Recombinant F. heparinum Heparinase I Protein, CF

Heparinase I digestion of Heparin Sulfate (200 μg) is assessed in a 5-minute kinetic assay atroom temperature by monitoring absorbance at 232 nm. R&D Systems Recombinant F.heparinum Heparinase I (Catalog #6145-GH) (orange) exhibits greater activity than the Bacteroides Heparinase I from the competition (green).

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

Recombinant F. heparinum Heparinase I Protein, CF Summary

• Details of Functionality: Measured by its ability to liberate oligosaccharides from heparin. The specific activity is >24,500 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived f. heparinum Heparinase I protein
  Gln22-Arg384, with an N-terminal Met and 6-His tag
• Accession #: Q05819
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 42 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: 40-42 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and DTT.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 100 mM NaCl, 2 mM CaCl₂, pH 7.5
  • Recombinant F. heparinum Heparinase I (rFhHeparinase I) (Catalog # 7897-GH)
  • Substrate: Heparin (Tocris, Catalog # 2812), 20 mg/mL stock in deionized water
  • 96-well Clear UV Plate (Costar, Catalog # 3635)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rFhHeparinase I to 0.5 µg/mL in Assay Buffer.
  2. Dilute Substrate to 0.75 mg/mL in Assay Buffer.
  3. Load 100 µL of diluted rFhHeparinase I into a UV plate, and start the reaction by adding 200 µL of 0.75 mg/mL Substrate. Include a Substrate Blank containing 100 µL of Assay Buffer and 200 µL of 0.75 mg/mL Substrate.
  4. Read plate in kinetic mode for 5 minutes at an absorbance of 232 nm.
  5. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x well volume (L) x 10^12 pmol/mol) / (ext. coeff** (M^-1cm^-1) x path corr.*** (cm) x amount of enzyme (µg))

  
  *Adjusted for Substrate Blank
  **Using the extinction coefficient 3800 M^-1cm^-1
  ***Using the path correction 0.92 cm
  Note: the output of many spectrophotometers is in mOD Per Reaction:
  • rFhHeparinase I: 0.05 µg
  • Substrate: 0.5 mg/mL

Notes

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

Alternate Names for Recombinant F. heparinum Heparinase I Protein, CF

• Heparinase I

Background

Heparin and heparan sulfate are sulfated glycosaminoglycans that share basic carbohydrate backbone structure with alternating uronic acid and N-acetylglucosamine residues (1, 2). Heparin is found in mast cells and has strong anticoagulation properties. Heparan sulfate is found on cell membrane and extracellular matrix and is involved in various biological events from cell growth, adhesion and migration to lipid metabolism. Heparin has a much higher degree of sulfation than heparan sulfate, which can be considered as a polysaccharide with regions similar to heparin interspaced with much less sulfated regions. Both heparin and heparan sulfate can be digested by heparinases, a group of bacterial lyases that are widely used as tools for processing and analyze these polysaccharides. Heparinases degrade heparin and heparan sulfate glycosaminoglycans through an eliminative mechanism (3). Heparinase I from Flavobacterium heparinum is highly active on heparin and has no activity against chondroitin sulfate and keratan sulfate (4). The enzyme readily releases highly sulfated oligosaccharides from heparin (5).

1. MacArthur, J. M. et al. (2007) J. Clin. Invest. 117:153.
2. Esko, J. D. and Selleck, S. B. (2002) Annu. Rev. Biochem. 71:435.
3. Linhardt, R. J. et al. (1986) Appl. Biochem. Biotech. 12:135.
4. Wu, Z.L. et al. (2011) Glycobiology 21:625.
5. Ernst, S. et al. (1996) Biochem. J. 315:589.

Publications for Heparinase I (7897-GH)(4)

Publications using 7897-GH

• Volpi, N;Galeotti, F;Gatto, F; High-throughput glycosaminoglycan extraction and UHPLC-MS/MS quantification in human biofluids Nature protocols 2024-11-14 [PMID: 39543382] (Bioassay, Human)
• Li, W;Plante, JA;Lin, C;Basu, H;Plung, JS;Fan, X;Boeckers, JM;Oros, J;Buck, TK;Anekal, PV;Hanson, WA;Varnum, H;Wells, A;Mann, CJ;Tjang, LV;Yang, P;Reyna, RA;Mitchell, BM;Shinde, DP;Walker, JL;Choi, SY;Brusic, V;Llopis, PM;Weaver, SC;Umemori, H;Chiu, IM;Plante, KS;Abraham, J; Shifts in receptors during submergence of an encephalitic arbovirus Nature 2024-07-24 [PMID: 39048821] (Bioassay, Human)
• Y Yu, L Fu, P He, K Xia, S Varghese, J Dordick, H Wang, F Zhang, RJ Linhardt Enzymatic synthesis of low molecular weight heparins from N-sulfo heparosan depolymerized by heparanase or heparin lyase Carbohydrate polymers, 2022-07-05;295(0):119825. 2022-07-05 [PMID: 35988993] (Enzyme Activity, N/A)
• M Puray-Chav, KM LaPak, TP Schrank, JL Elliott, DP Bhatt, MJ Agajanian, R Jasuja, DQ Lawson, K Davis, PW Rothlauf, Z Liu, H Jo, N Lee, K Tenneti, JE Eschbach, C Shema Mugi, EM Cousins, EW Cloer, HR Vuong, LA VanBlargan, AL Bailey, P Gilchuk, JE Crowe, MS Diamond, DN Hayes, SPJ Whelan, A Horani, SL Brody, D Goldfarb, MB Major, SB Kutluay Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell Cell Reports, 2021-06-23;0(0):109364. 2021-06-23 [PMID: 34214467] (Bioassay, Human)

Bioinformatics

• Uniprot:
  • Q05819()