Enzymatic Assay of Sulfatase (EC 3.1.6.1.)

Description

This procedure may be used for determination of sulfatase activity in products, except for Sulfatase products from Aerobacter aerogenes (Product No. S1629).

This spectrophotometric stop rate determination (A₅₁₅, Light path = 1 cm) is based on the following reaction:

Unit Definition: One unit will hydrolyze 1.0 µmol of p-nitrocatechol sulfate per hour at pH 5.0 at 37 °C.

Reagents and Equipment Required

Sodium acetate trihydrate, Product No. S8625

p-Nitrocatechol sulfate, dipotassium salt, Product No. N7251

1 N Sodium hydroxide solution, Product No. 319511

5.0 M Sodium chloride solution, Product No. S6546

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.

Buffer solution (200 mM Sodium Acetate Buffer, pH 5.0 at 37 °C) – Prepare a five‑fold dilution with ultrapure water of 1.0 M sodium acetate trihydrate stock solution (prepared using Product No. S8625 in ultrapure water). Adjust to pH 5.0 at 37 °C with 1 N Acetic acid or 1 N NaOH solution.

Substrate solution (6.25 mM p-Nitrocatechol Sulfate) – Prepare a 2.05 mg/mL solution using p-nitrocatechol sulfate, dipotassium salt, Product No. N7251, in ultrapure water.

NaOH solution (1 N Sodium Hydroxide) – Use 1 N sodium hydroxide solution, Product No. 319511.

NaCl solution (34.2 mM Sodium Chloride) – Prepare a 146-fold dilution of 5.0 M sodium chloride solution, Product No. S6546, with ultrapure water.

Test solution (Sulfatase) – Immediately before use, prepare a solution containing 2.5‑5.0 units/mL of sulfatase in cold NaCl solution.

Procedure

Final Assay Conditions – In a 1.00 mL reaction mix, the final concentrations are 100 mM sodium acetate, 2.5 mM p-nitrocatechol, 1.71‑3.42 mM sodium chloride, and 0.25‑0.50 units of sulfatase.

1. Pipette (in mL) the following reagents into suitable containers:

Reagent	Blank	Test 1	Test 2	Test 3
Buffer solution	0.50	0.50	0.50	0.50
Substrate solution	0.40	0.40	0.40	0.40

2. Mix by swirling and equilibrate to 37 °C in a suitably thermostatted water bath.

3. Then add (in mL):

Reagent	Blank	Test 1	Test 2	Test 3
Test solution	–	0.05	0.07	0.10

4. Mix by swirling and incubate at 37 °C for exactly 30 minutes.

5. Then add (in mL):

Reagent	Blank	Test 1	Test 2	Test 3
NaOH solution	5.00	5.00	5.00	5.00
Test solution	0.10	0.05	0.03	–

6. Immediately mix by swirling. Transfer all the Test and Blanks to suitable cuvettes and record the A₅₁₅ using a suitable spectrophotometer.

Results

Calculations

1.

Where:

2 = Time factor correction (Unit Definition Time = 1 hour)

df = Dilution factor

6.0 = Total volume (in mL) of the assay

12.6 = Millimolar extinction coefficient of p-nitrocatechol at 515 nm

0.10 = Volume (in mL) of Test used

2.