Detection of 100 molecules of product formed in a fucosyltransferase reaction.
We present the detection of 100 molecules of enzyme substrate and product. A fucosyltransferase and a fucosidase were used to add and remove, respectively, a monosaccharide from a synthetic oligosaccharide fluorescently labelled with tetramethylrhodamine. The reaction was followed by use of capillary zone electrophoresis, to separate the product and reactant, with laser-induced fluorescence detection. These are the most sensitive enzyme assays reported to date and six orders of magnitude more sensitive than any reported for these two enzymes. This simple technology allows high-sensitivity determination of the activity of any enzyme for which a fluorescent substrate can be synthesized, and brings within reach the ability to assay glycosyltransferase activities in single cells.
Zhao
J Y
JY
Department of Chemistry, University of Alberta, Edmonton, Canada.
Dovichi
N J
NJ
Hindsgaul
O
O
Gosselin
S
S
Palcic
M M
MM
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes; D005647:Fucosyltransferases / Q000378:metabolism*; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D012235:Rhodamines; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D014312:Trisaccharides / Q000096:biosynthesis* / Q000138:chemical synthesis / Q000737:chemistry*; D005644:alpha-L-Fucosidase / Q000378:metabolism*
D004187:Disaccharides; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D012235:Rhodamines; D014312:Trisaccharides; C005358:tetramethylrhodamine; D005647:Fucosyltransferases; D005644:alpha-L-Fucosidase