TY - JOUR
T1 - Assaying sialyltransferase activity with surface plasmon resonance
AU - Plath, Christian
AU - Weimar, Thomas
AU - Peters, Hannelore
AU - Peters, Thomas
N1 - Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2006/8
Y1 - 2006/8
N2 - 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 sialyl-transferase 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 enzymotic 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 sialyl-transferase 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.
AB - 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 sialyl-transferase 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 enzymotic 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 sialyl-transferase 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.
UR - http://www.scopus.com/inward/record.url?scp=33747191138&partnerID=8YFLogxK
U2 - 10.1002/cbic.200600012
DO - 10.1002/cbic.200600012
M3 - Journal articles
C2 - 16847845
AN - SCOPUS:33747191138
VL - 7
SP - 1226
EP - 1230
JO - Chembiochem : a European journal of chemical biology
JF - Chembiochem : a European journal of chemical biology
SN - 1439-4227
IS - 8
ER -