Flavobacterium Heparinase II

Heparinases II

Heparinase II, also known as Heparin Lyase II or Heparitinase II is catalytically active on both heparin and sulfated domains of heparan sulfate in an endolytic manner. It cleaves less selectively, via an elimination mechanism, sulfated polysaccharide chains containing 1-4 linkages between hexosamines and uronic acid residues (both iduronic and glucuronic acid residues) on the nonreducing end. The reaction yields oligosaccharide products (mainly disaccharides) containing unsaturated uronic acids which can be detected by UV spectroscopy at 232 nm. Complete depolymerization of polysaccharide chains is improved when Heparinase II is used in combination with Heparinase I and III for the disaccharide analysis.

The enzyme cleaves both heparin and heparan sulfate, with the heparan sulfate activity being 2-fold higher than the heparin activity.

Recombinant enzyme homologous to its native counterpart and expressed in the natural host, Pedobacter heparinus (formerly, Flavobacterium heparinum).

Heparinase II is O-glycosylated at Thr-134
Purity: ≥ 90 % by reversed phase HPLC analysis
IBEX liquid heparinase II has optimal activity and stability for up to 2.5 years when stored frozen at -70°C
IBEX lyophilized heparinase II has optimal activity and stability for up to two years when stored at 2-8^oC
Calcium is inhibitory

HEPARINASE I

HEPARINASE III

HEPARINASE I for DIAGNOSTICS

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APPLICATIONS

REFERENCES

Data Sheet - LIQUID


	DATA SHEET	Revised May 2019, R.06
Heparinase II	Research Grade	PN 50-011 50-011-001
Synonyms	Heparitinase
Source	Flavobacterium heparinum (recombinant)
EC Number	None assigned
CAS Number	149371-12-0
Catalyzed Reaction	The enzyme cleaves, via an elimination mechanism, sulfated polysaccharide chains containing 1-4 linkages between hexosamines and uronic acid residues (both iduronic and glucuronic acid residues). The reaction yields oligosaccharide products (mainly disaccharides) containing unsaturated uronic acids which can be detected by UV spectroscopy at 232 nm. The enzyme cleaves both heparin and heparan sulfate, with the heparan sulfate activity being about twice as high as the heparin activity.
Substrate Specificity	Heparin, heparan sulfate.
Properties	• Molecular weight: 85,765 Da • Isoelectric point: 9.1 – 9.2 • pH optimum for activity: 7 - 8 • pH range for activity: 5 – 9 • Optimal testing temperature range: 20 ºC – 37 ºC • Optimal storage temperature: – 70 ºC
Purity	≥90 % by reversed phase HPLC analysis.

Specific Activity	≥ 5 IU/mg – using heparan sulfate as substrate. One international unit (IU) is defined as the amount of enzyme that will liberate 1.0 µmole unsaturated oligosaccharides from heparin or heparan sulfate per minute at 30 ºC and pH 7.5
Stability	• PN 50-011 (vial of 0.5 IU): Expiration is 30 months from manufacturing date frozen at -70 ºC in aqueous buffer containing Sodium Chloride, Sodium Phosphate and Sucrose 5%. • PN 50-011-001 (vial of 0.1 IU): Expiration is 30 months from manufacturing date frozen at -70 ºC in aqueous buffer containing Sodium Chloride, Sodium Phosphate and Sucrose 5%.
Applications	• As research reagent (glycosaminoglycan degradation). • For the preparation of di- and oligo-saccharides of heparin and heparan sulfate and the preparation of oligosaccharide libraries.
Availability	A proprietary expression system for F. heparinum and the fermentation and isolation processes developed by IBEX Pharmaceuticals allow the production of large quantities of high purity product.
References	• Review: “Enzymatic Degradation of Glycosaminoglycans”. S. Ernst et al. in Critical Reviews in Biochemistry and Molecular Biology (1995), 30(5): 387-444. • “Purification and Characterization of Heparin Lyases from Flavobacterium heparinum”. D.L. Lohse and R.J. Linhardt in J. Biol. Chem. (1992) 267: 24347-24355. • “Substrate Specificity of the Heparin Lyases from Flavobacterium heparinum”. U.R.Desai, H.Wang and R.J. Linhardt in Archives of Biochemistry and Biophysics (1993) 306(2): 461-468. • “Heparinase-II-Catalyzed Degradation of N-Propionylated Heparin”. C.F. Moffat, W.F. Long, M.W. McLean and F.B. Williamson in Archives of Biochemistry and Biophysics (1997) 338 (2): 201-206. • “Isolation and Expression in Escherichia coli of hepB and hepC, Genes Coding for the Glycosaminoglycan-Degrading Enzymes Heparinase II and Heparinase III, Respectively, from Flavobacterium heparinum”. HongSheng Su, Françoise Blain, Roy A. Musil, Joseph J.F. Zimmermann, KangFu Gu and D. Clark Bennett, in Applied and Environmental Microbiology, (1996): 2723-2734. • US Patents 5,681,733 and 5,919,693 “Nucleic Acid sequences and Expression Systems for Heparinase II and Heparinase III derived from Flavobacterium heparinum”.

