Recombinant P. heparinus Heparinase II Protein, CF

Heparinase II digestion of Heparin Sulfate (200 μg)is assessed in a 5-minute kinetic assay by monitoring absorbance at 232 nm. R&D Systems P. heparinus Heparinase I (catalog #6336-GH) exhibits activity at 2595.6 pm/min/μg.

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

Recombinant P. heparinus Heparinase II Protein, CF Summary

• Details of Functionality: Measured by its ability to liberate oligosaccharides from heparin. The specific activity is >750 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived p. heparinus Heparinase II protein
  Ala26-Arg772, with an N-terminal Met and 6-His tag
• Accession #: YP_003092674
• 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: 86 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: 66-75 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 and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Assay Procedure:
  • Assay Buffer: 100 mM Tris, pH 7.5
  • Recombinant P. heparinus Heparinase II (rPhHeparinase II) (Catalog # 6336-GH)
  • Substrate: Heparin (Tocris, Catalog # 2812), 20 mg/mL stock in deionized water
  • 96 well clear UV-transparent microplate (Corning, Catalog # 3635)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rPhHeparinase II to 20 ng/µL in Assay Buffer.
  2. Dilute Substrate to 3.0 mg/mL in Assay Buffer.
  3. Load into a plate 50 µL of the diluted rPhHeparinase II, and start the reaction by adding 50 µL of 3.0 mg/mL Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 3.0 mg/mL Substrate.
  4. Read 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 0.32 cm
  Note: the output of many spectrophotometers is in mOD Per Well:
  • rPhHeparinase II: 1.0 µg
  • Substrate: 1.5 mg/mL

Notes

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

Alternate Names for Recombinant P. heparinus Heparinase II Protein, CF

• Heparinase II

Background

Heparan sulfate is a sulfated glycosaminoglycan with the repeating disaccharide units of ‑4HexA1,4GlcNAc beta 1-. It is usually attached to the protein cores of proteoglycans found on cell membrane and extracellular matrix, where it binds to a variety of protein ligands and regulates a wide range of biological activities, including developmental processes, angiogenesis, blood coagulation and tumor metastasis (1, 2). Heparan sulfate has a domain structure containing sulfated regions interspaced with less or non-sulfated regions (3, 4). Heparin shares the backbone structure with heparan sulfate but contains no non-sulfated regions. Heparinases are a family of lyases that release unsaturated oligosaccharides from heparin and heparan sulfate upon digestion (5). Heparinase I recognizes highly sulfated regions and is more specific for heparin. Heparinase II digests both heparin and heparan sulfate. Heparinase III prefers less-sulfated regions and is active only on heparan sulfate (6, 7).

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. Maccarana, M. et al. (1996) J. Biol. Chem. 271:17804.
4. Linker, A. and Hovingh, P. (1975) Biochim. Biophys. Acta. 385:324.
5. Linker, A. and Hovingh, P. (1965) J. Biol. Chem. 240:3724.
6. Su, H. et al. (1996) Appl. Environ. Microbiol. 62:2723.
7. Hovingh, P. and Linker, A. (1970) J. Biol. Chem. 245:6170.

Publications for Heparinase II (6336-GH)(10)

Publications using 6336-GH

• Yamashita, R;Tsutsui, S;Mizumoto, S;Watanabe, T;Yamamoto, N;Nakano, K;Yamada, S;Okamura, T;Furuichi, T; CANT1 Is Involved in Collagen Fibrogenesis in Tendons by Regulating the Synthesis of Dermatan/Chondroitin Sulfate Attached to the Decorin Core Protein International journal of molecular sciences 2025-03-10 [PMID: 40141107] (Bioassay, Mouse)
• 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)
• Beddows, CA;Shi, F;Horton, AL;Dalal, S;Zhang, P;Ling, CC;Yong, VW;Loh, K;Cho, E;Karagiannis, C;Rose, AJ;Montgomery, MK;Gregorevic, P;Watt, MJ;Packer, NH;Parker, BL;Brown, RM;Moh, ESX;Dodd, GT; Pathogenic hypothalamic extracellular matrix promotes metabolic disease Nature 2024-09-18 [PMID: 39294371] (Bioassay, Mouse)
• Ota, H;Sato, H;Mizumoto, S;Wakai, K;Yoneda, K;Yamamoto, K;Nakanishi, H;Ikeda, JI;Sakamoto, S;Ichikawa, T;Yamada, S;Takahashi, S;Ikehara, Y;Nishihara, S; Switching mechanism from AR to EGFR signaling via 3-O-sulfated heparan sulfate in castration-resistant prostate cancer Scientific reports 2023-07-18 [PMID: 37463954] (Bioassay, Human)
• S Saito, S Mizumoto, T Yonekura, R Yamashita, K Nakano, T Okubo, S Yamada, T Okamura, T Furuichi Mice lacking nucleotide sugar transporter SLC35A3 exhibit lethal chondrodysplasia with vertebral anomalies and impaired glycosaminoglycan biosynthesis PLoS ONE, 2023;18(4):e0284292. 2023 [PMID: 37053259] (Bioassay, Mouse)
• S Saito, S Mizumoto, T Yonekura, R Yamashita, K Nakano, T Okubo, S Yamada, T Okamura, T Furuichi Mice lacking nucleotide sugar transporter SLC35A3 exhibit lethal chondrodysplasia with vertebral anomalies and impaired glycosaminoglycan biosynthesis PLoS ONE, 2023-04-13;18(4):e0284292. 2023-04-13 [PMID: 37053259] (Bioassay, Mouse)
• YH Chu, WC Liao, YJ Ho, CH Huang, TJ Tseng, CH Liu Targeting Chondroitin Sulfate Reduces Invasiveness of Glioma Cells by Suppressing CD44 and Integrin beta1 Expression Cells, 2021-12-20;10(12):. 2021-12-20 [PMID: 34944101] (Bioassay, Human)
• 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)
• CH Liu, BR Wu, YJ Ho, YH Chu, WC Hsu, TJ Tseng, JP Li, WC Liao CHPF Regulates the Aggressive Phenotypes of Hepatocellular Carcinoma Cells via the Modulation of the Decorin and TGF-&amp;beta Pathways Cancers, 2021-03-12;13(6):. 2021-03-12 [PMID: 33809195] (Bioassay, Human)
• 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

• Uniprot:
  • YP_003092674()