Hyaluronidase from sheep testes, CAS No.37326-33-3

CAS: 37326-33-3 Cat. No.: H1492276 EG-Nummer: 253-464-3

GRADE & PURITY Bioactive, ActiBioPure™, Native, Endotoxin tested, High Performance, EnzymoPure™, ≥3000 IU/mg dry weight

Accession #

Q8SQG7

Endotoxin Concentration

<0.2 EU/IU

Bioactivity

≥3000 IU/mg dry weight

Übersicht

Enzymes extracted from mammalian testes (e.g., sheep testes) can hydrolyze mucopolysaccharides of the hyaluronic acid type. They may contain a suitable stabilizer. Potency: Minimum 3000 IU of hyaluronidase activity per milligram (of dry substance).

Production

Animals used for producing hyaluronidase must meet the health requirements for animals intended for human consumption.

Characteristics

1.Appearance: White or yellowish-white, amorphous powder.

2.Solubility: Soluble in water, almost insoluble in acetone and absolute ethanol.

Identification

A solution containing 100 IU of hyaluronidase in 1 mL of 9 g/L sodium chloride solution depolymerizes a 10 g/L sodium hyaluronate BRP solution at 20°C, resulting in a significant decrease in viscosity. Heating the hyaluronidase at 100°C for 30 minutes destroys this effect.

Tests

1.Appearance of Solution: The solution should be clear. Dissolve 0.10 g in water and dilute to 10 mL with the same solvent.

2.pH: 4.5 to 7.5. Dissolve 30 mg in carbon dioxide-free water and dilute to 10 mL with the same solvent.

3.Loss on Drying: Maximum 5.0%. Determine by drying 0.500 g at 60°C under a pressure not exceeding 670 Pa for 2 hours.

4.Bacterial Endotoxins: ≤ 0.2 EU/IU.

Assay

The activity of hyaluronidase is determined using a slope-ratio assay, by comparing the rate at which it hydrolyzes sodium hyaluronate BRP with the rate obtained using the International Standard or a reference preparation calibrated in International Units.

Substrate Solution

In a 25 mL conical flask, add 0.10 g of sodium hyaluronate BRP, then slowly add 20.0 mL of water at 4°C. The addition rate must be slow enough to allow the substrate particles to swell (approximately 5 minutes). Maintain at 4°C and stir for at least 12 hours. Store at 4°C and use within 4 days.
For both the test solution and the reference solution, prepare the solutions and perform dilutions at 0°C to 4°C.
1.Test Solution: Dissolve an appropriate amount of the substance in hyaluronidase diluent to obtain a solution containing 0.6 ± 0.3 IU of hyaluronidase per mL.

2.Reference Solution: Dissolve an appropriate amount of hyaluronidase BRP in hyaluronidase diluent to obtain a solution containing 0.6 IU of hyaluronidase per mL.
In a reaction vessel, mix 1.50 mL of phosphate buffer solution (pH 6.4) and 1.0 mL of the substrate solution, and equilibrate at 37 ± 0.1°C. At time t₀ = 0 (using the first timer), add 0.50 mL of the test solution containing E milligrams of the enzyme to be tested, mix well. Maintain the mixture at 37 ± 0.1°C using a suitable viscometer, record the flow time t using a second timer (with 0.1-second intervals), and perform multiple measurements over approximately 20 minutes (monitoring with the first timer). Use the following viscometer: microviscometer (DIN 51 562, Part 2), capillary type MII, with a viscometer constant of approximately 0.1 mm²/s².
Repeat the above procedure using 0.50 mL of the reference solution containing hyaluronidase BRP. Calculate the viscosity ratio using the following expression:

K = Viscometer constant (in mm²/s², indicated on the viscometer);

t₂ = Flow time of the solution (in seconds);

0.6915 = Kinematic viscosity of the buffer solution at 37°C (in mm²/s).
Since the enzymatic reaction continues during the flow time measurement, the actual reaction time is equal to t₀ + t/2 (i.e., half of the flow time (t/2) is added to the initial measurement time t₀). Plot (ln η)⁻¹ as a function of the reaction time (t₀ + t/2) (in seconds); a linear relationship should be obtained. Calculate the slope (b) of the substance to be tested and the slope (bᵣ) of the reference preparation. Determine the specific activity in International Units per milligram using the following expression:

A = Specific activity of hyaluronidase BRP (in International Units per milligram).
Perform at least three complete sets of the procedure and calculate the average activity of the substance to be tested.

Storage

Store in a tightly closed container at a temperature of 2°C to 8°C. If the substance is sterile, the container should also be sterile and tamper-proof.

