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

CAS: 37326-33-3 Cat. No.: H1492276 EC Number: 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

Overview

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

Synonyms

Hyaluronate 4-glycanohydrolase | Hyaluronoglucosaminidase | HYAL2 | oHyal2

Grade

ActiBioPure™, Bioactive, Endotoxin tested, High Performance, Native, EnzymoPure™

Specifications & Purity

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

Biochemical and Physiological Mechanisms

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.

Bioactivity

≥3000 IU/mg dry weight

Endotoxin Concentration

<0.2 EU/IU

Accession #

Q8SQG7

CAS

37326-33-3

Enzyme Commission Number

3.2.1.35

Molecule Type

Enzyme

Storage and Shipping

Concentration

≥3000 IU/mg dry weight

Reconstitution

Reconstitute in water.

Storage

Store at 2-8°C

Shipped In

Wet ice

Stability And Storage

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

Unit definition

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

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