| Size | Price | Stock | Qty |
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| 100mg |
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| 250mg |
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| 500mg |
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| 1g |
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| Other Sizes |
| Targets |
The enzymatic target of 4-Nitrocatechol sulfate dipotassium salt is sulfatase enzymes, specifically arylsulfatases (including arylsulfatase A, B, and C). More specifically, in clinical diagnostics it is used as a substrate to measure arylsulfatase B (ARSB, also known as N-acetylgalactosamine-4-sulfatase) activity. The sulfate group on the catechol ring is recognized and hydrolyzed by the active-site cysteine residue of sulfatases via a formylglycine-dependent mechanism. Upon cleavage, the chromogenic 4-nitrocatechol product is released and detected.
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| ln Vitro |
In vitro experiments use 4-Nitrocatechol sulfate as a substrate to quantify sulfatase activity in cell lysates or purified enzyme preparations. Upon incubation with arylsulfatase B (ARSB) or related sulfatases, the sulfate ester bond is hydrolyzed. The product, 4-nitrocatechol, develops a yellow color (λmax = 515 nm) under alkaline conditions, allowing for spectrophotometric quantification. Each molecule of substrate cleaved corresponds to one molecule of 4-nitrocatechol produced, enabling direct calculation of enzyme activity in units per mg protein.
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| ln Vivo |
This substrate is not administered to animals for therapeutic assessment. Instead, it is used in ex vivo assays on biological samples obtained from animals. For example, in a mouse model of MPS VI (arylsulfatase B knockout), tissue homogenates (liver, kidney, spleen) or cultured fibroblasts are incubated with 4-nitrocatechol sulfate to quantify residual ARSB activity. Activity levels can then be correlated with disease severity or used to evaluate the efficacy of enzyme replacement therapy (ERT). No in vivo PK/PD studies involve direct administration of this substrate.
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| Enzyme Assay |
Protocol for sulfatase activity measurement using 4-nitrocatechol sulfate dipotassium salt: Prepare a reaction mixture containing 50 mM sodium acetate buffer (pH 5.0-5.6), 10 mM substrate (dissolved in buffer), and 10-50 microL of enzyme sample (cell lysate or tissue homogenate, typically 50-200 microg protein) in a total volume of 200 microL. Incubate at 37degC for 1-4 hours. Stop reaction by adding 800 microL of 1.0 M NaOH to raise pH and develop color. Measure absorbance at 515 nm against a blank. One unit of enzyme activity is defined as the amount releasing 1 micromol of 4-nitrocatechol per hour. Include positive control (purified arylsulfatase) and negative control (boiled enzyme).
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| Cell Assay |
A standard in vitro cell-based protocol for measuring sulfatase activity: Culture cells (e.g., human fibroblasts, HeLa, or COS-7) to confluency in 6-well plates. Harvest cells by trypsinization, wash twice with PBS, and lyse in 0.1% Triton X-100 in water (or lysis buffer with protease inhibitors). Centrifuge at 14,000 rpm for 10 minutes at 4degC to remove debris. Measure protein concentration of the supernatant (BCA assay). For the activity assay, mix 10-50 microL of lysate (50-200 microg protein) with 50 mM sodium acetate buffer (pH 5.6) containing 10 mM 4-nitrocatechol sulfate in a final volume of 200 microL. Incubate at 37degC for 1-4 hours. Stop with 800 microL 1 N NaOH and read A515. Calculate specific activity (nmol/h/mg protein).
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| Animal Protocol |
For analysis of tissue samples from animal experiments: Sacrifice mouse, collect tissues (liver, kidney, spleen, brain). Homogenize 100 mg tissue in 500 microL of cold lysis buffer (0.1% Triton X-100 in water with protease inhibitors). Centrifuge at 10,000 × g for 15 minutes at 4degC. Collect supernatant, measure protein concentration. For the assay, incubate 20-50 microL of tissue homogenate (100-200 microg protein) with 10 mM substrate in 50 mM sodium acetate buffer (pH 5.6) at 37degC for 2-16 hours. Stop reaction with 1 N NaOH, measure absorbance at 515 nm. Alternatively, end-point can be measured continuously at 405 nm over time.
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| ADME/Pharmacokinetics |
No pharmacokinetic data are available for 4-Nitrocatechol sulfate dipotassium salt, as it is used as a non-absorbable in vitro substrate, not a drug. If the compound were administered in vivo, the sulfate group would likely be rapidly cleaved by tissue sulfatases, releasing 4-nitrocatechol. 4-Nitrocatechol is a known hepatotoxicant and nephrotoxicant in high doses. Plasma half-life of 4-nitrocatechol in rats is approximately 2-4 hours, with glucuronidation and sulfation as major metabolic pathways. No such data are available for the parent substrate.
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| Toxicity/Toxicokinetics |
The compound has low acute toxicity as it is poorly absorbed. However, the hydrolysis product 4-nitrocatechol has reported hepatotoxicity and nephrotoxicity. Avoid inhalation of powder and skin contact. Standard laboratory precautions should be used. The compound is stable under dry, cool storage conditions. Waste containing 4-nitrocatechol should be treated as hazardous chemical waste. No carcinogenicity classification is available. May cause eye and skin irritation.
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| References | |
| Additional Infomation |
4-Nitrocatechol sulfate dipotassium salt is not a drug; it is a diagnostic research reagent for enzymatic assays. It has not undergone clinical trials. However, the substrate is widely used in clinical diagnostic laboratories for the confirmation of mucopolysaccharidosis VI (MPS VI) from dried blood spots, leukocytes, or cultured fibroblasts. A positive diagnostic result (deficient ARSB activity) can lead to treatment with recombinant human ARSB (galsulfase, brand name Naglazyme), an FDA-approved enzyme replacement therapy. The compound is also used in high-throughput screening campaigns to identify novel sulfatase inhibitors or activators.
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| Molecular Formula |
C6H3K2NO7S
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|---|---|
| Molecular Weight |
311.35
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| Exact Mass |
310.89
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| CAS # |
14528-64-4
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| Related CAS # |
10485-66-2 (Parent)
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| PubChem CID |
16219779
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.87 g/cm3
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| Melting Point |
>300°C
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| LogP |
2.181
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
17
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| Complexity |
315
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1=CC(=C(C=C1[N+](=O)[O-])OS(=O)(=O)[O-])[O-].[K+].[K+]
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| InChi Key |
CKWWBDCAYRJAJB-UHFFFAOYSA-L
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| InChi Code |
InChI=1S/C6H5NO7S.2K/c8-5-2-1-4(7(9)10)3-6(5)14-15(11,12)13;;/h1-3,8H,(H,11,12,13);;/q;2*+1/p-2
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| Chemical Name |
dipotassium;(5-nitro-2-oxidophenyl) sulfate
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: 100 mg/mL (321.18 mM)
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|---|---|
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.2118 mL | 16.0591 mL | 32.1182 mL | |
| 5 mM | 0.6424 mL | 3.2118 mL | 6.4236 mL | |
| 10 mM | 0.3212 mL | 1.6059 mL | 3.2118 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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