Figure 3

(A). Chemical structure of the synthetic sugar donor substrate CMP-sialic acid-PEG ₄ -biotin utilized in our high-throughput assay for sialyltransferase activity. (B). Schematic of the developed assay for sialyltransferase activity. 96- or 384-well plates are coated initially with asialo bovine submaxillary mucin (aBSM). aBSM carries exposed terminal GalNAc moieties. Active ST6GalNAcI catalyzes the transfer of sialic acid- PEG₄-biotin from the sugar donor substrate CMP-sialic acid-PEG₄-biotin onto aBSM and immobilizes biotin on the plate. Biotinylation can be subsequently detected with europium (Eu)-labeled streptavidin and time-resolved fluorescence. (C). Time-resolved (TR) fluorescence counts plotted against different concentrations of chicken ST6 expressed in Sf9 insect cells (positive control enzyme). (D). Detection limit of the sialyltransferase assay. Calculated signal-to-background (no enzyme control) ratios for different concentrations of chicken ST6 expressed in Sf9 insect cells. Asterisks indicate enzyme concentrations that exhibited enzymic activity which was statistically different from the no enzyme control (Dunnett's MCT, p < 0.01). Experiments were carried out in triplicate and the error bars correspond to one standard deviation from the mean values. a.u.: arbitrary units; U: unit of sialyltransferase activity.