Recombinant Human Heparan Sulfate-6-O-Sulfotransferase-1, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human Heparan Sulfate-6-O-Sulfotransferase-1, CF Summary

• Details of Functionality: Measured by its ability to transfer sulfate from PAPS to heparan sulfate. The specific activity is >45 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human Heparan Sulfate 6-O-Sulfotransferase 1/HS6ST1 protein
  Pro28-Trp401, with an N-terminal 6-His tag
• Accession #: AAY14736
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Enzymes
• Gene: HS6ST1
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 45 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 56-60 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and EDTA.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 15 mM MgCl₂, pH 7.5
  • Recombinant Human Heparan Sulfate 6‑O‑Sulfotransferase 1/HS6ST1 (rhHS6ST1) (Catalog # 5057-ST)
  • Donor Substrate: 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) (Catalog # ES019)
  • Acceptor Substrate: Heparan Sulfate (Celsus Labs, Catalog # HO3105), 50 mg/mL stock in deionized water
  • Sulfotransferase Activity Kit (Catalog # EA003)
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Create Assay Buffer by Diluting 10X Phosphatase Buffer 3 (supplied in kit) to 1X in deionized water.
  2. Dilute 1 mM Phosphate Standard by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock.
  3. Continue standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
  4. Prepare a reaction mixture containing 0.4 mM PAPS, 4 mg/mL Heparan Sulfate, and 20 ug/mL Coupling Phosphatase 3 in Assay Buffer. *Note: Incorporate Coupling Phoshphatase 3 into reaction mixture right before the mixture is added to the plate in order to avoid unnecessary hydrolysis of PAPS.
  5. Dilute rhHS6ST1 to 20 µg/mL in Assay Buffer.
  6. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  7. Load 25 µL of the 40 µg/mL rhHS6ST1 into the plate. Include a control containing 25 µL of Assay Buffer.
  8. Add 25 µL of reaction mixture to the wells, excluding the standard curve.
  9. Seal plate and incubate at 37 °C for 20 minutes.
  10. Add 30 µL of the Malachite Green Reagent A to all wells.
  11. Add 100 µL of deionized water to all wells. Mix briefly.
  12. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  13. Read plate at 620 nm (absorbance) in endpoint mode.
  14. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Phosphate released* (nmol) x (1000 pmol/nmol)) / (Incubation time (min) x amount of enzyme (µg))

  *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.

  Per Reaction:
  • rhHS6ST1: 0.5 µg
  • Coupling Phosphatase 3: 0.5 µg
  • Heparan Sulfate: 100 ug (2 mg/mL)
  • PAPS: 0.2 mM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Heparan Sulfate-6-O-Sulfotransferase-1, CF

• DKFZp547H098
• EC 2.8.2
• EC 2.8.2.-
• FLJ25392
• heparan sulfate 6-O-sulfotransferase 1
• heparan-sulfate 6-sulfotransferase
• HS6ST1
• HS6ST-1
• HS6STheparan-sulfate 6-O-sulfotransferase 1
• MGC116899
• MGC116901

Background

Heparan Sulfate is a highly sulfated polysaccharide that can be found on cell surface and extracellular matrix. It is usually covalently attached to a protein core as the glycan component of a proteoglycan. Heparan Sulfate interacts with a variety of proteins, such as growth factors, protease inhibitors, cytokines, lipoprotein lipase and viral envelope proteins, thus plays roles from cell growth, cell differentiation, cell motility, blood coagulation, lipid metabolism to viral infection (1, 2). Heparan Sulfate consists of repeating residues of uronic acid and N‑acetylglucosamine. The uronic acid residues can be sulfated at 2-O position by Heparan Sulfate 2‑O‑Sulfotransferase (HS2ST). The N‑acetylglucosamine residues can be sulfated at N-, 3-O, and 6-O positions by N‑deacetylase/N‑sulfotransferases (NDSTs), Heparan Sulfate 3-O and 6-O sulfotransferases (HS3STs and HS6STs) respectively. However, the reactions catalyzed by these sulfotransferases are normally incomplete on the whole chain of Heparan Sulfate. As a result, Heparan Sulfate displays enormous sequence diversity that allows it to interact with a wide spectrum of proteins differently. Among three HS6STs, HS6ST1 is the first to be cloned (3). Mice deficient of the HS6ST1 homologue gene showed embryonic lethality (4).The enzymatic activity of the recombinant human HS6ST1 is measured using a phosphatase-coupled assay (5).

1. Bernfield, M. et al. (1999) Annu. Rev. Biochem. 68:729.
2. Esko, J. D. and Selleck, S. B. (2002) Annu. Rev. Biochem. 71:435.
3. Habuchi, H et al. (1998) J. Biol. Chem. 273:9208.
4. Habuchi, H et al. (2007) J. Biol. Chem. 282:15578.
5. Prather, B. et al. (2012) Anal. Biochem. 423:86.

Publications for Heparan Sulfate 6-O-Sulfotransferase 1/HS6ST1 (5057-ST)(2)

Publications using 5057-ST

• Qin Y, Ke J, Gu X, Fang J, Wang W, Cong Q, Li J, Tan J, Brunzelle J, Zhang C, Jiang Y, Melcher K, Li J, Xu H, Ding K Structural and functional study of D-glucuronyl C5-epimerase. J Biol Chem, 2015-01-07;290(8):4620-30. 2015-01-07 [PMID: 25568314] (Bioassay, Zebrafish)
• Wu ZL, Prather B, Ethen CM Detection of specific glycosaminoglycans and glycan epitopes by in vitro sulfation using recombinant sulfotransferases. Glycobiology, 2010-12-17;21(5):625-33. 2010-12-17 [PMID: 21169395] (Enzyme Assay, Bovine, Porcine)

Bioinformatics

• Gene Symbol: HS6ST1
• Uniprot:
  • AAY14736()