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| Targets |
MMP-3 MMP-10
The primary target of NFF-3 TFA is the active site of matrix metalloproteinases (MMPs), specifically MMP-3 and MMP-10. The peptide is hydrolyzed by these enzymes at a specific cleavage site. The high catalytic efficiency (kcat/Km) of MMP-3 for this substrate (218,000 s-¹ M-¹) indicates a strong interaction, making it a preferred substrate. This specificity allows researchers to measure the proteolytic activity of MMP-3 and MMP-10 in complex biological samples. The peptide also serves as a model substrate to study the mechanisms of other proteases, including trypsin, hepatocyte growth factor activator, and factor Xa, which are involved in various physiological and pathological processes. The CyDye-labeled version is used to visualize these proteolytic events in cell-based assays. |
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| ln Vitro |
In a cell-free enzymatic assay, NFF-3 TFA is highly active and selective for MMP-3 and MMP-10. The catalytic efficiency (kcat/Km) for MMP-3 is 218,000 s-¹ M-¹, which is excellent for a fluorogenic substrate. For MMP-9, the efficiency is much lower (kcat/Km = 10,100 s-¹ M-¹), and it is not cleaved by MMP-1 or MMP-2. This specificity makes it ideal for monitoring MMP-3 activity in the presence of other MMPs. In cell-based assays, when labeled with a CyDye pair (Cy3/Cy5Q), the intact peptide shows no fluorescence due to FRET. Upon cleavage by active MMP-3, the Cy3 and Cy5Q are separated, resulting in a strong increase in Cy3 fluorescence, allowing real-time monitoring of protease activity. This provides a direct readout of MMP-3 activity in live cells.
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| ln Vivo |
No specific in vivo data for the NFF-3 TFA peptide is provided in the search results. However, it is used as a molecular tool in vivo to monitor MMP-3 activity. When administered locally (e.g., intratumorally) or systemically, the fluorescently labeled (Cy3/Cy5Q) NFF-3 peptide can be used to image areas of high MMP-3 activity in live animals. For example, in a xenograft mouse model of fibrosarcoma (HT-1080), injection of the labeled NFF-3 probe results in a strong fluorescent signal at the tumor site due to cleavage by MMP-3 and MMP-10. This signal can be detected using in vivo fluorescence imaging systems (e.g., IVIS). The TFA salt is primarily for in vitro and ex vivo use due to the high cost and sensitivity of the peptide. It serves as a direct chemical probe for protease activity.
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| Enzyme Assay |
A standard cell-free protease activity assay is conducted in a 96-well black plate (for fluorescence). The reaction mixture (100 uL) contains 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM CaCl2, 0.05% Brij-35, and 10 uM of the NFF-3 TFA substrate. The reaction is initiated by the addition of 10-100 ng of recombinant active human MMP-3 or MMP-10 enzyme. The plate is immediately placed in a fluorescence plate reader and incubated at 37degC. The increase in fluorescence (e.g., excitation at 320/40 nm, emission at 405/20 nm for MCA) is monitored every 1-2 minutes for 30-60 minutes. The initial linear part of the fluorescence vs. time curve is used to calculate the reaction velocity. To perform an inhibition assay, the enzyme is pre-incubated with varying concentrations of a test compound (MMP inhibitor) for 15 minutes before adding the substrate. The IC50 is determined by plotting the percent inhibition versus the log concentration of the inhibitor. The reaction can be terminated by adding EDTA (final 25 mM) to chelate the calcium ions required for MMP activity.
