As an endogenous protein, Cytochrome c interacts with several molecular partners. It transfers electrons to Cytochrome c oxidase (CcO, Complex IV). The reaction with isolated CcO showed half-maximal turnover at a substrate concentration of 2.5 μM for unphosphorylated cytochrome c, whereas Tyr97-phosphorylated cytochrome c required 5.5 μM. Maximal turnover for Tyr48-phosphorylated cytochrome c was more than 50% reduced compared to the unphosphorylated form. [1]
It binds to cardiolipin via two sites (A-site and C-site) and acts as a cardiolipin peroxidase. The phosphomimetic substitution Tyr48Glu decreased cytochrome c-cardiolipin binding by about 30% compared to wild-type. [1]
Upon release, it binds to Apaf-1 to form the apoptosome. A mutant containing the phosphomimetic amino acid Glu48 (instead of Tyr48) was completely incapable of inducing downstream caspase activation. [1]
Cytochrome c targets two key cellular processes: mitochondrial electron transport and apoptosis. In the electron transport chain, cytochrome c acts as an electron carrier, transferring electrons from Complex III to Complex IV, contributing to the generation of ATP through oxidative phosphorylation. In apoptosis, cytochrome c is released from the mitochondria into the cytoplasm, where it binds to apoptotic protease-activating factor 1 (Apaf-1), forming the apoptosome, which activates caspase-9 and downstream effector caspases, leading to programmed cell death. Cytochrome c's dual role in cellular energy production and apoptosis makes it a critical regulator of cell fate.

Mammalian Cytochrome C oxidizes cardiolipin, produces reactive oxygen species (ROS) during apoptosis with the cofactor p66Shc, and scavenges ROS under normal circumstances [1].
In vitro, cytochrome c is used as a standard in biochemical assays to study electron transport and apoptosis. The protein's electron transfer activity can be measured spectrophotometrically by following the reduction or oxidation of the heme group. Cytochrome c is also used in cell-free apoptosis assays, where its ability to activate caspases in the presence of Apaf-1 and dATP is assessed. In cell-based assays, the release of cytochrome c from mitochondria is used as a marker of apoptosis. Cytochrome c is a key player in the intrinsic apoptosis pathway, and its release from mitochondria is a critical step in the execution of programmed cell death.

In vivo, cytochrome c is essential for normal cellular function, as it is a critical component of the mitochondrial electron transport chain. The protein is ubiquitously expressed in eukaryotic cells and is required for ATP production through oxidative phosphorylation. Cytochrome c's role in apoptosis is also important for normal development and tissue homeostasis, as well as for the elimination of damaged or infected cells. Dysregulation of cytochrome c function has been implicated in various diseases, including neurodegenerative disorders, cardiovascular disease, and cancer. Cytochrome c is used in clinical diagnostics as a marker of mitochondrial dysfunction and apoptosis.

CcO activity assay: The reaction of cytochrome c with isolated cytochrome c oxidase (CcO) was measured. For Tyr97-phosphorylated cytochrome c, enhanced signal kinetics were observed, and half-maximal turnover was observed at a substrate concentration of 5.5 μM compared to 2.5 μM for unphosphorylated cytochrome c. For Tyr48-phosphorylated cytochrome c, the kinetics were hyperbolic but maximal turnover was more than 50% reduced for the phosphorylated form. [1]
Cardiolipin peroxidase assay: The peroxidase activity of cytochrome c was measured using electron paramagnetic resonance (EPR) to detect tyrosyl radical formation. In the presence of non-oxidizable cardiolipin (TOCL) and H₂O₂, a strong EPR signal for tyrosyl radical was detected. In the presence of oxidizable cardiolipin (TLCL), the magnitude of the tyrosine radical EPR signal was significantly less (30 ± 7% decrease), attributed to quenching of the radical by its involvement in cardiolipin peroxidation. [1]
Mitochondrial membrane potential measurement: The mitochondrial membrane potential (ΔΨₘ) under physiological conditions (80-140 mV) was measured in perfused rat hearts, intact cultured fibroblasts, neuroblastoma cells, etc., using various methods. It was noted that higher ΔΨₘ (>140 mV) leads to exponential ROS production. [1]
In vitro enzyme assays for cytochrome c involve measuring its electron transfer activity. The assay typically uses purified cytochrome c and cytochrome c oxidase (Complex IV) or other electron acceptors. The reduction of cytochrome c by a reducing agent (e.g., dithionite) or by Complex III is monitored spectrophotometrically at 550 nm. The oxidation of reduced cytochrome c by cytochrome c oxidase is also monitored. The activity of cytochrome c is expressed as the rate of electron transfer. For apoptosis assays, cytochrome c is added to cell lysates or purified Apaf-1 and procaspase-9, and caspase activation is measured using fluorogenic substrates.

In vitro cell-based assays for cytochrome c involve assessing its release from mitochondria as a marker of apoptosis. Cells are treated with apoptotic stimuli (e.g., staurosporine, etoposide, or UV irradiation) and harvested at various time points. Mitochondrial and cytoplasmic fractions are prepared by differential centrifugation. Cytochrome c levels in the cytoplasmic fraction are assessed by Western blotting or ELISA. The release of cytochrome c is correlated with the activation of caspases and other markers of apoptosis. The mitochondrial membrane potential is assessed using fluorescent dyes such as JC-1 or TMRM. The integrity of the mitochondrial outer membrane is assessed by measuring the release of other mitochondrial proteins.

