Measurement of Aryl and Alcohol Sulfotransferase Activity

Jonathan J. Sheng1, Vyas Sharma1, Michael W. Duffel1

1 University of Iowa, Iowa City, Iowa
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.5
DOI:  10.1002/0471140856.tx0405s08
Online Posting Date:  August, 2001
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Two methods are presented for determining the catalytic activity of aryl and alcohol sulfotransferases. The first assay is a simple and rapid procedure that is based on the extraction of a paired ion formed between the product organic sulfate and methylene blue. The second method employs HPLC analysis of the substrate‚Äźdependent formation of PAP, an assay that is particularly useful when the sulfuric acid ester product is chemically unstable.

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Table of Contents

  • Basic Protocol 1: Methylene Blue Extraction Assay for Aryl or Alcohol Sulfotransferase Activity
  • Basic Protocol 2: HPLC Assay for Sulfotransferase Activity
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Methylene Blue Extraction Assay for Aryl or Alcohol Sulfotransferase Activity

  • Reaction buffer: 41.0 g/liter sodium acetate, adjusted to pH 5.5 with acetic acid
  • 0.1 M 2‐mercaptoethanol (2‐ME)
  • 4 mM 3′‐phosphoadenosine 5′‐phosphosulfate (PAPS; see recipe)
  • 10 mM 2‐naphthol (recrystallized from water) in acetone (ACS reagent grade)
  • 2.0 mM dehydroepiandrosterone (DHEA) in acetone (ACS reagent grade)
  • Enzyme sample of known protein concentration
  • Methylene blue reagent (see recipe)
  • Chloroform (ACS spectrophotometric grade)
  • Sodium sulfate (anhydrous)
  • 13 × 100–mm glass test tubes
  • Spectrophotometer and quartz cuvettes

Basic Protocol 2: HPLC Assay for Sulfotransferase Activity

  • HPLC mobile phase (see recipe)
  • 4.0 mM 3′‐phosphoadenosine 5′‐phosphosulfate(PAPS; see recipe) or 4.0 mM adenosine 3′,5′‐diphosphate (PAP; see recipe)
  • HPLC‐grade water
  • PAP standards (see recipe)
  • Reaction premix (see recipe)
  • Substrate solution (see recipe)
  • Solvent used for substrate solution
  • Enzyme sample of known protein concentration
  • HPLC‐grade methanol
  • C18 5‐µM reversed‐phase HPLC column (i.d. = 4.6 mm, length = 250 mm)
  • High‐performance liquid chromatograph (HPLC) with 20‐µl sample‐injection loop, uv/vis detector, and integrator
  • 6 × 50–mm glass test tubes
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Literature Cited

Literature Cited
   Beckmann, J.D. 1991. Continuous fluorometric assay of phenol sulfotransferase. Anal Biochem. 197:408‐411.
   Borchardt, R.T., Baranczyk‐Kuzma, A., and Pinnick, C.L. 1983. An ecteola‐cellulose chromatography assay for 3′‐phosphoadenosine‐5′‐phosphosulfate:phenol sulfotransferase. Anal. Biochem. 130:334‐338.
   Burkart, M.D. and Wong, C.H. 1999. A continuous assay for the spectrophotometric analysis of sulfotransferases using aryl sulfotransferase IV. Anal. Biochem. 274:131‐137.
   Duffel, M.W. 1997. Sulfotransferases. In Comprehensive Toxicology, Vol. 3: Biotransformation (F.P. Guengerich, ed.) pp. 365‐383. Elsevier Science Publishing, Oxford.
   Duffel, M.W. and Jakoby, W.B. 1981. On the mechanism of aryl sulfotransferase. J. Biol. Chem. 256:11123‐11127.
   Duffel, M.W. and Janss, M.N. 1986. Aryl sulfotransferase IV catalyzed sulfation of 1‐naphthalenemethanol. In Biological Reactive Intermediates III—Mechanisms of Action in Animal Models and Human Disease (J.J. Kocsis, D.J. Jollow, C.M. Witmer, J.O. Nelson, and R. Snyder, eds.) pp. 197:415‐422. Plenum, New York.
   Duffel, M.W., Binder, T.P., and Rao, S.I. 1989. Assay of purified aryl sulfotransferase suitable for reactions yielding unstable sulfuric acid esters. Anal. Biochem. 183:320‐324.
   Falany, C.N. 1997. Enzymology of human cytosolic sulfotransferases. FASEB J. 11:206‐216.
   Foldes, A. and Meek, J.L. 1973. Rat brain sulfotransferase: Partial purification and some properties. Biochim. Biophys. Acta 327:365‐374.
   Gregory, J.D. and Lipmann, F. 1957. The transfer of sulfate among phenolic compounds with 3′,5′‐diphosphoadenosine as coenzyme. J. Biol. Chem. 229:1081‐1090.
   Honkasalo, T. and Nissinen, E. 1988. Determination of phenol sulfotransferase activity by high‐performance liquid chromatography. J. Chromatogr. 424:136‐140.
   Jakoby, W.B., Sekura, R.D., Lyon, E.S., Marcus, C.J., and Wang, J.L. 1980. Sulfotransferases. In Enzymatic Basis of Detoxication, Vol. 2 (W.B. Jakoby, ed.) pp. 199‐228. Academic Press, New York.
   Mulder, G.J. and Jakoby, W.B. 1990. Sulfation. In Conjugation Reactions in Drug Metabolism (G.J. Mulder, ed.) pp. 107‐161. Taylor & Francis, New York.
   Nose, Y. and Lipmann, F. 1958. Separation of steroid sulfokinases. J. Biol. Chem. 233:1348‐1351.
   Ramaswamy, S.G. and Jakoby, W.B. 1987. Sulfotransferase assays. Methods Enzymol. 143:201‐207.
   Sekura, R.D. 1981. Adenosine 3′‐phosphate 5′‐phosphosulfate. Methods Enzymol. 77:413‐415.
   Sekura, R.D., Marcus, C.J., Lyon, E.S., and Jakoby, W.B. 1979. Assay of sulfotransferases. Anal. Biochem. 95:82‐86.
   Sekura, R.D., Duffel, M.W., and Jakoby, W.B. 1981. Aryl sulfotransferases. Methods Enzymol. 77:197‐206.
   Weinshilboum, R. and Otterness, D. 1994. Sulfotransferase enzymes. In Handbook of Experimental Pharmacology (F.C. Kauffman, ed.) pp. 112:45‐78. Springer‐Verlag, Berlin.
   Yamazoe, Y., Nagata, K., Ozawa, S., and Kato, R. 1994. Structural similarity and diversity of sulfotransferases. Chemico‐Biol. Interact. 92:107‐117.
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