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Phalloidin [Mast Cell Degranulating (MCD) Peptide] is a novel and potent toxin derived from mushroom and may be used to label F-actin of the cytoskeleton with fluorochrome (λex=495 nm, λem=520 nm). It has a sequence of Ala-{d-Thr}-Cys-{Hyp}-Ala-Trp-Leu (Disulfide bridge: Cys3-Cys6); and sequence shortening of A-{d-Thr}-C-{Hyp}-AWL (Disulfide bridge: Cys3-Cys6).
| Targets |
Fluorescent Dye; F-actin
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|---|---|
| ln Vitro |
Phalloidin staining works well on cells fixed in PBS containing 0.2% glutaraldehyde, but not on fixed cells (possibly because of defects in disrupted actin filaments) [1].
The eukaryotic cell has evolved to compartmentalize its functions and transport various metabolites among cellular compartments. Therefore, in cell biology, the study of organization and structure/function relationships is of great importance. The cytoskeleton is composed of a series of filamentous structures, including intermediate filaments, actin filaments, and microtubules. Immunofluorescent staining has been most frequently used to study cytoskeletal components. However, it is also possible to fluorescently label isolated cytoskeletal proteins and either microinject them back into the cell or add them to fixed, permeabilized cells. Alternatively, it is possible to use the mushroom-derived fluorescinated toxins, phalloidin or phallacidin, to label F-actin of the cytoskeleton, as is described in this article. Phalloidin is available labeled with different fluorophores. The choice of the specific fluorophore should depend on whether phalloidin labeling for actin is part of a double-label experiment. In most cells, the abundance of actin filaments should provide a very strong signal. In double-label experiments, the fluorophore should be chosen to take this into account. In general, rhodamine labels are more resistant to photobleaching and can be subjected to the longer exposures required for finer structures[1]. |
| Cell Assay |
Phalloidin Staining Protocol [1]
1. Dilution: Prepare a 1:200 dilution of fluorescein or rhodamine Phalloidin (300 units/mL stock) in PBS. 2. Washing: Aspirate the cell culture medium from cells grown on glass coverslips and rinse three times with PBS. 3. Fixation: Fix cells for 10 minutes in 3.7% formaldehyde solution. 4. Post-Fixation Wash: Rinse fixed cells three times with PBS (5 min per wash). 5. Permeabilization: Treat cells with 0.2% Triton X-100 for 5 minutes. 6. Post-Permeabilization Wash: Rinse cells three times with PBS. 7. Staining: Incubate cells with fluorescein or rhodamine Phalloidin for 5–10 minutes at room temperature. 8. Final Wash: Rinse cells three times with PBS (5 min per wash). 9. Mounting & Imaging: Mount coverslips and observe cells using the appropriate filter set (fluorescein or rhodamine, depending on the probe). |
| ADME/Pharmacokinetics |
Absorption, Distribution and Excretion
This study compared the absorption, binding, and excretion of phalloidin in the livers of adult rats and young rats (17-19 days old) in vivo and in vitro. No correlation was found between the concentration of the toxicant in the liver and its toxicity in either group of rats. Although young rats showed significantly higher tolerance to phalloidin than adult rats, the concentration of the toxicant in the liver of young rats was higher, and the excretion rate was significantly slower than in adult rats. The extremely high binding affinity and concentration of phalloidin in the liver can be attributed to its extremely low dissociation constant. Following administration of Amanita phalloidin to rats, both phalloidin and γ-amaminine were detectable in urine on day 1, and phalloidin was detectable in urine on day 2. Metabolism/Metabolites Various observations indicate that phalloidin (a toxic component of Amanita phalloides) itself is not toxic, but rather undergoes bioactivation in the liver. Using (3)H-demethylphalanxane (a toxic phalanxane derivative), /it was concluded/...(3)H-demethylphalanxane, and likely also phalanxane, is not metabolized in the livers of rats and mice. Secondary excretion products found in the urine of the test animals were self-degrading compounds of the (3)H-labeled derivative. |
| Toxicity/Toxicokinetics |
Interactions
Pre-oral administration of rifampin (100 or 300 mg/kg) protected mice from the toxicity of phalloidin (3 mg/kg, intraperitoneal injection), resulting in a reduction in mortality. In mice, treatment with silybin (a plant compound isolated from milk thistle) prevented phalloidin-induced acute hemorrhagic necrosis of the liver. A single administration of silybin pretreatment completely eliminated the morphological changes induced by the toxin and significantly reduced serum enzyme activity. Toxicity Overview It binds to actin, preventing its depolymerization and leading to cytotoxicity. Phaloidin specifically binds to the interface between F-actin subunits, locking adjacent subunits together. Phaloidin is a bicyclic heptapeptide that binds to actin filaments much more strongly than to actin monomers, resulting in a reduced rate constant for the dissociation of actin subunits from the filament ends, thereby stabilizing actin filaments by preventing filament depolymerization. Furthermore, the study found that phalloidin can also inhibit the ATP hydrolytic activity of F-actin (L1183). LD50: 2 mg/kg (mice) (T258) |
| References | |
| Additional Infomation |
Phalloidin is a homobicyclic heptapeptide containing a sulfur bridge. It has been reported to exist in Amanita suballiacea, Amanita phalloides, and other organisms with relevant data. It is a highly toxic polypeptide, primarily isolated from Amanita phalloides, also known as the "death cup"; it can cause mushroom poisoning, resulting in fatal liver, kidney, and central nervous system damage; it is commonly used in liver injury research. Mechanism of Action Unlike normal hepatocytes, AS-30D rat hepatocellular carcinoma cells are insensitive to phalotoxin. Hepatocellular carcinoma cells apparently do not consume this toxin like normal hepatocytes. Tolerant doses of phalotoxin can protect mice from lethal doses. Tolerance is induced at 1/10 LD95 of phalotoxin, appearing approximately 8 hours after pretreatment.