Specifications

Product Name

Hyaluronidase from sheep testes, CAS No.37326-33-3

Synonyme

Hyaluronate 4-glycanohydrolase | Hyaluronoglucosaminidase | HYAL2 | oHyal2

Note

ActiBioPure™, Auf Endotoxine geprüft, Bioactive, High Performance, Native, EnzymoPure™

Spezifikationen & Reinheit

Bioactive, ActiBioPure™, Native, endotoxin tested, High Performance, EnzymoPure™, ≥3000 IU/mg dry weight

Biochemische und physiologische Mechanismen

Catalyzes hyaluronan degradation into small fragments that are endocytosed and degraded in lysosomes by HYAL1 and exoglycosidases. Essential for the breakdown of extracellular matrix hyaluronan.

Bioaktivität

≥3000 IU/mg dry weight

Endotoxin-Konzentration

<0.2 EU/IU

Beitritt #

Q8SQG7

CAS

37326-33-3

Enzym Kommissionsnummer

3.2.1.35

Molekültyp

Enzyme

Product Line

Biologische Produkte

Lagerung und Versand

Konzentration

≥3000 IU/mg dry weight

Rekonstitution

Reconstitute in water.

Storage

Store at 2-8°C

Verschickt in

Wet ice

Stabilität und Lagerung

Store at 2-8℃ long term (2 years). Upon receipt, it is recommended to aliquot. 

Definition der Einheit

The activity of hyaluronidase is determined by comparing the rate at which it hydrolyzes sodium hyaluronate BRP with the rate obtained using an international standard, or by using a reference preparation calibrated in international units, using a slope-ra

Application Protocols

Item-specific tested applications and protocols: Not specified for this item; refer to CoA/Spec Sheet.

Guidance

• Because protocols depend strongly on the specific activity, formulation, and intended application, users should validate conditions locally. Use the Reaction Conditions tab for general literature-based starting points, and adjust enzyme units, pH, and time to achieve the desired extent of HA degradation.
• For cell- or tissue-adjacent workflows, ensure buffers and exposure times preserve target viability/epitopes and comply with institutional biosafety practices.

Biological Roles

General/literature overview (context; not product specs)

• Substrate specificity: Mammalian hyaluronidases predominantly degrade hyaluronan in the extracellular matrix (ECM); some isoforms can act on chondroitin sulfates with lower efficiency.
• Physiological roles: Participate in ECM turnover, tissue remodeling, and modulation of interstitial fluid viscosity. In reproduction biology, testicular hyaluronidase contributes to cumulus matrix dispersion surrounding oocytes.
• Cellular localization: Depending on isoform, hyaluronidases are found in lysosomes, on the cell surface, or secreted; acidic pH optima align with lysosomal environments.
• Products of action: HA oligosaccharides that can have distinct biological signaling effects in vitro (size-dependent interactions with receptors such as CD44 and TLRs; literature context, research only).
• Pathway context: HA synthesis by HAS1–3 and degradation by hyaluronidases maintain HA homeostasis; dysregulation is a topic of research in development, inflammation, and matrix biology.

Note: The above describes biological context for research planning only. This product is designated For research use only; no clinical or diagnostic use is intended or implied.

Buffer Applications

Item-specific buffer composition: Not specified for this item; refer to CoA/Spec Sheet.

General/literature guidance for hyaluronidase handling (context; not product specs)

• Activity buffers: Mildly acidic buffers such as 50–100 mM sodium acetate or citrate at pH ~4.0–5.5 often support higher activity. For gentler, slower digestion compatible with cells or neutral conditions, phosphate or HEPES near pH 7.0 can be used with adjusted incubation times.
• Ionic strength: 0.1–0.2 M total ionic strength is common; optimize empirically to balance enzyme stability and substrate binding.
• Stabilizers: Carrier proteins (e.g., 0.1% BSA) and 5–10% glycerol are sometimes included to reduce adsorption and preserve activity during storage and low-concentration work.
• Inhibitors/denaturants to avoid: SDS and strong nonionic detergents, chaotropes (urea, guanidinium), high concentrations of organic solvents, and extreme pH.
• Assay readouts: Viscosity reduction, turbidity decrease in HA–albumin complexes, reducing ends (e.g., PAHBAH/DNS assays), or HA-specific dye methods.
• Sterility: For cell-associated workflows, sterile-filter buffers (0.22 µm) and endotoxin-aware practices are recommended; consult the lot’s endotoxin data.

Green Alternatives

Context and positioning

• Enzymatic depolymerization with hyaluronidase is inherently a greener alternative to harsh chemical depolymerization of hyaluronan (e.g., strong acid/base hydrolysis or oxidative degradation), offering aqueous operation, mild conditions, and superior selectivity.

