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| 1mg |
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| 5mg |
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| 10mg |
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| Targets |
HD-CHA-Ala-Arg-pNA diacetate targets proteolytic enzymes, particularly serine proteases or other amidolytic enzymes that cleave the peptide bond before the p-nitroanilide group. The substrate sequence (D-CHA-Ala-Arg) is specifically recognized and cleaved by certain proteases. Upon enzymatic cleavage, the p-nitroanilide (pNA) chromophore is released, generating a yellow color that can be detected spectrophotometrically.
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| ln Vitro |
In vitro, HD-CHA-Ala-Arg-pNA diacetate is used as a chromogenic substrate to measure proteolytic enzyme activity in biochemical assays. The substrate is cleaved by active proteases, releasing the yellow p-nitroanilide chromophore, which absorbs at approximately 405 nm. The increase in absorbance over time is directly proportional to enzyme activity. The substrate can be used to determine enzyme kinetics (Km, Vmax), inhibitor potency (IC50), and to screen for protease inhibitors in drug discovery programs.
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| ln Vivo |
In vivo activity is not directly measured for HD-CHA-Ala-Arg-pNA diacetate, as it is a substrate used in biochemical assays rather than a therapeutic agent. However, the substrate can be used to assess protease activity in tissue homogenates, plasma, or other biological samples collected from animal models of disease. Elevated protease activity detected using this substrate can serve as a biomarker of disease progression or treatment response in preclinical studies.
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| Enzyme Assay |
Non-cellular enzyme assays using HD-CHA-Ala-Arg-pNA diacetate are performed in buffer systems optimized for the target protease. The substrate is incubated with purified enzyme or tissue lysates at the appropriate pH and temperature. The reaction is monitored continuously using a spectrophotometer or plate reader at 405 nm, and the initial rate of absorbance increase is calculated. Inhibitors are added to the reaction to determine their potency. Controls including enzyme blanks and substrate blanks are included to account for background absorbance.
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| Cell Assay |
In vitro cellular experiments using HD-CHA-Ala-Arg-pNA diacetate are limited due to the substrate's inability to cross cell membranes. However, cell lysates can be prepared from treated cells, and protease activity in the lysates can be measured using the substrate. This approach allows researchers to assess the effects of test compounds on protease activity in cellular contexts. Cells are treated with compounds of interest, lysed, and the lysates are incubated with the substrate to measure enzymatic activity.
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| Animal Protocol |
In vivo animal experiments using HD-CHA-Ala-Arg-pNA diacetate involve collecting tissue samples or biological fluids from animals and measuring protease activity ex vivo. The substrate is incubated with the samples, and absorbance at 405 nm is measured to quantify enzyme activity. This approach can be used to monitor disease progression or evaluate the pharmacodynamic effects of protease inhibitors in preclinical models of cancer, inflammation, or other diseases where proteases play a role.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties are not relevant for HD-CHA-Ala-Arg-pNA diacetate as it is a substrate used in biochemical assays and not a therapeutic compound. The molecular weight is 638.7 g/mol. The molecular formula is C28H46N8O9. The diacetate salt form enhances the compound's solubility and handling in aqueous buffers. The compound should be stored according to the manufacturer's recommendations, typically at -20°C protected from light.
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| Toxicity/Toxicokinetics |
Toxicity data for HD-CHA-Ala-Arg-pNA diacetate are not typically characterized as the compound is a research reagent used in enzymatic assays, not a therapeutic agent. Standard laboratory safety precautions should be followed when handling the compound. The p-nitroanilide chromophore is generally considered to have low toxicity in the context of in vitro assays. Researchers should consult the safety data sheet for specific handling and disposal guidelines.
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| References | |
| Additional Infomation |
HD-CHA-Ala-Arg-pNA diacetate is a chromogenic substrate for amidolytic assays. It is also known as H-D-CHA-Ala-Arg-pNA (diacetate). The diacetate salt form enhances solubility. The substrate is ideal for use in biochemical assays, enzyme kinetics, and drug screening applications where precise detection of proteolytic activity is essential. All products are for research use only.
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| Molecular Formula |
C28H46N8O9
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| Molecular Weight |
638.71
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| Appearance |
Typically exists as solid at room temperature
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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 (e.g. under nitrogen), avoid exposure to moisture and light. |
| 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) |
H2O :≥ 50 mg/mL (~78.28 mM)
DMSO :≥ 50 mg/mL (~78.28 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 | 1.5657 mL | 7.8283 mL | 15.6566 mL | |
| 5 mM | 0.3131 mL | 1.5657 mL | 3.1313 mL | |
| 10 mM | 0.1566 mL | 0.7828 mL | 1.5657 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.
Products are for research use only; We do not sell to patients
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