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

CAS: 37326-33-3 Cat. No.: H128644 EC Number: 253-464-3

GRADE & PURITY Bioactive, ActiBioPure™, Native, High Performance, EnzymoPure™, ≥300 USP/NF units/mg dry weight

Accession #

Q5E985,Q8SQG8

Bioactivity

≥300 USP/NF units/mg dry weight

Overview

Testicular hyaluronidase is a glycoprotein containing 5% mannose and 2.7% glucosamine. Optimum pH range is 4.5-6.0. The enzyme catalyzes the hydrolysis of endo-N-acetylhexosaminic bonds of hyaluronic acid and chondroitin sulfate A and C (but not B), primarily to tetrasaccharide residues.

Hyaluronidase catalyzes the random hydrolysis of 1,4-linkages between 2-acetamido-2-deoxy-b-D-glucose and D-glucose residues in hyaluronate.

Characteristics of Hyaluronidase from Bovine Testes:

Specificity

Testicular hyaluronidase hydrolyzes the endo-N-acetylhexosaminic bonds of hyaluronic acid and chondroitin sulfuric acids A and C (but not B), primarily to tetrasaccharide residues. Monosaccharides are not liberated.

Composition

The enzyme is a glycoprotein containing 5% mannose and 2.17% glucosamine. The amino acid composition has been determined.

Characteristics of Hyaluronidase

Molecular Weight: Four subunits of 14,000 each and a total molecular weight of 55,000.

Optimal pH: 4.5 - 6.

Extinction Coefficient: extinction coefficient= approximately 8.

Inhibitors: Fe2+ and Fe3+ are inhibitory as are Mn2+ and Cu2+.

Stability: Hyaluronidase is stable for 1 - 2 years and the purified preparation for 6 - 12 months when stored at -20°C.

Stabilizers: Sodium chloride

Hyaluronidase Assay

Method

Hyaluronic acid is measured by its ability to form turbidity with an acid albumin solution. Turbidity is a function of hyaluronic acid concentration and can hence be related to enzyme activity. One unit is based on the change in absorbency (turbidity) at 540nm of an internal standard assayed concurrently with each lot.  Internal standard replaces USP/NF reference no longer available.

Reagents

1. 0.1 M Sodium phosphate buffer, pH 5.3 with 0.15 M sodium chloride (HSE buffer)

2. 0.5 M Sodium acetate buffer, pH 4.2

3. Albumin reagent: Prepare by dissolving 2.5 grams of bovine serum albumin, Fraction V in 250 ml of 0.5 M sodium acetate buffer, pH 4.2. Adjust pH to 3.0 with 2 N HCl and heat at 93°C for 30 minutes. Cool and adjust final volume to 1000 ml with 0.5 ml sodium acetate buffer, pH 4.2.

4. Standard: Prepare stock solutions of 1.0 and 0.5 mg/ml.

5. Hyaluronic acid (HA): Dissolve 10 mg Worthington hyaluronic acid in 25 ml 0.1 M sodium phosphate buffer: pH 5.3 with 0.15 M sodium chloride. Note: This solution can be prepared by allowing VHHA to dissolve overnight. Heating in a boiling water bath for 10 - 15 minutes is the preferred method if the material is not immediately soluble.

Enzyme

Prepare stock solution of enzyme at one mg/ml in 0.1 M sodium phosphate buffer pH 5.3 with 0.15 M sodium chloride. Immediately prior to use dilute further in the same buffer. For crude grade material concentrations of 0.01 - 0.05 mg/ml are recommended. For purified grade concentration of 0.001 - 0.01 mg/ml are recommended.

Procedure:

I. Standard Curve

Into a series of numbered tubes, pipette as follows:

Place all tubes in a boiling water bath for 5 minutes. Cool to room temperature. Add 9.0 ml of albumin reagent and allow to stand for 10 minutes. Read absorbance at 540 nm. Plot absorbance at 540 nm versus mg HA to form standard curve.

Hyaluronic acid should be soluble under the defined conditions and should produce a standard curve with a slope of 1.5 or greater.

II. Test Procedure

Pipette 0.5 ml of a 0.4 mg/ml hyaluronic acid solution into a series of test tubes. Incubate at 37°C for 4 - 5 minutes to achieve temperature equilibrium. Incubate one blank tube with one ml of 0.1 M sodium phosphate buffer, pH 5.3 with 0.15 M sodium chloride. At timed intervals add 0.5 ml of appropriately diluted enzyme or standard to respective tubes. Incubate each tube exactly 10 minutes and cool in an ice bath to room temperature. Add 9.0 ml of albumin reagent to each tube and incubate at room temperature for 10 minutes. Read A540 of each tube versus the blank.

