Sulfonation and sulfation are important processes in the metabolism of compounds such as hormones, neurotransmitters, and drugs. Sulfonation and sulfation are catalyzed by various sulfotransferases. In turn, desulfonation and desulfation occur via the action of sulfatase.
Several sulfatases occur in Helix pomatia (also known as Roman Snail). Early studies indicated the presence of at least two such sulfatases. One publication on two sulfatases isolated from Helix pomatia indicated molecular mass values of ~85 kDa by gel filtration chromatography. More recent work has postulated that Helix pomatia contains at least three sulfatases.
In vitro, this Helix pomatia sulfatase product has been used for deconjugation studies of various compounds, including:
• Environmental contaminants, such as bisphenol A
• Hirudin
• Vitamin E metabolites
• β-adrenoreceptor agonists
• Lignans from carob germ and carob seed
This product is a lyophilized powder. It is known to contain β-glucuronidase activity. For this reason, β-glucuronidase activity of this product is also determined.
Biochem/physiol Actions
Sulfatase from Helix pomatia facilitates the synthesis of thiohydroximates. It causes the hydrolysis of β-naphthyl sulfate much faster than a-naphthyl sulfate and possesses regiospecificity in the hydrolysis of ortho and para substituted phenyl sulfates.
Enzymatic Assay of SULFATASE
PRINCIPLE:
CONDITIONS: T = 37°C, pH = 5.0, A515nm, Light path = 1 cm
METHOD: Colorimetric
REAGENTS:
A. 200 mM Sodium Acetate Buffer, pH 5.0 at 37°C(Prepare 50 ml in deionized water using Sodium Acetate, Trihydrate. Adjust to pH 5.0 at 37°C with 5 M HCl.)
B. 6.25 mM p-Nitrocatechol Sulfate Solution (PNCS)(Prepare 10 ml in deionized water using p-Nitrocatechol Sulfate, Dipotassium Salt.)
C. 1 M Sodium Hydroxide (NaOH)(Prepare 50 ml in deionized water using Sodium Hydroxide, Anhydrous.)
D. 0.2% (w/v) Sodium Chloride Solution(Prepare 50 ml in deionized water using Sodium Chloride.)
E. Sulfatase Enzyme Solution(Immediately before use, prepare a solution containing 2.5 - 5.0 units/ml of Sulfatase in cold Reagent D.)
PROCEDURE:
Pipette (in milliliters) the following reagents into suitable containers:
| Test | Blank |
|---|
| Reagent A (Buffer) | 0.50 | 0.50 |
| Reagent B (PNCS) | 0.40 | 0.40 |
Mix by swirling and equilibrate to 37°C. Then add:
Reagent E (Enzyme Solution)
| 0.10
| ------
|
Mix by swirling and incubate at 37°C for exactly 30 minutes. Then add:
| Reagent C (NaOH) | 5.00 | 5.00 |
|---|
| Reagent E (Enzyme Solution) | ------ | 0.10 |
Immediately mix by swirling. Transfer the solutions to suitable cuvettes and record the A515nm for both the Test and Blank using a suitable spectrophotometer.
CALCULATIONS:
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 milliliter) of enzyme used
UNIT DEFINITION:
One unit will hydrolyze 1.0 µmole of p-nitrocatechol sulfate per hour at pH 5.0 at 37°C.
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 - 0.50 unit of sulfatase.
NOTES:
This enzyme assay is not to be used to assay Sulfatase from Aerobacter aerogenes.
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