Sulfotransferase and Glycosyltransferase Analyses Using a 96-Well Filtration Plate

L. V. Hooper, J. U. Baenziger

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

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 (US)
Pages (from-to)128-133
Number of pages6
JournalAnalytical Biochemistry
Volume212
Issue number1
DOIs
StatePublished - Jul 1993

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Sulfotransferases
Glycosyltransferases
Transferrin
Assays
Sulfates
Biotin
Oligosaccharides
Cytidine Monophosphate N-Acetylneuraminic Acid
Phosphoadenosine Phosphosulfate
Sialyltransferases
Substrates
Avidin
Transferases
Physical vapor deposition
Reaction products
Enzymes
Fluids

ASJC Scopus subject areas

  • Molecular Biology
  • Biophysics
  • Biochemistry

Cite this

Sulfotransferase and Glycosyltransferase Analyses Using a 96-Well Filtration Plate. / Hooper, L. V.; Baenziger, J. U.

In: Analytical Biochemistry, Vol. 212, No. 1, 07.1993, p. 128-133.

Research output: Contribution to journalArticle

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