrophenol liberated from the substrate by lysozyme through a coupled reaction involving beta-N-acetylhexosaminidase (NAHase). The assay system gave a linear dose-response curve in the range of 2-120 micrograms of lysozyme in a 15-60 min incubation. The present assay was not significantly influenced by the ionic strength of the medium and was reproducible. This method using PNP-(GlcNAc)5 as a substrate was shown to be useful for lysozyme assay.
Nanjo
F
F
NFI Laboratories, Yaizu Suisan Kagaku Industry Co., Ltd., Shizuoka.
Sakai
K
K
Usui
T
T
D000818:Animals; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D003124:Colorimetry; D005960:Glucosides; D006027:Glycosides; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009113:Muramidase / Q000032:analysis* / Q000378:metabolism; D009844:Oligosaccharides; D013379:Substrate Specificity; D013696:Temperature; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D005960:Glucosides; D006027:Glycosides; D009844:Oligosaccharides; C055288:4-nitrophenyl-N,N',N'',N''',N''''-pentaacetyl-beta-chitopentaoside; D009113:Muramidase; D001619:beta-N-Acetylhexosaminidases