Comparison (literature; not product specs)

• Process conditions: Enzymatic methods operate in water at ambient to 37 °C versus chemical methods requiring elevated temperatures and corrosive reagents.
• Selectivity: Enzymes yield defined oligosaccharide profiles; chemical methods often produce broader, less controlled distributions and potential byproducts.
• Safety and waste: Reduced use of corrosives and oxidants lowers hazard potential and neutralization waste streams.

Alternative sourcing considerations

• Recombinant hyaluronidases (where available) can reduce animal-derived variability and support supply sustainability. However, native preparations may retain post-translational features important for some applications.

Trade-offs

• Enzyme cost and stability may be limiting; activity depends on pH/temperature windows. Chemical methods, while less selective, can be faster at scale and insensitive to biological storage constraints.

Pharmaceutical Uses

No therapeutic claims. The following are research/manufacturing context only.

General/literature roles (context; not product specs)

• Processing aid: Hyaluronidase is used in research to modulate the viscosity of hyaluronan-containing solutions and hydrogels, facilitating mixing, filtration, or diffusion studies during formulation development.
• Analytical support: Enzymatic digestion of HA-containing excipient matrices (e.g., sodium hyaluronate) to characterize molecular weight distributions and assess raw material consistency.
• Permeation modeling: In vitro studies may use hyaluronidase to study matrix permeability and mass transport in model tissues or hydrogels.
• Source considerations: Native animal-derived versus recombinant sources can have different impurity profiles and regulatory acceptance; selection depends on intended research context. For any GMP-related work, consult relevant pharmacopeial monographs and quality system requirements.

Item-specific regulatory status: Not specified for this item; refer to CoA/Spec Sheet. This product is designated For research use only.

Physical Properties

Item-specific (from Product Data)

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.
• Physical constants (BP/MP/density/refractive index): Not applicable to a protein preparation; not specified for this item.
• Solubility: Not specified for this item; refer to CoA/Spec Sheet.

General/literature values and notes (context; not product specs)

• Form: Enzyme preparations are commonly supplied lyophilized or in buffered aqueous solution; solubility is in aqueous buffers (e.g., acetate, citrate, phosphate) rather than organic solvents.
• pH activity profile: Mammalian hyaluronidases typically show maximal activity in the acidic range (approx. pH 4–5; literature), with reduced activity near neutral pH.
• Temperature: Many mammalian hyaluronidases are active at 37 °C (literature) but exhibit thermal lability at elevated temperatures; ice-cold handling is recommended to preserve activity.
• Cofactors: Mammalian hyaluronidases generally do not require metal cofactors (literature); activity may be influenced by ionic strength and stabilizing proteins (e.g., albumin) in solution.
• Substrate: High-molecular-weight hyaluronan; the reaction decreases solution viscosity (a hallmark readout).

Quality and Grades

Item-specific designations (from Product Data)

• Bioactive; Native; Endotoxin tested; High Performance; ActiBioPure™; EnzymoPure™.

What these designations generally imply (explanatory; not additional specs)

• Native: Derived from biological (sheep testis) source with native post-translational features. Useful when native glycosylation/disulfide pattern matters for activity or specificity.
• Bioactive / High Performance: Indicates the preparation retains functional enzymatic activity suitable for research applications (e.g., HA depolymerization, matrix modulation) and is processed to minimize contaminants that impair function.
• Endotoxin tested: Each lot is evaluated for endotoxin burden; consult the specific lot CoA for the numerical value and test method.
• ActiBioPure™ / EnzymoPure™: Vendor quality marks denoting tightened process controls, purification steps aimed at high specific activity, and reduced adventitious proteins. Refer to the CoA/Spec Sheet for lot-specific purity, specific activity units, and residual-protein profile.

User guidance

• For quantitative work, request and review the CoA for: specific activity definition and units (e.g., U/mg), assay conditions (substrate, pH, temperature), protein concentration determination method, bioburden/endotoxin data, and any stabilizers or excipients present.

Reaction and Applications

Item-specific use domain (from Product Data)

• Research use only. Native hyaluronidase from sheep testes for enzymatic depolymerization of hyaluronan and related glycosaminoglycans.

General/literature applications (context; not product specs)

• HA depolymerization: Reduces viscosity of hyaluronan solutions for rheological studies, biomaterials processing, and ECM remodeling assays.
• Analytical sample prep: Generation of defined HA oligosaccharides for standards, HPAEC-PAD, or LC–MS workflows; digestion of HA-rich matrices prior to glycomics or proteomics.
• Tissue processing: Facilitation of tissue dissociation in combination with collagenases/proteases in ex vivo workflows; optimize ratios and exposure times to protect target cell-surface epitopes.
• Biomaterials and viscoelasticity: Tuning of hydrogel pore size and viscoelastic properties by partial HA degradation.
• Mechanistic studies: Probing HA-dependent cell adhesion/signaling by controlled removal of pericellular HA (in vitro research).

