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.
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
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.
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.
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:
2. Mix by swirling and equilibrate to 37 °C in a suitably thermostatted water bath.
3. Then add (in mL):
4. Mix by swirling and incubate at 37 °C for exactly 30 minutes.
5. Then add (in mL):
6. Immediately mix by swirling. Transfer all the Test and Blanks to suitable cuvettes and record the A515 using a suitable spectrophotometer.
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