Enzymatic Assay of Hyaluronidase (3.2.1.35)

Description

This procedure is a turbidimetric determination (% Transmittance at 600 nm, Light path = 1 cm) of Hyaluronidase activity. It is based on the following reaction:

                        Hyaluronidase

Hyaluronic acid ––––––––––––> Di and monosaccharides + smaller hyaluronic acid fragments

Unit Definition: One unit of Hyaluronidase activity will cause a change in A₆₀₀ of 0.330 per minute at pH 5.35 at 37 °C in a 2.0 ml reaction mixture (45 minute assay).

Reagents and Equipment Required

5.0 M Sodium phosphate monobasic solution (Catalog Number 74092)

Hyaluronic acid (Catalog Number H7630 or H5388)

0.5 M Sodium phosphate dibasic solution (Catalog Number 94046)

5.0 M Sodium chloride solution (Catalog Number S6546)

Bovine Serum Albumin (Catalog Number A4503)

Sodium acetate (Catalog Number S8625)

Acetic acid (Catalog Number 695092)

Cuvettes

Precautions – Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Preparation Instructions

Use ultrapure water (≥18 MΩ×cm resistivity at 25 °C) for the preparation of reagents.

Phosphate Buffer (300 mM Sodium Phosphate, pH 5.35 at 37 °C) – Prepare a 1:16.7 dilution using 5.0 M Sodium phosphate monobasic solution (Catalog Number 74092) in ultrapure water. Adjust the pH to 5.35 at 37 °C with 1 M NaOH or 1 M HCl.

Hyaluronic Acid Solution [0.03% (w/v)]

1. Prepare a 0.3 mg/mL solution in Phosphate Buffer using hyaluronic acid (Catalog Number H7630 or H5388).
2. Heat the solution to 90–95 °C with stirring until the hyaluronic acid is dissolved. Fifteen to thirty minutes of heating and stirring is required for this material to completely dissolve. Do NOT boil.
3. After dissolution, allow the solution to cool to 37 °C in a water bath. Adjust the solution to original volume using ultrapure water. Adjust the pH to 5.35 at 37 °C with 1 M NaOH or 1 M HCl.

Enzyme Diluent [20 mM Sodium Phosphate with 77 mM Sodium Chloride and 0.01% (w/v) Bovine Serum Albumin, pH 7.0 at 37 °C] – To prepare 200 mL:

1. Add 8.46 mL of 0.2 M Sodium phosphate monobasic (prepared using Catalog Number 74092 in ultrapure water).
2. Add 11.54 mL of 0.2 M Sodium phosphate dibasic (prepared using Catalog Number 94046 in ultrapure water.
3. Add 3.08 mL of 5.0 M Sodium chloride solution (Catalog Number S6546).
4. Add 20 mg of Bovine Serum Albumin (Catalog Number A4503).
5. Add ultrapure water to make up the final volume.
6. Adjust the pH to 7.0 at 37 °C with 1 M NaOH or 1 M HCl.

Acidic Albumin Solution [24 mM Sodium Acetate, 79 mM Acetic Acid with 0.1% (w/v) Bovine Serum Albumin, pH 3.75 at 25 °C] – To prepare 150 mL:

1. Add 3.6 mL of stock 1.0 M Sodium Acetate, pH 4.8, solution (prepared using Catalog Number S8625).
2. Add 0.68 mL of Acetic Acid (Catalog Number 695092).
3. Add 150 mg of Bovine Serum Albumin (Catalog Number A4503).
4. Add ultrapure water to make up the final volume.
5. Adjust the pH to 3.75 at 25 °C with 5 M HCl.

Enzyme Solution (Hyaluronidase) – Immediately before use, prepare a 1,000 units/mL enzyme stock solution in cold Enzyme Diluent. Dilute the enzyme stock solution with cold Enzyme Diluent to obtain a working solution of ~6 units/mL

Procedure

Final Assay Concentrations – In a 2.0 mL reaction mix the final concentrations are 0.015% (w/v) hyaluronic acid, 150 mM sodium phosphate, and 2‑5 units of hyaluronidase.
Note: Stagger all Tests and Blank by at least 30 seconds such that there is sufficient time to mix each Test and Blank by inversion before incubating at room temperature.

1. Pipette the following reagents into suitable containers:

Reagent	Test 1 (mL)	Test 2 (mL)	Test 3 (mL)	Test 4 (mL)	Test 5 (mL)	Test 6 (mL)	Blank (mL)
Enzyme Solution	0.750	0.665	0.585	0.500	0.415	0.335	0.00
Enzyme Diluent	0.250	0.335	0.415	0.500	0.585	0.665	1.00

2. Mix by swirling and equilibrate to 37 °C for 10 minutes. Then add:

Reagent	Test 1 (mL)	Test 2 (mL)	Test 3 (mL)	Test 4 (mL)	Test 5 (mL)	Test 6 (mL)	Blank (mL)
Hyaluronic Acid Solution	1.00	1.00	1.00	1.00	1.00	1.00	1.00

3. Immediately mix by swirling and incubate at 37 °C for exactly 45 minutes.  

4. After 45 minutes, transfer 0.5 mL of each Test and Blank into suitable cuvettes containing 2.5 mL of Acidic Albumin
    Solution and mix immediately by inversion.  

5. Allow each cuvette to stand for 10 minutes at room temperature.
   Note: Results will change significantly if incubation time is not exactly 10 minutes.

6. Determine the % transmittance at 600 nm.

• Zero the spectrophotometer against the Blank. Read the % transmittance for the Tests and the Blank.
  Note: When batching samples together, blank the spectrophotometer against the respective Blank solutions prepared.  
• The uncorrected % transmittance for each Test must be between 130–170%. A minimum of three valid values for each Test is required to calculate the activity.
  Note: The concentration of the Enzyme Solution can be adjusted to achieve results within the valid % transmittance range.

Results

Calculations

1.

                                              (%T Test – %T Blank) (df)
Units/mL enzyme =  -----------------------------------------------
                                                (14.84) (ml of enzyme solution)

where:
df = dilution factor of enzyme
14.84 = Sigma-Aldrich Determined Extinction Coefficient
mL of enzyme solution = Volume of enzyme solution used in reaction

2.