Enzymatic Assay of Sulfatase
1. OBJECTIVE
To standardize a procedure for the enzymatic assay of Sulfatase
2. SCOPE
This procedure applies to all products that have a specification for Sulfatase activity, except Sulfatase from Aerobacter aerogenes, Sigma-Aldrich Product Number S1629.
3.1. Purified Water – Water from a deionizing system, resistivity > or = 18MΩ•cm @ 25ºC 3.2. Unit Definition – One unit will hydrolyze 1.0 μmole of p-nitrocatechol sulfate per hour at pH 5.0 at 37ºC.
4. DISCUSSION
p - Nitrocatechol Sulphate + H2O sulfatase > p - Nitrocatechol + Sulphate
5. RESPONSIBILITIES
It is the responsibility of all Analytical Services personnel to follow this protocol as written.
6. SAFETY
Refer to the Material Safety Data Sheet (MSDS) for hazards and appropriate handling precautions.
7.1. CONDITIONS
T = 37ºC, pH = 5.0, A515nm, Light path = 1 cm
7.2. METHOD
Colormetric
7.3. REAGENTS
7.3.1. 200 mM Sodium Acetate Buffer, pH 5.0 at 37ºC (Buffer)
Prepare a 27.2 mg/ml solution in purified water using Sodium Acetate, Trihydrate, Sigma-Aldrich Product Number S8625. Adjust to pH 5.0 at 37ºC with 5M HCl.
7.3.2. 6.25 mM p-Nitrocatechol Sulfate Solution (PNCS)
Prepare a 2.05 mg/ml solution in purified water using p-Nitrocatechol Sulfate, Dipotassium Salt, Sigma-Aldrich Product Number N7251.
7.3.3. 1 N Sodium Hydroxide (NaOH)
Use Sodium Hydroxide Solution, 1.0 N, Sigma-Aldrich Product Number S2567 or prepare a 40 mg/ml solution using Sodium Hydroxide, Reagent Grade, Sigma-Aldrich Number S5881.
7.3.4. 0.2% (w/v) Sodium Chloride Solution (NaCl)
Prepare a 2 mg/ml solution in purified water using Sodium Chloride, Sigma-Aldrich Product Number S9888.
7.3.5. Sulfatase Enzyme Solution (Enzyme)
Immediately before use, prepare a solution containing 2.5 to 5.0 units/mL of Sulfatase in cold NaCl (Reagent 7.3.4)
7.4. TEST METHOD
7.4.1. Pipette (in milliliters) the following reagents into suitable containers:
| Test | Blank | |
| Buffer (Reagent 7.3.1) | 0.50 | 0.50 |
| PCNS (Reagent 7.3.2) | 0.40 | 0.40 |
7.4.2. Mix by swirling and equilibrate to 37ºC. Then add:
| Enzyme (Reagent 7.3.5) | 0.10 | ------ |
7.4.3. Mix by swirling and incubate at 37ºC for exactly 30 minutes. Then add:
| NaOH (Reagent 7.3.3) | 5.00 | 5.00 |
| Enzyme (Reagent 7.3.5) | ------ | 0.10 |
7.4.4. Immediately mix by swirling. Transfer the solutions to suitable cuvettes and record the A515nm for both the Test and Blank using a suitable spectrophotometer.
7.5. CALCULATIONS
| 7.5.1. | Units/mL enzyme = | (A515nm Test - A515nm Blank)(3.0)(df)(6) |
| (12.6)(0.1) |
7.5.2. 2 = Time factor correction (Unit Definition Time = 1 hour)
df = Dilution factor
6 = Total volume (in milliliters) of the assay
12.6 = Millimolar extinction coefficient of 4-nitrocatechol at 515 nm
0.1 = Volume (in milliliters) of enzyme used
| 7.5.2. | Units/mL solid = | Units/mL enzyme |
| mg solid/mL enzyme |
7.6. FINAL ASSAY CONCENTRATION
In a 1.00 mL reaction mix, the final concentrations are 100 mM sodium acetate, 2.5 mM p-nitrocatechol, 0.02% (w/v) sodium chloride, and 0.25 to 0.50 units of sulfatase.
9. APPROVAL
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