In vivo animal studies with cytochrome c are not typically performed, as the protein is an endogenous component of cells rather than a drug. However, the effects of cytochrome c release on apoptosis and disease progression are studied in animal models. In models of ischemia-reperfusion injury, neurodegeneration, and cancer, the release of cytochrome c from mitochondria is assessed as a marker of cell death. The effects of compounds that modulate cytochrome c release (e.g., inhibitors of the mitochondrial permeability transition pore) are studied in vivo. Cytochrome c levels in tissues and body fluids are measured as biomarkers of mitochondrial dysfunction and apoptosis.

Cytochrome c is an endogenous protein, and its pharmacokinetics are not applicable as a drug. However, the protein's turnover and degradation are regulated by cellular mechanisms. Cytochrome c is synthesized in the cytoplasm and imported into mitochondria, where it is incorporated into the electron transport chain. The protein's stability and half-life depend on cellular conditions. In research applications, cytochrome c is typically supplied as a purified protein from bovine or equine heart tissue. The protein is stored at -20°C and is stable for extended periods under appropriate conditions.

Cytochrome c is an endogenous protein and is not associated with toxicity in its normal physiological context. However, the release of cytochrome c from mitochondria into the cytoplasm triggers apoptosis, which can be beneficial (e.g., elimination of damaged cells) or detrimental (e.g., excessive cell death in ischemia-reperfusion injury or neurodegeneration). As a research reagent, cytochrome c is generally considered safe to handle with standard laboratory precautions. The protein is for research use only and not for human therapeutic applications.

[1]. The multiple functions of cytochrome c and their regulation in life and death decisions of the mammalian cell: From respiration to apoptosis. Mitochondrion. 2011 May;11(3):369-81.

Type C cytochromes are found in eukaryotic mitochondria. They act as redox intermediates, accepting electrons from mitochondrial electron transport complex III and transferring them to mitochondrial electron transport complex IV.

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Multi-functionality: Cytochrome c is a multi-functional enzyme involved in electron transfer (respiration), radical scavenging, ROS production (via p66Shc), cardiolipin peroxidation, and apoptosome formation (apoptosis). [1]
Phosphorylation regulation: Cytochrome c is phosphorylated in vivo in a tissue-specific manner. Four phosphorylation sites have been identified: Tyr97 (cow heart), Tyr48 (cow liver), and Thr28/Ser47 (human skeletal muscle). These phosphorylations inhibit respiration and affect apoptosis. [1]
Cardiolipin oxidation: During apoptosis, cardiolipin (normally in the inner mitochondrial membrane) translocates to the outer membrane. Cytochrome c binds to cardiolipin via the C-site (involving Phe10 and Tyr97) and, in the presence of H₂O₂, catalyzes its oxidation. This oxidation decreases the affinity between cardiolipin and cytochrome c, facilitating its release. Tyr67 is identified as the major contributor to the peroxidase activity. [1]
Therapeutic potential: In a mouse model of sepsis, intravenous injection of exogenous cytochrome c improved mitochondrial function and increased survival from 15% (controls) to about 50%. Injected cytochrome c was taken up into cardiomyocytes. The mechanism may involve cell-penetrating peptide epitopes in cytochrome c. [1]
Isoforms: A testis-specific isoform of cytochrome c (T-Cytc) exists in rodents (but is a pseudogene in humans). T-Cytc shows a three-fold increased H₂O₂ reduction activity and about a four-fold increased apoptotic activity compared to the somatic isoform. [1]
Cytochrome c is a well-characterized protein that has been studied extensively for its roles in cellular energy metabolism and apoptosis. The protein's structure and function have been elucidated in detail, making it a model system for studying electron transfer proteins and apoptosis. Cytochrome c is used as a standard in biochemical research and as a marker in clinical diagnostics for the assessment of mitochondrial function and apoptosis. The protein is available from chemical suppliers for research purposes, typically purified from bovine or equine heart tissue. Cytochrome c's dual role in energy production and cell death makes it a fascinating molecule for studying the intersection of metabolism and cell fate.

13000 (average)

9007-43-6

16057918

Brown to reddish brown solid powder

1323.5ºC at 760 mmHg

300ºC

754.2ºC

6

14

16

59

1420

0

CC1=C(C2=CC3=C(C(=C([N-]3)C=C4C(=C(C(=N4)C=C5C(=C(C(=N5)C=C1[N-]2)C)C(C)SCC(C(=O)NC)N)C)C(C)SCC(C(=O)NC)N)C)CCC(=O)O)CCC(=O)O.[Fe+2]

WFVBWSTZNVJEAY-UHFFFAOYSA-L

InChI=1S/C42H54N8O6S2.Fe/c1-19-25(9-11-37(51)52)33-16-34-26(10-12-38(53)54)20(2)30(48-34)14-35-40(24(6)58-18-28(44)42(56)46-8)22(4)32(50-35)15-36-39(21(3)31(49-36)13-29(19)47-33)23(5)57-17-27(43)41(55)45-7;/h13-16,23-24,27-28H,9-12,17-18,43-44H2,1-8H3,(H6,45,46,47,48,49,50,51,52,53,54,55,56);/q;+2/p-2

3-[8,13-bis[1-[2-amino-3-(methylamino)-3-oxopropyl]sulfanylethyl]-18-(2-carboxyethyl)-3,7,12,17-tetramethylporphyrin-21,24-diid-2-yl]propanoic acid;iron(2+)

9007-43-6; Cytochrome C [JAN]; Cytochrome C (JAN); Myohematin; Cromoci;

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : 100 mg/mL

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)