Rate assay of N-acetyl-beta-D-hexosaminidase with 4-nitrophenyl N-acetyl-beta-D-glucosaminide as an artificial substrate.
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.
Shibata
H
H
Iatron Laboratory, Tokyo, Japan.
Yagi
T
T
D000117:Acetylglucosamine / Q000031:analogs & derivatives*; D002021:Buffers; D003505:Cyclodextrins; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009393:Nephritis / Q000652:urine; D009596:Nitrophenols / Q000378:metabolism; D015203:Reproducibility of Results; D047391:alpha-Cyclodextrins; D001619:beta-N-Acetylhexosaminidases / Q000032:analysis* / Q000652:urine
D002021:Buffers; D003505:Cyclodextrins; D009596:Nitrophenols; D047391:alpha-Cyclodextrins; C103606:diethylaminoethyl alpha-cyclodextrin; C014124:4-nitrophenyl-N-acetyl-2-deoxyglucopyranoside; D001619:beta-N-Acetylhexosaminidases; D005944:Glucosamine; D000117:Acetylglucosamine; C024836:4-nitrophenol