Practical notes (literature)

• Activity is typically highest at acidic pH (∼4–5); near-neutral conditions can be used for gentler, slower digestion.
• Pre-warm substrates and buffers to the assay temperature (often 37 °C) and keep the enzyme on ice until use.
• Viscosity loss can be monitored by rheometry or simple flow-time assays; reducing-sugar assays and HA-specific dyes (e.g., carbazole method for uronic acids) provide quantitative endpoints.

Reaction Conditions

Item-specific conditions: Not specified for this item; refer to CoA/Spec Sheet for lot-defined activity assay conditions and units.

General/literature guidance (context; not product specs)

• Buffer and pH: Sodium acetate or citrate buffers at pH 4.0–5.5 commonly yield high activity. For applications requiring near-neutral pH, expect slower rates.
• Temperature: 25–37 °C incubation is typical; maintain the enzyme on ice before addition. Avoid prolonged exposure above 40 °C.
• Substrate loading: HA concentrations from 0.05–5 mg/mL are common for kinetic and viscosity studies; higher concentrations may require longer times due to mass transport.
• Enzyme dosing: Defined in units (U) relative to an activity assay; start with a broad range (e.g., 0.001–1 U/mL) and titrate to the desired extent of digestion.
• Time course: Minutes to hours depending on pH, temperature, and target fragment sizes. Time-course sampling with quench (e.g., rapid cooling, pH shift, or denaturant) enables profile control.
• Compatibility: Avoid detergents, chaotropes, and organic solvents. Divalent cations are generally not required for mammalian hyaluronidase.
• Analytics: Monitor by viscosity, SEC-MALS for molecular weight reduction, CE or HPAEC-PAD for oligosaccharide profiling, or colorimetric uronic acid assays.

Safety and Handling

Item-specific (from Product Data)

• GHS classification, signal word, pictograms, H-statements: Not specified for this item; refer to SDS.
• Storage conditions: Store at 2–8 °C. Shipped on wet ice.

General safety guidance (consult SDS for authoritative information)

• Potential hazards: Proteins/enzymes may cause allergic sensitization upon inhalation or skin contact; avoid aerosol generation. Not volatile.
• PPE: Laboratory coat, nitrile gloves, and safety glasses. Use a biological safety cabinet for manipulations likely to generate aerosols or when preparing concentrated stock solutions.
• Handling: Keep on ice during use to preserve activity. Avoid repeated freeze–thaw cycles if frozen aliquots are prepared. Do not expose to proteases or harsh denaturants (SDS, strong acids/bases, chaotropes) unless intentionally inactivating.
• Incompatibilities: Strong oxidizers, surfactants/denaturants, and extreme pH or temperature may irreversibly inactivate the enzyme.
• First aid (overview): If inhaled, move to fresh air; in case of skin or eye contact, rinse with water for several minutes; if ingested, rinse mouth. Seek medical attention according to institutional policy. Always follow institutional biosafety procedures.
• Disposal: Treat as biologically active protein waste; decontaminate by appropriate chemical/thermal inactivation and dispose per local regulations.

Solvent Selection

Not typically applicable. This product is a protein enzyme intended for use in aqueous buffers rather than organic solvents.

General/literature guidance (context)

• Aqueous compatibility: Hyaluronidase is compatible with common biological buffers (e.g., acetate, citrate, phosphate), with activity typically favored in mildly acidic buffers.
• Avoid: Organic solvents, strong detergents, and chaotropes that denature proteins will diminish activity. Low percentages of benign additives (e.g., glycerol, BSA) are sometimes used to stabilize enzymes (literature) but consult the CoA for any formulation constraints.
• Ionic strength: Moderate ionic strength can improve stability; excessive salt may reduce substrate–enzyme interactions.
• If viscosity reduction of HA solutions is the goal, choose buffers that maintain HA solubility (e.g., acetate or phosphate) and the enzyme’s pH optimum.

Storage and Reconstitution

Item-specific (from Product Data)

• Storage: Store at 2–8 °C.
• Shipping: Shipped on wet ice.
• Reconstitution details, excipients, concentration: Not specified for this item; refer to CoA/Spec Sheet.

