Direct assay for alpha-amylase using fluorophore-modified cyclodextrins.
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.
Murayama
Takuya
T
Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-B-44 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
Tanabe
Tetsuya
T
Ikeda
Hiroshi
H
Ueno
Akihiko
A
D001236:Aspergillus oryzae / Q000187:drug effects / Q000201:enzymology; D001393:Azoles / Q000737:chemistry*; D003505:Cyclodextrins / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D006868:Hydrolysis; D008968:Molecular Conformation; D015394:Molecular Structure; D009578:Nitrobenzenes / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods; D013329:Structure-Activity Relationship; D013997:Time Factors; D000516:alpha-Amylases / Q000737:chemistry* / Q000187:drug effects*
C507677:4-amino-7-nitrobenz-2-oxa-1,3-diazole; D001393:Azoles; D003505:Cyclodextrins; D005456:Fluorescent Dyes; D009578:Nitrobenzenes; D000516:alpha-Amylases