Two time-resolved fluorometric high-throughput assays for quantitation of GDP-L-fucose.

Two rapid and simple procedures for the quantitative analysis of GDP-l-fucose (GDP-Fuc) are described. The methods are based on time-resolved fluorescence and microplate assay technology. The first assay relies on measuring the enzyme activity of alpha1, 3-fucosyltransferase. In this assay, transfer of fucose from GDP-Fuc converts sialyllactosamine to sialyl Lewis x tetrasaccharide, which is detected and quantified by relevant antibodies on a microplate. The formation of the reaction product is directly dependent on the presence of GDP-Fuc in the concentration range of 10-10,000 nM. In the second method GDP-Fuc inhibits the binding of fucose-specific Aleuria aurantia lectin to fucosylated glycan on a microwell. The lectin-based assay is less sensitive than the enzyme assay, but it is cheaper and faster. We used these assays in monitoring the amount of GDP-Fuc in crude lysates of transgenic yeast, which expresses the enzymes producing GDP-Fuc. The newly developed assays are versatile and applicable to measure also other nucleotide sugars or glycosyltransferase activities in a high-throughput manner.