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| Cell Assay |
A cell-based MMP-3 activity assay using the fluorescent NFF-3 substrate (Cy3/Cy5Q-NFF-3) is performed in a 96-well plate. Human fibrosarcoma HT-1080 cells (which express high levels of MMP-3) are seeded at 20,000 cells per well in serum-free DMEM and allowed to attach overnight. The cells are then washed with PBS. The Cy3/Cy5Q-NFF-3 peptide is added to the cells at a final concentration of 1-10 uM in serum-free medium. The plate is incubated at 37degC for 2-6 hours. Fluorescence is measured at regular intervals using a fluorescence plate reader with excitation at 544 nm and emission at 590 nm. Cells treated with a broad-spectrum MMP inhibitor (e.g., GM6001, 10 uM) serve as a negative control to determine the background fluorescence. The increase in fluorescence over time is directly proportional to the MMP-3/10 activity in the cell culture. This protocol allows for the screening of potential MMP inhibitors in a live-cell environment. For a standard plate reader, the Cy3/Cy5Q pair is used. The MCA/DNP pair used in the cell-free assay is not suitable for cell-based assays due to background autofluorescence.
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| Animal Protocol |
For an in vivo imaging study, fluorescently labeled NFF-3 (Cy5.5-NFF-3) can be used. Athymic nude mice (6-8 weeks old) are injected subcutaneously with 5×10⁶ HT-1080 fibrosarcoma cells in the right flank. When the tumor volume reaches 200-300 mm3, the mice are anaesthetized with isoflurane. The Cy5.5-NFF-3 peptide (100 ug in 100 uL PBS) is injected directly into the tumor (intratumoral, IT) or intravenously via the tail vein. For intravenous injection, the mice are imaged using an in vivo fluorescence imaging system (e.g., IVIS Spectrum) at 0, 1, 2, 4, 8, 12, and 24 hours post-injection. The excitation filter is set to 640 nm and the emission filter to 700 nm. Images are acquired with consistent exposure time, binning, and f-stop. The region of interest (ROI) is drawn around the tumor to quantify the average radiance (photons/sec/cm2/sr). A group of mice pre-treated with an MMP inhibitor (e.g., batimastat, 30 mg/kg IP) 1 hour before the probe injection serves as a control for signal specificity. The signal intensity in the tumor indicates the level of MMP-3 activity in the tumor microenvironment. This protocol provides a non-invasive readout of tumor biology.
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| ADME/Pharmacokinetics |
NFF-3 TFA has a molecular formula of C80H111F3N22O22 and a molecular weight of 1789.87 g/mol. The lyophilized powder should be stored at -20degC in a sealed container, protected from light and moisture, where it is stable for up to 3 years. For solution storage, it should be kept at -80degC for up to 1 year. It is soluble in DMSO and water. The TFA salt is the standard form for peptide synthesis and storage. The FRET-based design (MCA/DNP) allows for continuous monitoring of enzyme kinetics with high sensitivity. The peptide sequence includes Nva (norvaline) and DNP (2,4-dinitrophenyl)-labeled Lys. The Kcat/Km value for MMP-3 is 218,000 s-¹ M-¹.
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| References | |
| Additional Infomation |
The product is for research use only, not for human use. It is a highly specific substrate for MMP-3 and MMP-10 and is used for drug discovery and basic research. It can be cleaved by other proteases, but at much lower rates. The MCA group has an excitation peak at 325 nm and an emission peak at 395 nm. The peptide is extremely sensitive and specific. It is a chemical tool, not a drug. Handling should be done with care to avoid contamination by other proteases. Always use protease-free tips and tubes. The peptide is soluble in DMSO and water. Avoid repeated freeze-thaw cycles.
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| Molecular Formula |
C80H111F3N22O22
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| Molecular Weight |
1789.87
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| Related CAS # |
NFF-3;158584-09-9
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| Appearance |
Light yellow to yellow 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 (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) |
DMSO :~100 mg/mL (~55.87 mM)
H2O :< 0.1 mg/mL |
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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 | 0.5587 mL | 2.7935 mL | 5.5870 mL | |
| 5 mM | 0.1117 mL | 0.5587 mL | 1.1174 mL | |
| 10 mM | 0.0559 mL | 0.2793 mL | 0.5587 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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