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Enzymatic Assay of Hyaluronidase (3.2.1.35)

Description

This procedure may be used for all Hyaluronidase products.

The turbidimetric determination (% Transmittance at 600 nm, Light path = 1 cm) 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).

Precautions

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

Reagents and Equipment Required

Sodium phosphate monobasic (Catalog Number S0751)

Hyaluronic acid (Catalog Numbers H7630 or H5388)

Sodium chloride (Catalog Number S9888)

Bovine Serum Albumin (Catalog Number A4503)

Sodium acetate (Catalog Number S8625)

Acetic acid (Catalog Number 320099 or 695092)

Cuvettes

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 36.0 mg/ml solution in ultrapure water using sodium phosphate monobasic (Catalog Number S0751). Adjust the pH to 5.35 at 37 °C with 1 N NaOH or 1 N 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. Once at the required temperature, adjust the pH to 5.35 at 37 °C with 1 N NaOH or 1 N HCl. Maintain the Hyaluronic Acid Solution temperature at 37 °C by keeping it in the water bath.

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) – Prepare a 2.4 mg/ml solution in ultrapure water using sodium phosphate monobasic (Catalog Number S0751) containing 4.5 mg/ml sodium chloride (Catalog Number S9888) and 0.1 mg/ml bovine serum albumin (Catalog Number A4503). Adjust the pH to 7.0 at 37 °C with 1 N NaOH or 1 N 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) – Prepare a 3.27 mg/ml solution in ultrapure water using sodium acetate (Catalog Number S8625) containing 4.5 µl/ml of acetic acid (Catalog Number 320099 or 695092) and 1 mg/ml bovine serum albumin (Catalog Number A4503). Adjust the pH to 3.75 at 25 °C with 5 N 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 2–4 units of hyaluronidase.

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.

6. Determine the % transmittance at 600 nm.

  • Zero the spectrophotometer against the Blank. Read the % transmittance for the Tests and the Blank.
  • The uncorrected % transmittance for each Test must be between 130170%.

 

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 Determined Extinction Coefficient
ml of enzyme solution = Volume of enzyme solution used in reaction

2.

                          units/ml enzyme
Units/mg solid=   ---------------------------
                          mg solid/ml enzyme

Materials

     

06/15-1

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