Hyaluronidase Assay

Method: Based on that of Tolksdorf et al. (1949) and Kass and Seastone (1944). 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

• 0.1 M Sodium phosphate buffer, pH 5.3 with 0.15 M sodium chloride (HSE buffer)
• 0.5 M Sodium acetate buffer, pH 4.2
• 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.
• Standard: Prepare stock solutions of 1.0 and 0.5 mg/ml.
• Hyaluronic acid (HA): Dissolve 10 mg Worthington hyaluronic acid (Code: VHHA) 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:

		Corresponding to
Tube #1	ml HA	mg HA	ml HSE buffer
1	0.00	0.00	1.00
2	0.10	0.04	0.90
3	0.20	0.08	0.80
4	0.25	0.10	0.75
5	0.30	0.12	0.70
6	0.40	0.16	0.60
7	0.50	0.20	0.50
8	0.60	0.24	0.40
9	0.70	0.28	0.30
10	0.80	0.32	0.20

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:

Calculate turbidity reducing units/mg of enzyme or standard as follows:

Calculate  units/mg enzyme as follows: