Sulfotransferase Activity Assays

Introduction

Sulfation is an important post-translational modification that occurs on glycans, proteins, steroids, hormones, neurotransmitters, and xenobiotics. It can result in changes in their chemical, physical, and biological properties such as solubility, activity, and affinity. In humans, there are 48 sulfotransferases. More than half of them are Golgi resident enzymes involved in sulfation on carbohydrates. R&D Systems currently offers several recombinant carbohydrate-specific sulfotransferases (CHST; Table 1). Activities of these recombinant sulfotransferases have been tested on various oligosaccharides, polysaccharides, and proteoglycans.

Warnings and Precautions

These assays involve the use of radioactive material. Use appropriate precautions.

Materials

Note: The assay buffers, enzyme dilutions, and substrates vary depending on the enzyme that is being assayed. Consult the specific assay protocol (Steps 1 and 2) for the assay buffer and enzyme and substrate dilutions that should be used in each assay.

Assay Buffers

• Assay Buffer 1: 25 mM MES, 0.5% (w/v) Triton® X-100, 2.5 mM MgCl₂, 2.5 mM MnCl₂, 1.25 mM CaCl₂, 0.75 mg/mL BSA, pH 7.0
• Assay Buffer 2: 25 mM MES, 0.5% (w/v) Triton X-100, 2.5 mM MgCl₂, 2.5 mM MnCl₂, 1.25 mM CaCl₂, 0.75 mg/mL BSA, 0.3 mg/mL Protamine (Sigma, Catalog # P4005), pH 7.0
• Assay Buffer 3: 0.1 M Sodium Acetate, pH 5.0
• Gel Running Buffer: 40 mM Tris, 40 mM Acetic Acid, 1 mM EDTA, pH 8.0
• Recombinant Enzyme (See table below)
• Substrate (See table below)
• Adenosine 3’-phosphate 5’-phosphosulfate lithium salt hydrate (PAPS) (Sigma, Catalog # A5508), 1 mM stock solution in 5% ethanol, 95% deionized water
• 35S-PAPS, ~0.1 mM, 1 mCi/mL, prepared in-house (1-3)
• 2X or 5X SDS gel loading buffer
• 8% SDS-PAGE (approximately 15 cm x 20 cm, 20 lanes per gel)
• Blotting paper
• Gel dryer
• Glogos® II autorad markers (Stratagene, Catalog # 420202) or equivalent
• Blue sensitive medical X-ray film
• X-ray film cassette
• Film developer (Konica SRX-101A Medical Film Processor) or equivalent
• Liquid scintillation counter (Beckman Coulter, Model # LS5000TD) or equivalent (Note: Sensitivity of the scintillation counter may affect linearity when measuring low levels of activity)
• Liquid scintillation fluid (Beckman Coulter, Catalog # 141349)

Assay Protocols

References

1. Robbins, P.W. (1962) Methods in Enzymology, Vol. V, New York: Academic Press, Inc., p. 964.
2. MacRae, I.J. et al. (2000) Biochemistry 39:1613.
3. Wu, Z.L. et al. (2002) Faseb J. 16:539.

Triton is a registered trademark of Union Carbide Corp.
Glogos is a registered trademark of Stratagene.

Protocol Category

Enzyme Assays