General/literature guidance (context; not product specs)

• Short-term handling: Keep on ice during use. Minimize time at room temperature to preserve activity.
• Reconstitution (if lyophilized): Use cold, sterile buffer appropriate for intended pH (e.g., acetate or phosphate). Gentle inversion to dissolve; avoid vigorous vortexing or foaming.
• Working stocks: Prepare aliquots to avoid repeated freeze–thaw. If freezing is acceptable for your workflow, snap-freeze small aliquots at −80 °C with stabilizers such as 10–50% glycerol or carrier protein (e.g., 0.1% BSA), as compatible with downstream applications.
• Storage stability: Refrigerated stability depends on formulation; verify lot-specific guidance. Monitor activity periodically using a standard assay.
• Compatibility: Avoid exposure to proteases and denaturants. Use low-protein-binding plastics to reduce adsorption losses at low concentrations.
• Documentation: Record reconstitution buffer, pH, and concentration for reproducibility; retain lot CoA for activity and endotoxin traceability.

Structure and Identity

Item-specific (from Product Data)

• Product: Hyaluronidase from sheep testes (SKU: H1492276)
• CAS: 37326-33-3
• InChIKey: Not specified for this item; refer to CoA/Spec Sheet.
• SMILES: Not applicable for proteins/enzymes; not specified for this item.
• Molecular formula: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.
• Identity notes: Native, bioactive, endotoxin-tested enzyme preparation.

General/literature description (for context; not item-specific specs)

• Hyaluronidases are glycosidic hydrolases that degrade hyaluronan (hyaluronic acid), a linear polysaccharide of repeating β-1→4-linked N-acetyl-D-glucosamine and β-1→3-linked D-glucuronic acid.
• Mammalian testicular hyaluronidase typically acts as an endo-β-N-acetylhexosaminidase (EC 3.2.1.35), cleaving internal β-1→4 linkages to reduce HA viscosity and generate oligosaccharides.
• Structural features: Protein enzyme (polypeptide), typically contains disulfide bonds and N-glycosylation in native sources; no small-molecule 2D structure applies. No defined single stereocenter representation as for small molecules.

Synthetic Utility

Not a traditional small-molecule building block. However, in bioprocess and analytical settings hyaluronidase has distinctive utility.

General/literature utility (context; not product specs)

• Biopolymer tailoring: Controlled enzymatic cleavage of hyaluronan to generate defined oligosaccharide sizes for standards, structure–activity studies, and biomaterials engineering.
• Sample cleanup: Pre-treatment of ECM-rich samples to reduce viscosity and improve extraction efficiency for downstream LC–MS or electrophoretic analyses.
• Selective depolymerization: Compared to chemical hydrolysis, hyaluronidase offers site-selective cleavage under aqueous mild conditions, aiding mechanistic studies of HA interactions and receptor binding.
• Coupled workflows: Sequential or combined treatments with collagenase, dispase, or DNase in tissue dissociation protocols to achieve efficient yet gentle matrix breakdown.

Limits and considerations

• Substrate specificity is largely confined to HA (and to a lesser extent some chondroitin variants), making it unsuitable for broad glycan hydrolysis. Enzyme activity is sensitive to pH, temperature, and denaturants; empirical optimization is essential.

Target Specificity

Item-specific target/epitope details: Not applicable; this is an enzyme, not an antibody.

From Product Data

• No target specificity data (clone, isotype, species reactivity, epitope) are provided for this item.

Guidance

• Substrate specificity (literature context): Hyaluronidase targets hyaluronan polymers in the extracellular matrix; activity on other glycosaminoglycans is isoform- and context-dependent. For quantitative specificity data (KM, kcat) for a given lot or assay, consult literature methods and the CoA where available.

Häufig gestellte Fragen

What is ActiBioPure??

ActiBioPure? is one of Aladdin Scientific's premium product lines, formulated for high-purity applications across multiple workflows. Compared to standard grades in the same workflow, ActiBioPure? products typically offer tighter specifications, more rigorous QC, and stronger lot-to-lot consistency.

What does Bioactive mean?

Bioactive indicates that the product has been processed and tested for biological and biochemical applications. Specifications may include endotoxin limits, microbial controls, sterility, low DNase/RNase/protease activity, and verified biological activity where relevant.

How should this product be stored?

Store at 2–8 °C. Refrigerated storage is required to maintain the specified shelf life.

How is this product shipped?

This product ships chilled on wet ice. Unpack on arrival and transfer it to the storage condition stated above.

What are the CAS number, molecular formula and molecular weight?

The CAS Number is 37326-33-3.

What documentation is provided?

Available product documentation, including Certificates of Analysis (COA), Safety Data Sheets (SDS), and specification sheets, is shown in the product document area. Document availability and access follow the current site policy.