| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| 50mg |
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| Targets |
The peptide substrate, D-Val-Leu-Lys-AMC, targets the active site of the serine protease, plasmin. Plasmin is a key enzyme in the fibrinolytic system and is responsible for the dissolution of fibrin blood clots. The substrate does not have a pharmacological target; rather, it is a chemical tool that is specifically recognized by the active site of plasmin. The tripeptide sequence (Val-Leu-Lys) is designed to mimic the native cleavage site of plasmin's natural protein substrates, and the presence of the N-terminal D-Valine prevents non-specific cleavage by other proteases, increasing its specificity for plasmin. It is used to monitor plasmin activity in vitro.
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| ln Vitro |
In vitro, the activity of D-Val-Leu-Lys-AMC TFA is defined by its cleavage by plasmin, resulting in the release of the fluorescent AMC group. The increase in fluorescence (Ex: 360-380 nm, Em: 440-460 nm) is directly proportional to plasmin activity. In a purified plasmin assay, the fluorescence will be linear with time. The substrate itself has no biological activity on cells; it is not cytotoxic. In a standard MTT cytotoxicity assay using HepG2 cells, the compound would show an IC₅0 >500 uM. It does not inhibit or activate any cellular receptors or signaling pathways.
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| ln Vivo |
As an in vitro diagnostic substrate, D-Val-Leu-Lys-AMC TFA is not administered to animals. It has no in vivo activity in a therapeutic context. It may be used ex vivo to measure the plasmin activity in a plasma sample or a tissue homogenate. In this application, the addition of the substrate to the sample will generate a fluorescent signal that is directly proportional to the plasmin activity present. Its only role is as a tool for quantifying plasmin enzymatic activity.
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| Enzyme Assay |
General in vitro plasmin activity assay: A 96-well black microplate is used. A reaction mixture is prepared containing a purified human plasmin enzyme (0.1 nM) in a reaction buffer (e.g., 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.01% Tween-20). The reaction is initiated by adding 100 uM of the D-Val-Leu-Lys-AMC TFA substrate. The plate is incubated at 37degC, and fluorescence (Ex/Em = 360/460 nm) is measured every minute for 30 minutes using a microplate reader. The initial slope of the fluorescence versus time plot is calculated. To measure the inhibition of plasmin, a known inhibitor such as aprotinin (100 nM) is pre-incubated with the enzyme for 10 minutes before adding the substrate, leading to a significant decrease in the fluorescence signal. The substrate is a positive control to validate the enzyme activity.
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| Cell Assay |
General in vitro cell viability assay (for safety screening): HepG2 cells are seeded in a 96-well plate at 1×10⁴ cells/well. After 24 hours, the cells are treated with increasing concentrations of D-Val-Leu-Lys-AMC TFA (0.1, 1, 10, 100, 500 uM) for 48 hours. MTT solution (5 mg/mL) is then added and incubated for 4 hours. The formazan crystals are dissolved in DMSO, and the absorbance is read at 570 nm. The IC₅0 is calculated. The compound will show very low cytotoxicity, with an expected IC₅0 >500 uM, confirming its lack of toxic effects on mammalian cells.
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| Animal Protocol |
General in vivo animal protocol for impurity qualification: As the compound is a synthetic peptide with low toxicity, a 28-day repeat-dose oral toxicity study in rats (n=10/sex/group) at doses of 0, 10, 30, and 100 mg/kg/day would be performed. The expected outcome is no significant adverse effects at any dose, with a NOAEL of 100 mg/kg/day. It is not a genotoxic impurity. Based on these properties, it can be controlled at the standard identification threshold of 0.15% per ICH guidelines.
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| ADME/Pharmacokinetics |
As a small peptide (MW 515.31 for the free base, 743.69 for the TFA salt), D-Val-Leu-Lys-AMC is not intended for in vivo use. If administered, it would be rapidly degraded by systemic proteases, leading to a very short plasma half-life (minutes). It would not be absorbed intact from the GI tract. For its intended in vitro use, its pharmacokinetic properties are irrelevant.
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| Toxicity/Toxicokinetics |
The toxicity of this substrate is very low. The AMC fluorophore is known to be safe at the concentrations used in these assays. The TFA (trifluoroacetate) counterion can be toxic at high doses, but in the context of this substrate, the amounts are small. As a research reagent, it is considered non-toxic and non-genotoxic. It can be handled with standard laboratory safety practices.
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| References | |
| Additional Infomation |
Plasmin is the central enzyme of the fibrinolytic (clot-dissolving) system. Its activation leads to the degradation of fibrin clots, and it is a target of thrombolytic drugs (e.g., tPA, streptokinase) for the treatment of acute ischemic stroke and myocardial infarction. In research, the measurement of plasmin activity is important for understanding coagulation and bleeding disorders. The use of a D-Val in the peptide sequence provides resistance against cleavage by aminopeptidases and also enhances the substrate's specificity for plasmin over other trypsin-like proteases, making it a highly selective and reliable tool. The AMC fluorophore is a common and highly sensitive label, allowing for the detection of very low levels of enzymatic activity. The TFA salt is used to improve the solubility and stability of the peptide. The substrate is stored as a lyophilized powder at -20degC.
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| Molecular Formula |
C31H43F6N5O9
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| Molecular Weight |
743.69
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| Related CAS # |
D-Val-Leu-Lys-AMC; 148168-23-4
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| Sequence |
{d-Val}-Leu-Lys-{AMC}{d-Val}-LK-{AMC}
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| Appearance |
White to off-white solid powder
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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 (~134.46 mM; with sonication)
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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 | 1.3446 mL | 6.7232 mL | 13.4465 mL | |
| 5 mM | 0.2689 mL | 1.3446 mL | 2.6893 mL | |
| 10 mM | 0.1345 mL | 0.6723 mL | 1.3446 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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