Direct assay for alpha-amylase using fluorophore-modified cyclodextrins

Bioorg Med Chem. 2006 Jun 1;14(11):3691-6. doi: 10.1016/j.bmc.2006.01.037. Epub 2006 Feb 7.

Abstract

A simple assay method for alpha-amylase was developed based on fluorophore-modified cyclodextrins (CDs). Four kinds of CD derivatives bearing a 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD-amine) moiety were prepared as artificial substrates for the assay method. The fluorescence intensity of all the NBD-amine-modified CDs decreased upon addition of Aspergillus oryzae alpha-amylase, indicating a reduction in hydrophobicity near the NBD-amine moiety induced by hydrolysis of the CD ring. NC4gammaCD, having a gamma-CD and an amino-tetramethylene spacer, was the most sensitive substrate for the alpha-amylase assay. The initial rate of hydrolysis of NC4gammaCD displayed a liner correlation to the concentration of the alpha-amylase. NC4gammaCD was sensitive to the alpha-amylase but was not sensitive to guest compounds that were accommodated by the native CDs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aspergillus oryzae / drug effects
  • Aspergillus oryzae / enzymology
  • Azoles / chemistry*
  • Cyclodextrins / chemical synthesis
  • Cyclodextrins / chemistry
  • Cyclodextrins / pharmacology*
  • Fluorescent Dyes / chemical synthesis
  • Fluorescent Dyes / chemistry
  • Fluorescent Dyes / pharmacology*
  • Hydrolysis
  • Molecular Conformation
  • Molecular Structure
  • Nitrobenzenes / chemistry*
  • Sensitivity and Specificity
  • Spectrometry, Fluorescence / methods
  • Structure-Activity Relationship
  • Time Factors
  • alpha-Amylases / chemistry*
  • alpha-Amylases / drug effects*

Substances

  • 4-amino-7-nitrobenz-2-oxa-1,3-diazole
  • Azoles
  • Cyclodextrins
  • Fluorescent Dyes
  • Nitrobenzenes
  • alpha-Amylases