A continuously monitored spectrophotometric assay of glycosidases with nitrophenyl glycosides

A general method for a continuously monitored spectrophotometric assay of glycosidases at all values of pH using p-nitrophenyl glycosides is presented. The method is demonstrated specifically by the development of a routine assay for α-galactosidase from fig and Mortierella vinacea using p-nitrophen...

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Published inAnalytical biochemistry Vol. 54; no. 1; pp. 120 - 128
Main Authors Ford, James R., Nunley, James A., Li, Yu-Teh, Chambers, Robert P., Cohen, William
Format Journal Article
LanguageEnglish
Published United States Elsevier Inc 01.07.1973
Subjects
Acetamides
Autoanalysis
Galactosidases - analysis
Galactosidases - metabolism
Glycosides
Hexosaminidases - analysis
Hexosaminidases - metabolism
Hydrogen-Ion Concentration
Kinetics
Methods
Nitrophenols
Plants - enzymology
Spectrophotometry, Ultraviolet
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Summary:A general method for a continuously monitored spectrophotometric assay of glycosidases at all values of pH using p-nitrophenyl glycosides is presented. The method is demonstrated specifically by the development of a routine assay for α-galactosidase from fig and Mortierella vinacea using p-nitrophenyl galactopyranoside (NPG) at pH 3.9 and 5.8, respectively, and also for jack bean meal β- N-acetylhexosaminidase using p-nitrophenyl-β-2-acetamido-2-deoxy- d-glucopyranoside (NPADG) at pH 5.0. A number of different wavelengths may be used for the assay depending upon the criterion of the user; maximum sensitivity at a selected pH, determination of enzyme pH optima with a pH-independent difference extinction coefficient, or the reduction of background absorbance for kinetic studies at high substrate concentrations.
Bibliography:ObjectType-Article-1
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ISSN:0003-2697
1096-0309
DOI:10.1016/0003-2697(73)90254-6