FIGURE 1.
Fluorescently labeled primers for the separate testing of hexosyl- and sialyltransferase activity and chromatographic behavior in anion exchange chromatography. Starting from 4-MU-Sia, the monofunctional active site mutants of SiaDW/SiaDY were used in iterative steps to generate labeled acceptors of defined length. Reaction samples were analyzed on a CarboPac® PA-100 column, and products eluted with a curved NaNO3 gradient as shown. A, structures of the labeled SiaDW glycans (top) and their running behavior on anion exchange chromatography (bottom) are shown. Note that the retention time increases with the addition of a Sia residue and decreases with the addition of Hex, leading to the elution order DP2, DP1, DP4, and then DP3. B, the elution behavior of labeled SiaDY glycans is similar to that of SiaDW. Of note, the chromatograms show that the conversion of 4-MU-DP1 to 4-MU-DP2 was incomplete under the conditions used, whereas all other reactions proceeded to completion.