This procedure is a turbidimetric determination (% Transmittance at 600 nm, Light path = 1 cm) of Hyaluronidase activity. It is based on the following reaction:
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).
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)
Precautions – Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.
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)]
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:
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:
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
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:
2. Mix by swirling and equilibrate to 37 °C for 10 minutes. Then add:
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.
(%T Test – %T Blank) (df)
Units/mL enzyme = -----------------------------------------------
(14.84) (ml of enzyme solution)
df = dilution factor of enzyme
14.84 = Sigma-Aldrich Determined Extinction Coefficient
mL of enzyme solution = Volume of enzyme solution used in reaction
To continue reading please sign in or create an account.Don't Have An Account?