Calculation

Determine the amount of hyaluronic acid remaining after digestion from the standard curve.

Calculate the amount of hyaluronic acid digested as follows:

mg HA digested = 0.2 mg - mg HA remaining

Specifications

Product Name

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

Synonyms

Hyaluronidase from Bovine Testes, | s4424 | Hyaluronoglucosaminidase | Hyaluronidase | 6-(3, 3-dimethyl-2-methylideneindol-1-yl)hexanoic acid;hydrobromide | AKOS025293742 | chondroitinase | chondroitinase I | chondroitinase I | hyaluronoglucosidase | Hyalu

Grade

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

Specifications & Purity

Bioactive, ActiBioPure™, Native, High Performance, EnzymoPure™, ≥300 USP/NF units/mg dry weight

Biochemical and Physiological Mechanisms

Hyaluronidase degrades hyaluronan and has been found to be inappropriately regulated during cancer progression. These enzymes randomly cleave β-N-acetylhexosamine-[1→4] glycosidic bonds in hyaluronic acid, chondroitin, and chondroitin sulfates.

Bioactivity

≥300 USP/NF units/mg dry weight

Sequence

Belongs to the glycosyl hydrolase 56 family.

Accession #

Q5E985, Q8SQG8

Predicted molecular weight

~55 kDa

Action Type

ACTIVATOR

CAS

37326-33-3

Enzyme Commission Number

3.2.1.35

Molecule Type

Enzyme

Storage and Shipping

Concentration

≥300 USP/NF units/mg dry weight

Reconstitution

Reconstitute in 0.1M sodium phosphate buffer, pH 5.3 with 0.15M sodium chloride.

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

Unit definition

One unit is based on the change in absorbency (turbidity) at 540nm of an internal standard assayed concurrently with each lot. Internal standard replaces USP/NF reference no longer available.

Taxonomic Classification

Taxonomy Tree

• Kingdom Organic compounds
  • Superclass Organoheterocyclic compounds
    • Class Indoles and derivatives
      • Subclass Indolyl carboxylic acids and derivatives

• Kingdom: Organic compounds
• Superclass: Organoheterocyclic compounds
• Class: Indoles and derivatives
• Subclass: Indolyl carboxylic acids and derivatives
• Intermediate Tree Nodes: Not available
• Direct Parent: Indolyl carboxylic acids and derivatives
• Alternative Parents: Indoles Tertiary alkylarylamines Medium-chain fatty acids Heterocyclic fatty acids Amino fatty acids Benzenoids Amino acids Monocarboxylic acids and derivatives Enamines Carboxylic acids Azacyclic compounds Organic oxides Hydrocarbon derivatives Hydrobromides Carbonyl compounds
• Molecular Framework: Aromatic heteropolycyclic compounds
• Substituents: Indolyl carboxylic acid derivative - Indole - Medium-chain fatty acid - Tertiary aliphatic/aromatic amine - Amino fatty acid - Heterocyclic fatty acid - Fatty acyl - Benzenoid - Amino acid or derivatives - Amino acid - Tertiary amine - Carboxylic acid derivative - Carboxylic acid - Azacycle - Enamine - Monocarboxylic acid or derivatives - Amine - Organonitrogen compound - Organooxygen compound - Hydrobromide - Hydrocarbon derivative - Organic oxide - Carbonyl group - Organic oxygen compound - Organic nitrogen compound - Aromatic heteropolycyclic compound
• Description: This compound belongs to the class of organic compounds known as indolyl carboxylic acids and derivatives. These are compounds containing a carboxylic acid chain (of at least 2 carbon atoms) linked to an indole ring.
• External Descriptors: Not available

Data sources

1. Djoumbou Feunang Y, Eisner R, Knox C, Chepelev L, Hastings J, Owen G, Fahy E, Steinbeck C, Subramanian S, Bolton E, Greiner R, and Wishart DS. ClassyFire: Automated Chemical Classification With A Comprehensive, Computable Taxonomy. Journal of Cheminformatics, 2016, 8:61.

DOI: 10.1186/s13321-016-0174-y

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