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
L2215645
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