p-Nitrophenyl Penta-N-Acetyl-β-Chitopentaoside as a Novel Synthetic Substrate for the Colorimetric Assay of Lysozyme
1988 Volume 104 Issue 2 Pages 255-258
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1988 Volume 104 Issue 2 Pages 255-258
p-Nitrophenyl β-glycosides of N-acetylchitooligosaccharides (PNP-(G1cNAc)n n=3-5) were examined as substrates for lysozyme [EC 3.2.1.17]. The enzyme released predominantly p-nitrophenyl N-acetyl-β-D-glucosaminide (PNP-G1cNAc) from each substrate. Furthermore, the initial rate of PNP-GleNAc formation in lysozyme-catalyzed hydrolysis of p-nitrophenyl penta-N-acetyl-β-chitopentaoside (PNP-(G1cNAc)5) was about 350 and 25 times faster than those of p-nitrophenyl tri-N-acetyl-β-chitotrioside (PNP-(GleNAc)3) and p-nitrophenyl tetra-N-acetyl-β-chitotetraoside (PNP-(G1cNAc)4), respectively. From these results, a new colorimetric assay method of lysozyme using PNP-(G1cNAc)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 β-N-acetylhexosaminidase (NAHase). The assay system gave a linear dose-response curve in the range of 2-120 μg 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-(G1cNAc)5 as a substrate was shown to be useful for lysozyme assay.