Safety Data Sheets - LIQUID


Safety Data Sheet - Heparinase II - Liquid
SECTION 1 – IDENTIFICATION
Release Date	May 2019 R. 01
Product Identity	Heparinase II, recombinant in sucrose, Research Grade
Part Number	50-011
Manufacturer	IBEX Pharmaceuticals Inc. 5485 Rue Paré, Suite 100 Montréal, Québec, H4P 1P7 Canada Tel : (514) 344-4004 Fax : (514) 344-8827
Product use	Research

SECTION 2 – HAZARD IDENTIFICATION
Emergency overview	Solution in water. Will not support burning. Not explosive.
Potential Health Effects
Skin	May cause skin irritation
Eyes	May cause irritation or burning sensation
Inhalation	May cause irritation to the respiratory tract
Swallowed	Not known to cause toxic effects
Injection	Not known to cause toxicity with acute exposure, but may produce toxicity with repeated injections.
Carcinogenicity	Not determined
SECTION 3 – COMPOSITION/INFORMATION ON INGREDIENTS
CAS No.	149371-12-0
EC No.	None assigned
Systematic name	Heparin lyase II
Synonyms	Heparinase II
Origin	Recombinant, non-pathogenic microorganism
Ingredients	No components need to be disclosed according to the applicable regulations.
SECTION 4 – FIRST AID MEASURES
Skin contact	Flush with copious amounts of water.
Eye contact	Rinse opened eye for several minutes under running water. Then consult a doctor.
Inhalation	Supply the victim with fresh air; consult doctor in case of complaints.
Swallowing	Rinse mouth with plenty of water.
On all of the above	Consult a doctor if symptoms develop.
SECTION 5 – FIRE FIGHTING MEASURES
Suitable extinguishing agents	Noncombustible.
Protective equipment	No special measures required. Prevent contact with skin and eyes
SECTION 6 – ACCIDENTAL RELEASE MEASURES
Person-related safety precautions	Prevent contact with skin and eyes. Ventilate area.
Measures for environmental protection	No special measures required.
Measures for cleaning/collecting	Spilled material should be carefully wiped up and disposed as biomedical waste as per applicable waste disposal regulations. Wash spill site after material pickup is complete.
SECTION 7 – HANDLING AND STORAGE
Handling
Information for safe handling	Prevent contact with skin and eyes and prevent inhalation and swallowing. No other special precautions are necessary if used correctly.
Information about protection against explosions and fires	No special measures required.
Storage
Requirements to be met by storerooms and receptacles	No special requirements except that product should be stored frozen (preferably at -70 °C).
Information about storage in one common storage facilit	Not required.
Further information about storage conditions	None
Storage class	None assigned
Class according to regulation on flammable liquids	Void
SECTION 8 – EXPOSURE CONTROLS / PERSONAL PROTECTION
General protective and hygienic measures	The usual precautionary measures for handling chemicals and potentially biohazardous materials should be followed. Use clothing sufficient to avoid skin contact. Safety shower and eye wash should be available in proximity.
Protection of hands	Use synthetic water resistant gloves.
Eye protection	Use safety glasses.
SECTION 9 – PHYSICAL AND CHEMICAL PROPERTIES
Physical form	Frozen, colorless, odorless aqueous solution. (Heparinase II is a protein)
Melting point	0 °C (melting point of aqueous solution)
Boiling point	Not applicable
Flash point	Not applicable
Flammability	Product is not flammable
Danger of explosion	Not determined
Solubility	Heparinase II and ingredients are water soluble
