Sulfotransferase and glycosyltransferase analyses using a 96-well filtration plate

Lora V. Hooper, Jacques U. Baenziger

Research output: Contribution to journalArticlepeer-review

12 Scopus citations


Previously, we identified and characterized a sulfotransferase responsible for the 4-O-sulfation of Asnlinked oligosaccharides terminating with the sequence GalNAcβ1,4GlcNAcβ1,2Manα (GGnM). Here we present a rapid and sensitive method to assay for this sulfotransferase which is significantly more convenient than existing procedures. The acceptor substrate in this assay is human transferrin (Tfn) which has been enzymatically modified such that its N-linked oligosaccharides bear the terminal sequence GGnM. GGnM-Tfn is biotinylated, and a fluid-phase transferase reaction is done which utilizes [35S]3′-phosphoadenosine 5′-phosphosulfate ([35S]PAPS) as the sulfate donor. The reaction product [35S]SGGnM-Tfn-biotin is separated from unreacted [35S]PAPS and radiolabeled endogenous acceptors by capture onto avidin immobilized onto a PVD F-based 96-well filtration plate. Sulfate incorporation in this assay is linear with respect both to the donor and to the acceptor substrates and is also dependent upon enzyme input and time. With this assay, we can detect as little as 0.1 pmol of sulfate transfer. In addition, by using asialo-Tfn-biotin as an acceptor and [3H]CMP-sialic acid as a donor substrate, we demonstrate that this assay can be modified for use with the α2,6-sialyltransferase.

Original languageEnglish
Pages (from-to)128-133
Number of pages6
JournalAnalytical Biochemistry
Issue number1
StatePublished - Jul 1993


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