Assaying sialyltransferase activity with surface plasmon resonance.
Here, we describe an activity assay for sialyltransferases based on surface plasmon resonance (SPR). Different natural and synthetic oligosaccharides serving as acceptor substrates for the sialyltransferase ST3Gal-III (EC 2.4.99.6) were immobilized or synthesized on SPR chips. The chip was then exposed to different concentrations of a reaction mixture of ST3Gal-III and CMP-Neu5Ac either by injection or by external application of the reaction mixture to the chip surface. The binding of two lectins, one that specifically recognizes the unmodified acceptor, the other the sialylated oligosaccharide, was utilized to determine the extent of enzymatic turnover. In order to obtain enzymatic activities, the SPR data were correlated to data obtained from a classical radio assay. After regeneration, that is, cleavage of the sialic acid residues by using a sialidase, the chip is available for new experiments. The technique allows the rapid determination of sialyltransferase activity with only nanomolar quantities of acceptor substrates and should be of particular value in cases in which a large variety of samples, including cell lysates, have to be screened for their enzymatic activities.
Plath
Christian
C
Institute for Chemistry, University of Lübeck, 23538 Lübeck, Germany.
Weimar
Thomas
T
Peters
Hannelore
H
Peters
Thomas
T
D015394:Molecular Structure; D011134:Polysaccharides / Q000378:metabolism; D012799:Sialyltransferases / Q000378:metabolism*; D019809:Streptavidin; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance / Q000379:methods*
D011134:Polysaccharides; D019809:Streptavidin; D012799:Sialyltransferases