Organic solvents	0 %
Solids content	0 %
SECTION 10 – STABILITY AND REACTIVITY
Thermal decomposition / conditions to be avoided	No decomposition if used according to specifications.
Dangerous reactions	No dangerous reactions known.
Dangerous products of decomposition	No dangerous decomposition products known.
SECTION 11 – TOXICOLOGICAL INFORMATION
Effects of Acute Exposure	Low acute toxicity expected following inhalation, ingestion, injection or skin exposure. The potential eye irritation effects of Heparinase II are unknown and it should be considered to be potentially irritating to the eyes.
Effects of Chronic Exposure	Not determined for oral ingestion or skin exposure
Development Toxicity	Not determined
Reproductive Toxicity	Not determined
Carcinogenicity	Not determined
SECTION 12 – ECOLOGICAL INFORMATION
Toxicity	No data available
Persistence and degradability	No data available
Bioaccumulative potential	No data available
Mobility in soil	No data available
PBT and vPvB assessment	No data available
Other adverse effects	No data available
SECTION 13 – DISPOSAL INFORMATION
Waste Disposal	Incinerate or bury as a solid in a licensed facility. Do not discharge into waterways or sewer systems without proper authority.
Container Disposal	Dispose of in a licensed facility. Recommend crushing or other means to prevent unauthorized reuse.
SECTION 14 – TRANSPORT INFORMATION
DOT (US)	Not dangerous goods
IMDG	Not dangerous goods
IATA	Not dangerous goods
SECTION 15 – REGULATORY INFORMATION
Not a dangerous substance or mixture according to the Globally Harmonised System (GHS).
SECTION 16 – OTHER INFORMATION
This information is given without any warranty or representation. It is believed to be correct but does not claim to be all inclusive and shall be used only as a guide. IBEX Pharmaceuticals Inc. shall not be held liable for any damage resulting from handling or contact with the above product. It is offered solely for your consideration, investigation and verification.

Data Sheet - LYOPHILIZED


	DATA SHEET	Revised May 2019, R.01
Heparinase II Lyophilized	Research Grade	PN 60-018 60-019
Synonyms	Heparin Lyase II
Source	Flavobacterium heparinum (recombinant)
EC Number	None assigned
CAS Number	149371-12-0
Catalyzed Reaction	The enzyme cleaves, via an elimination mechanism, sulfated polysaccharide chains containing 1-4 linkages between hexosamines and uronic acid residues (both iduronic and glucuronic acid residues). The reaction yields oligosaccharide products (mainly disaccharides) containing unsaturated uronic acids which can be detected by UV spectroscopy at 232 nm. The enzyme cleaves both heparin and heparan sulfate, with the heparan sulfate activity being about twice as high as the heparin activity.
Substrate Specificity	Heparin, heparan sulfate.
Properties	• Lyophilized powder • Molecular weight: 85,765 Da • Isoelectric point: 9.1 – 9.2 • pH optimum for activity: 7 - 8 • pH range for activity: 5 – 9 • Optimal testing temperature range: 20 ºC – 37 ºC • Optimal storage temperature: 5 +/-3ºC
Purity	≥90 % by reversed phase HPLC analysis, made from Heparinase II (PN 50-004).
Enzymatic Activity	One international unit (IU) is defined as the amount of enzyme that will liberate 1.0 µmole unsaturated oligosaccharides from heparin or heparan sulfate per minute at 30 ºC.
Reconstitution	Add 250 µL of water to reconstitute to its original formulation.
Stability	Expiration is 24 months from manufacturing date when stored at 5 +/- 3ºC.