Within 1 hour of exposure to 50 μg/ml phalloidin, primary hepatocyte cultures from adult rats showed 60-70% cell death (trypan blue staining). In Ca2+-free media, the same cells exposed to phalloidin did not show loss of viability. Phaloidin initially interacts with cell membrane-associated actin via a Ca2+-independent process. The second step is a Ca2+-dependent process, likely representing Ca2+ influx into the cell through a damaged cell membrane permeability barrier and downward flow along the steep concentration gradient between the intracellular and extracellular spaces. These results reinforce the hypothesis of in vivo Ca2+ homeostasis disturbance. Seven consecutive days (500 μg/kg/day) of phalloidin injection into male rats increased the mean content of hepatic filamentous actin. Bile flow and bile acid excretion were correspondingly reduced. Microfilaments may affect the permeability of tight junctions between hepatocytes. In phalloidin-induced cholestasis, bile components may reflux from bile canaliculi into the intercellular space. |
| Molecular Formula |
C35H48N8O12S
|
|---|---|
| Molecular Weight |
804.867020000001
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| Exact Mass |
788.316
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| CAS # |
17466-45-4
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| PubChem CID |
441542
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| Appearance |
White to off-white solid powder
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| Density |
1.51g/cm3
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| Boiling Point |
1370.5ºC at 760mmHg
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| Melting Point |
MAXIMUM ABSORPTION (WATER): 295 NM (E= 0.597, 1%, 1 CM); MELTING POINT: 280-282 °C /HEXAHYDRATE/
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| Flash Point |
782.6ºC
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| Vapour Pressure |
0mmHg at 25°C
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| Index of Refraction |
1.683
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| LogP |
-1.7
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| Hydrogen Bond Donor Count |
11
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| Hydrogen Bond Acceptor Count |
12
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
55
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| Complexity |
1510
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| Defined Atom Stereocenter Count |
10
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| SMILES |
C[C@H]1C(=O)N[C@H]2CC3=C(NC4=CC=CC=C34)SC[C@@H](C(=O)N5C[C@H](C[C@H]5C(=O)N1)O)NC(=O)[C@H](NC(=O)[C@@H](NC(=O)[C@@H](NC2=O)C[C@](C)(CO)O)C)[C@H](C)O
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| InChi Key |
KPKZJLCSROULON-QKGLWVMZSA-N
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| InChi Code |
InChI=1S/C35H48N8O11S/c1-15-27(47)38-22-10-20-19-7-5-6-8-21(19)41-33(20)55-13-24(34(53)43-12-18(46)9-25(43)31(51)37-15)40-32(52)26(17(3)45)42-28(48)16(2)36-30(50)23(39-29(22)49)11-35(4,54)14-44/h5-8,15-18,22-26,41,44-46,54H,9-14H2,1-4H3,(H,36,50)(H,37,51)(H,38,47)(H,39,49)(H,40,52)(H,42,48)/t15-,16-,17-,18-,22-,23-,24-,25-,26+,35+/m0/s1
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| Chemical Name |
(1S,14R,18S,20S,23S,28S,31S,34R)-28-[(2R)-2,3-dihydroxy-2-methylpropyl]-18-hydroxy-34-[(1S)-1-hydroxyethyl]-23,31-dimethyl-12-thia-10,16,22,25,27,30,33,36-octazapentacyclo[12.11.11.03,11.04,9.016,20]hexatriaconta-3(11),4,6,8-tetraene-15,21,24,26,29,32,35-heptone
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| Synonyms |
17466-45-4; Phalloidin from Amanita phalloides; 28-(2,3-Dihydroxy-2-methylpropyl)-18-hydroxy-34-(1-hydroxyethyl)-23,31-dimethyl-12-thia-10,16,22,25,27,30,33,36-octazapentacyclo[12.11.11.03,11.04,9.016,20]hexatriaconta-3(11),4,6,8-tetraene-15,21,24,26,29,32,35-heptone; SCHEMBL39329; GTPL4736; KPKZJLCSROULON-UHFFFAOYSA-N; NSC523214; SMP1_000234;
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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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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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 | 1.2424 mL | 6.2122 mL | 12.4244 mL | |
| 5 mM | 0.2485 mL | 1.2424 mL | 2.4849 mL | |
| 10 mM | 0.1242 mL | 0.6212 mL | 1.2424 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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