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Rate Assay of N-acetyl-beta-D-hexosaminidase With 4-nitrophenyl N-acetyl-beta-D-glucosaminide as an Artificial Substrate

Rate Assay of N-acetyl-beta-D-hexosaminidase With 4-nitrophenyl N-acetyl-beta-D-glucosaminide as an Artificial Substrate

H Shibata, T Yagi

Clin Chim Acta. 1996 Jul 15;251(1):53-64.

PMID: 8814350

Abstract:

A rapid and accurate rate assay method for N-acetyl-beta-D-hexosaminidase (EC 3.2.1.52, also known as N-acetyl-beta-D-glucosaminidase, or NAGase) using 4-nitrophenyl N-acetyl-beta-D-glucosaminide (NP-GlcNAc) as an artificial substrate was developed using diethylaminoethyl-alpha-cyclodextrin (DEn-CD, where n is the number of diethylaminoethyl groups introduced to alpha-cyclodextrin), as an additive to ionize 4-nitrophenol to yellow-colored 4-nitrophenoxide at pH near 5, where the enzyme acts optimally. A possible recipe for the rate assay of NAGase is as follows. Prepare a stock solution containing 4.8 mmol/l NP-GlcNAc and 1% DEn-CD (n is preferably near 17) in 0.1 mol/l glycolate buffer, pH 5.50. Introduce the stock solution and a properly diluted sample (urine or other body fluid) to a reaction cell placed in a spectrophotometer at a ratio of 1:1, and monitor the absorbance at 400 or 420 nm. The reaction rate (enzymatic activity) can be conveniently read directly from calibration plots prepared for a given lot of DEn-CD sample, or can be calculated from the rate of the absorbance increase, ionization degree of 4-nitrophenol at pH 5.50, and the millimolar absorbance coefficient of 4-nitrophenoxide in the presence of 0.5% DEn-CD.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP3459185	4-Nitrophenyl N-acetyl-β-D-glucosaminide		3459-18-5	Price

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