Applications	• As research reagent (glycosaminoglycan degradation). • For the preparation of di- and oligo-saccharides of heparin and heparan sulfate and the preparation of oligosaccharide libraries.
Availability	A proprietary expression system for F. heparinum and the fermentation and isolation processes developed by IBEX Pharmaceuticals allow the production of large quantities of high purity product.
References	• Review: “Enzymatic Degradation of Glycosaminoglycans”. S. Ernst et al. in Critical Reviews in Biochemistry and Molecular Biology (1995), 30(5): 387-444. • “Purification and Characterization of Heparin Lyases from Flavobacterium heparinum”. D.L. Lohse and R.J. Linhardt in J. Biol. Chem. (1992) 267: 24347-24355. • “Substrate Specificity of the Heparin Lyases from Flavobacterium heparinum”. U.R.Desai, H.Wang and R.J. Linhardt in Archives of Biochemistry and Biophysics (1993) 306(2): 461-468. • “Heparinase-II-Catalyzed Degradation of N-Propionylated Heparin”. C.F. Moffat, W.F. Long, M.W. McLean and F.B. Williamson in Archives of Biochemistry and Biophysics (1997) 338 (2): 201-206. • “Isolation and Expression in Escherichia coli of hepB and hepC, Genes Coding for the Glycosaminoglycan-Degrading Enzymes Heparinase II and Heparinase III, Respectively, from Flavobacterium heparinum”. HongSheng Su, Françoise Blain, Roy A. Musil, Joseph J.F. Zimmermann, KangFu Gu and D. Clark Bennett, in Applied and Environmental Microbiology, (1996): 2723-2734. • US Patents 5,681,733 and 5,919,693 “Nucleic Acid sequences and Expression Systems for Heparinase II and Heparinase III derived from Flavobacterium heparinum”.

Safety Data Sheet - Lyophilized


Safety Data Sheet - Heparinase II - Lyophilized
SECTION 1 – IDENTIFICATION
Release Date	February 28, 2018
Product Identity	Heparinase II, Lyophilized
Part Number	60-018, 60-019
Manufacturer	IBEX Pharmaceuticals Inc. 5485 Rue Paré, Suite 100 Montréal, Québec, H4P 1P7 Canada Tel : (514) 344-4004 Fax : (514) 344-8827
Product use	Research

