Specialists in the Isolation and Purification of Horseradish Peroxidase and a Select Range of Enzymes

Assay Procedure (Ref. No. FGAP045) - Hyaluronidase (International Unit)

The method of assay is that of Dorfman in which the enzymatic reduction in turbidity, resulting when hyaluronic acid is mixed with serum albumin at an acid pH, is measured spectrophotometrically at 600 nm and 37^°C.

Unit Definition

That amount of enzyme which causes a reduction in turbidity under specified conditions similar to that caused by one unit of an international standard.

Reagents

1.	0.02 M Sodium Phosphate Buffer pH 6.9 / 0.45% NaCl: Dissolve 1.40 g NaH2PO4 . 2H2O [MM 156.01], or 1.24 g NaH2PO4.H2O [MM137.99]1.56 g Na2HPO4 [MM 142.0] and 4.5 g NaCl [MM 58.44] in ± 800 ml distilled H2O. Check pH 6.8 - 7.0 and dilute to 1000 ml in a volumetric flask.
2.	0.3 M Phosphate Buffer pH 5.3: Dissolve 36.77 g KH2PO4 [MM 136.09] and {(0.873 g Na2HPO4 [MM 142.0] ) or (1.65 g Na2HPO4.7H2O [MM 268.25]) or (2.2 g Na2HPO4.10H2O [MM 358.50])} in ± 800 ml distilled H2O. Check pH 5.3 and dilute to 1000 ml in a volumetric flask.
3.	50% Hydrochloric Acid: To 50 ml distilled water, carefully add 50 ml conc. HCl [MM 36.46]. Mix well.
4.	Acid Albumin Solution pH 3.72 - 3.78: Add 4.56 ml glacial acetic acid [MM 60.05] to ± 900 ml distilled H2O. Mix well. Dissolve 3.26 g CH3COONa[MM 82.03] OR 5.4 g CH3COONa.3H2O [MM136.08] in the diluted acid. Adjust pH to 3.72 - 3.78 with HCl [3]. Dissolve 1 mg Bovine Serum Albumin (BSA) / ml pH 3.72 - 3.78 buffer solution. (PREPARE FRESH DAILY).
5.	Enzyme Diluent: Dissolve 1 mg Bovine Serum Albumin (BSA) / ml pH 6.9 buffer [3A]. Store on ice. (PREPARE FRESH DAILY).

Substrate:

Dissolve 4 mg hyaluronic acid potassium salt / ml phosphate buffer pH 5.3 [2]. Add 0.1 ml toluene [MM 92.14] / 10 ml substrate. [This stock solution with toluene can be stored for ± 4 weeks at 5^°C.]

Immediately before assay, prepare a series of substrate dilutions in buffer [2] between ¹/₁₈ and ¹/₂₅. Mix 0.5 ml of each dilution with 0.5 ml buffer [1]; incubate at 37^°C for 5 minutes and add 5 ml acid albumin [4] at t_o (zero time).

Read A_{600 nm} after exactly 10 minutes. Use dilution yielding 0.38 £ A_{600 nm} £ 0.40 for preparation of stock solution. Store at 37^°C. (PREPARE FRESH DAILY)

Enzyme Sample:

Dissolve 5 mg enzyme/ml ice-cold diluent [5]. Immediately before assay, dilute solution to yield expected 4 units/ml diluent [5].

Enzyme Standard:

Dissolve International Standard (USP or BP) or Internal House Standard to yield exactly 4 units/ml diluent [5]

Procedure

Into 125 x 16 mm test-tubes, pipette the following:

Mix and equilibrate at 37^°C.

At 1 minute intervals, start reaction by adding 0.5 ml substrate at 37^°C to tubes 2 to 10. Incubate for exactly 30 minutes. Stop reaction by adding 5 ml acid albumin [4] at minute intervals. Incubate for 10 minutes at 37^°C, and measure A_{600 nm} at minute intervals. Subtract blank value from all enzyme values.
Record all values. Draw a Standard Curve by plotting absorbance values against enzyme activity. Use this Standard Curve to determine the activity of the unknown sample.

Calculation

	sample units from standard curve x dilution
Units/mg material =
	volume enzyme x mg enzyme/ml original solution

At least two sample readings should intersect the standard curve.

Reference

Dorfman A.: (1955) Methods in Enzymology Vol. 1, 166. Ed by Colowick S.P. and Kaplan N.O. Academic Press, New York.