SECTION 2 – HAZARD IDENTIFICATION
Emergency overview	Powder. Will not support burning. Not explosive
Potential Health Effects
Skin	May cause skin irritation
Eyes	May cause irritation or burning sensation
Inhalation	May cause irritation to the respiratory tract
Swallowed	Not known to cause toxic effects
Injection	Not known to cause toxicity with acute exposure, but may produce toxicity with repeated injections.
Carcinogenicity	Not determined
SECTION 3 – COMPOSITION/INFORMATION ON INGREDIENTS
CAS No.	149371-12-0
EC No.	None assigned
Systematic name	Heparin lyase II
Synonyms	Heparinase II
Origin	Recombinant, non-pathogenic microorganism
Ingredients	No components need to be disclosed according to the applicable regulations.
SECTION 4 – FIRST AID MEASURES
Skin contact	Flush with copious amounts of water.
Eye contact	Rinse opened eye for several minutes under running water. Then consult a doctor.
Inhalation	Supply the victim with fresh air; consult doctor in case of complaints.
Swallowing	Rinse mouth with plenty of water.
On all of the above	Consult a doctor if symptoms develop.
SECTION 5 – FIRE FIGHTING MEASURES
Suitable extinguishing agents	Noncombustible.
Protective equipment	No special measures required. Prevent contact with skin and eyes
SECTION 6 – ACCIDENTAL RELEASE MEASURES
Person-related safety precautions	Prevent contact with skin and eyes. Ventilate area.
Measures for environmental protection	No special measures required.
Measures for cleaning/collecting	Spilled material should be carefully wiped up and disposed as biomedical waste as per applicable waste disposal regulations. Wash spill site after material pickup is complete.
SECTION 7 – HANDLING AND STORAGE
Handling
Information for safe handling	Prevent contact with skin and eyes and prevent inhalation and swallowing. No other special precautions are necessary if used correctly.
Information about protection against explosions and fires	No special measures required.
Storage
Requirements to be met by storerooms and receptacles	No special requirements except that product should be stored refrigerated 2 - 8 °C
Information about storage in one common storage facilit	Not required.
Further information about storage conditions	None
Storage class	None assigned
Class according to regulation on flammable liquids	Void
SECTION 8 – EXPOSURE CONTROLS / PERSONAL PROTECTION
General protective and hygienic measures	The usual precautionary measures for handling chemicals and potentially biohazardous materials should be followed. Use clothing sufficient to avoid skin contact. Safety shower and eye wash should be available in proximity.
Protection of hands	Use synthetic water resistant gloves.
Eye protection	Use safety glasses.
SECTION 9 – PHYSICAL AND CHEMICAL PROPERTIES
Physical form	Lyophilized white cake in amber glass vial closed with cap covered with aluminum seal.
Melting point	Not applicable
Boiling point	Not applicable
Flash point	Not applicable
Flammability	Product is not flammable
Danger of explosion	Not determined
Solubility	Heparinase II and ingredients are water soluble
Organic solvents	0 %
Solids content	100 %
SECTION 10 – STABILITY AND REACTIVITY
Thermal decomposition / conditions to be avoided	No decomposition if used according to specifications.
Dangerous reactions	No dangerous reactions known.
Dangerous products of decomposition	No dangerous decomposition products known.
SECTION 11 – TOXICOLOGICAL INFORMATION
Effects of Acute Exposure	Low acute toxicity expected following inhalation, ingestion, injection or skin exposure. The potential eye irritation effects of Heparinase II are unknown and it should be considered to be potentially irritating to the eyes.
Effects of Chronic Exposure	Not determined for oral ingestion or skin exposure
Development Toxicity	Not determined
Reproductive Toxicity	Not determined
Carcinogenicity	Not determined
SECTION 12 – ECOLOGICAL INFORMATION
Toxicity	No data available
Persistence and degradability	No data available
Bioaccumulative potential	No data available
Mobility in soil	No data available
PBT and vPvB assessment	No data available
Other adverse effects	No data available
SECTION 13 – DISPOSAL INFORMATION
Waste Disposal	Incinerate or bury as a solid in a licensed facility. Do not discharge into waterways or sewer systems without proper authority.
Container Disposal	Dispose of in a licensed facility. Recommend crushing or other means to prevent unauthorized reuse.
SECTION 14 – TRANSPORT INFORMATION
DOT (US)	Not dangerous goods
IMDG	Not dangerous goods
IATA	Not dangerous goods
SECTION 15 – REGULATORY INFORMATION
Not a dangerous substance or mixture according to the Globally Harmonised System (GHS).
SECTION 16 – OTHER INFORMATION
This information is given without any warranty or representation. It is believed to be correct but does not claim to be all inclusive and shall be used only as a guide. IBEX Pharmaceuticals Inc. shall not be held liable for any damage resulting from handling or contact with the above product. It is offered solely for your consideration, investigation and verification.
Date of revision	February 28, 2018

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